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Sample records for activatable cell-penetrating peptides

  1. Single-cell resolution imaging of retinal ganglion cell apoptosis in vivo using a cell-penetrating caspase-activatable peptide probe.

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    Xudong Qiu

    Full Text Available Peptide probes for imaging retinal ganglion cell (RGC apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in

  2. Enhancing siRNA-based cancer therapy using a new pH-responsive activatable cell-penetrating peptide-modified liposomal system

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    Xiang B

    2017-03-01

    Full Text Available Bai Xiang,1,* Xue-Li Jia,1,* Jin-Long Qi,2 Li-Ping Yang,1 Wei-Hong Sun,1 Xiao Yan,1 Shao-Kun Yang,1 De-Ying Cao,1 Qing Du,1 Xian-Rong Qi3 1Department of Pharmaceutics, School of Pharmaceutical Sciences, 2Department of Pharmacology, Hebei Medical University, Shijiazhuang, Hebei, 3School of Pharmaceutical Sciences, Peking University, Beijing, China *These authors contributed equally to this work Abstract: As a potent therapeutic agent, small interfering RNA (siRNA has been exploited to silence critical genes involved in tumor initiation and progression. However, development of a desirable delivery system is required to overcome the unfavorable properties of siRNA such as its high degradability, molecular size, and negative charge to help increase its accumulation in tumor tissues and promote efficient cellular uptake and endosomal/lysosomal escape of the nucleic acids. In this study, we developed a new activatable cell-penetrating peptide (ACPP that is responsive to an acidic tumor microenvironment, which was then used to modify the surfaces of siRNA-loaded liposomes. The ACPP is composed of a cell-penetrating peptide (CPP, an acid-labile linker (hydrazone, and a polyanionic domain, including glutamic acid and histidine. In the systemic circulation (pH 7.4, the surface polycationic moieties of the CPP (polyarginine are “shielded” by the intramolecular electrostatic interaction of the inhibitory domain. When exposed to a lower pH, a common property of solid tumors, the ACPP undergoes acid-catalyzed breakage at the hydrazone site, and the consequent protonation of histidine residues promotes detachment of the inhibitory peptide. Subsequently, the unshielded CPP would facilitate the cellular membrane penetration and efficient endosomal/lysosomal evasion of liposomal siRNA. A series of investigations demonstrated that once exposed to an acidic pH, the ACPP-modified liposomes showed elevated cellular uptake, downregulated expression of polo

  3. Blocking hepatic metastases of colon cancer cells using an shRNA against Rac1 delivered by activatable cell-penetrating peptide.

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    Bao, Ying; Guo, Huihui; Lu, Yongliang; Feng, Wenming; Sun, Xinrong; Tang, Chengwu; Wang, Xiang; Shen, Mo

    2016-11-22

    Hepatic metastasis is one of the critical progressions of colon cancer. Blocking this process is key to prolonging survival time in cancer patients. Studies on activatable cell-penetrating peptides (dtACPPs) have demonstrated their potential as gene carriers. It showed high tumor cell-targeting specificity and transfection efficiency and low cytotoxicity in the in vitro settings of drug delivery. However, using this system to silence target genes to inhibit metastasis in colorectal cancer cells has not been widely reported and requires further investigation. In this study, we observed that expression of Rac1, a key molecule for cytoskeletal reorganization, was higher in hepatic metastatic tumor tissue compared with prime colon cancer tissue and that patients with high Rac1-expressing colon cancer showed shorter survival time. Base on these findings, we created dtACPP-PEG-DGL (dtACPPD)/shRac1 nanoparticles and demonstrated that they downregulated Rac1 expression in colon cancer cells. Moreover, we observed inhibitory effects on migration, invasion and adhesion in HCT116 colorectal cancer cells in vitro, and our results showed that Rac1 regulated colon cancer cell matrix adhesion through the regulation of cytofilament dynamics. Moreover, mechanically, repression of Rac1 inhibiting cells migration and invasion by enhancing cell to cell adhesion and reducing cell to extracellular matrix adhesion. Furthermore, when atCDPPD/shRac1 nanoparticles were administered intravenously to a HCT116 xenograft model, significant tumor metastasis to the liver was inhibited. Our results suggest that atCDPP/shRac1 nanoparticles may enable the blockade of hepatic metastasis in colon cancer.

  4. Cell-penetrating peptides for drug delivery across membrane barriers

    DEFF Research Database (Denmark)

    Foged, Camilla; Nielsen, Hanne Moerck

    2008-01-01

    During the last decade, cell-penetrating peptides have been investigated for their ability to overcome the plasma membrane barrier of mammalian cells for the intracellular or transcellular delivery of cargoes as diverse as low molecular weight drugs, imaging agents, oligonucleotides, peptides......, proteins and colloidal carriers such as liposomes and polymeric nanoparticles. Their ability to cross biological membranes in a non-disruptive way without apparent toxicity is highly desired for increasing drug bioavailability. This review provides an overview of the application of cell......-penetrating peptides as transmembrane drug delivery agents, according to the recent literature, and discusses critical issues and future challenges in relation to fully understanding the fundamental principles of the cell-penetrating peptide-mediated membrane translocation of cargoes and the exploitation...

  5. Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections

    DEFF Research Database (Denmark)

    Bahnsen, Jesper S; Franzyk, Henrik; Sayers, Edward J

    2015-01-01

    PURPOSE: To investigate the suitability of three antimicrobial peptides (AMPs) as cell-penetrating antimicrobial peptides. METHODS: Cellular uptake of three AMPs (PK-12-KKP, SA-3 and TPk) and a cell-penetrating peptide (penetratin), all 5(6)-carboxytetramethylrhodamine-labeled, were tested in He......La WT cells and analyzed by flow cytometry and confocal microscopy. Furthermore, the effects of the peptides on eukaryotic cell viability as well as their antimicrobial effect were tested. In addition, the disrupting ability of the peptides in the presence of bilayer membranes of different composition...... the cellular viability to an unacceptable degree. TPk showed acceptable uptake efficiency, high antimicrobial activity and relatively low toxicity, and it is the best potential lead peptide for further development....

  6. Bioportide: an emergent concept of bioactive cell-penetrating peptides

    Czech Academy of Sciences Publication Activity Database

    Howl, J.; Matou-Nasri, S.; West, D. C.; Farquhar, M.; Slaninová, Jiřina; Ostenson, C. G.; Zorko, M.; Ostlund, P.; Kumar, S.; Langel, U.; McKeating, J.; Jones, S.

    2012-01-01

    Roč. 69, č. 17 (2012), s. 2951-2966 ISSN 1420-682X Institutional research plan: CEZ:AV0Z40550506 Keywords : angiogenesis * bioportide * cell-penetrating peptide * second messenger * insulin secretion Subject RIV: CE - Biochemistry Impact factor: 5.615, year: 2012

  7. [Potential of cell penetrating peptides for cell drug delivery].

    Science.gov (United States)

    Poillot, Cathy; De Waard, Michel

    2011-05-01

    The interest of the scientific community for cell penetrating peptides (CPP) has been growing exponentially for these last years, and the list of novel CPP is increasing. These peptides are powerful tools for the delivery of cargoes to their site of action. Indeed, several drugs that cannot translocate through the cell plasma membrane have been successfully delivered into cells when grafted to a CPP. Various cargoes have been linked to CPP, such as oligonucleotides, pharmacologically active drugs, contrast agents for imaging, or nanoparticles as platforms for multigrafting purposes… This review illustrates the fabulous potential of CPP and the diversity of their use, but their most interesting application appears their future clinical use for the treatment of various pathological conditions. © 2011 médecine/sciences - Inserm / SRMS.

  8. Translocation of cell-penetrating peptides into Candida fungal pathogens.

    Science.gov (United States)

    Gong, Zifan; Karlsson, Amy J

    2017-09-01

    Cell-penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF) 3 K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration-dependent manner, and an additional peptide (TP-10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF) 3 K, and TP-10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens. © 2017 The Protein Society.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  10. Electrochemistry of a ferrocene-grafted cell-penetrating peptide

    International Nuclear Information System (INIS)

    Messina, Pierluca; Hallais, Géraldine; Labbé, Eric; Béranger, Marie; Chassaing, Gérard; Lavielle, Solange; Mansuy, Christelle; Buriez, Olivier

    2012-01-01

    A cationic cell-penetrating peptide (CPP) labeled with both a ferrocenyl (Fc) moiety and a biotin (B) was successfully synthesized and investigated by electrochemistry. This original CPP derivative noted as Fc-CPP-B could be electrochemically detected, at a micromolar concentration, at a naked gold bead electrode. The presence of a biotin tag in the Fc-CPP-B complex allowed its complexation with avidin, which was itself tethered to a thiolated self-assembled monolayer. Such an avidin-modified gold surface, characterized by atomic force microscopy (AFM), allowed the immobilization of Fc-CPP-B onto the electrode surface, which greatly enhanced its electrochemical detection. Nevertheless, under these conditions the electrogenerated ferrocenium cation could not be reduced during the backward scan, indicating its unexpected reactivity when tethered within the avidin environment. In terms of detection and redox probe regeneration the best results were obtained at a glassy carbon electrode modified with a cation-exchange polymer. Ion-exchange voltammetry, performed under these conditions, allowed the pre-concentration of the peptide at the electrode surface thanks to the net positive charge of the CPP derivative. Interestingly, the anionic character of the polymer contributed to retain the electrogenerated cation Fc + in the film so that it could be reduced back to its original neutral form during the reverse voltammetric scans.

  11. Internalisation of cell-penetrating peptides into tobacco protoplasts.

    Science.gov (United States)

    Mäe, Maarja; Myrberg, Helena; Jiang, Yang; Paves, Heiti; Valkna, Andres; Langel, Ulo

    2005-05-20

    Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.

  12. Translocation of Cell Penetrating Peptide Engrafted Nanoparticles Across Skin Layers

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    Patlolla, Ram R; Desai, Pinaki; Belay, Kalayu; Singh, Mandip

    2010-01-01

    The objective of the current study was to evaluate the ability of cell penetrating peptides (CPP) to translocate the lipid payload into the skin layers. Fluorescent dye (DID-oil) encapsulated nano lipid crystal nanoparticles (FNLCN) were prepared using Compritol, Miglyol and DOGS-NTA-Ni lipids by hot melt homogenization technique. The FNLCN surface was coated with TAT peptide (FNLCNT) or control YKA peptide (FNLCNY) and in vitro rat skin permeation studies were performed using Franz diffusion cells. Observation of lateral skin sections obtained using cryotome with a confocal microscope demonstrated that skin permeation of FNLCNT was time dependent and after 24 h, fluorescence was observed upto a depth of 120 µm which was localized in the hair follicles and epidermis. In case of FNLCN and FNLCNY formulations fluorescence was mainly observed in the hair follicles. This observation was further supported by confocal Raman spectroscopy where higher fluorescence signal intensity was observed at 80 and 120 µm depth with FNLCNT treated skin and intensity of fluorescence peaks was in the ratio of 2:1:1 and 5:3:1 for FNLCNT, FNLCN, and FNLCNY treated skin sections, respectively. Furthermore, replacement of DID-oil with celecoxib (Cxb), a model lipophilic drug showed similar results and after 24 h, the CXBNT formulation increased the Cxb concentration in SC by 3 and 6 fold and in epidermis by 2 and 3 fold as compared to CXBN and CXBNY formulations respectively. Our results strongly suggest that CPP can translocate nanoparticles with their payloads into deeper skin layers. PMID:20413152

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

    KAUST Repository

    Haidar, Malak; de Laté , Perle Latré ; Kennedy, Eileen J.; Langsley, Gordon

    2017-01-01

    One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way

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

    Science.gov (United States)

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

    2017-01-04

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

  15. Self-association of a highly charged arginine-rich cell-penetrating peptide

    Czech Academy of Sciences Publication Activity Database

    Tesei, G.; Vazdar, M.; Jensen, M. R.; Cragnell, C.; Mason, Philip E.; Heyda, J.; Skepö, M.; Jungwirth, Pavel; Lund, M.

    2017-01-01

    Roč. 114, č. 43 (2017), s. 11428-11433 ISSN 0027-8424 R&D Projects: GA ČR(CZ) GA16-01074S Institutional support: RVO:61388963 Keywords : cell-penetrating peptide * self-association * MD simulations * SAXS * NMR Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 9.661, year: 2016

  16. Metabolic cleavage of cell-penetrating peptides in contact with epithelial models

    DEFF Research Database (Denmark)

    Tréhin, Rachel; Nielsen, Hanne Mørck; Jahnke, Heinz-Georg

    2004-01-01

    We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) a...

  17. The uptake of arginine-rich cell-penetrating peptides: putting the puzzle together

    NARCIS (Netherlands)

    Brock, R.E.

    2014-01-01

    Over the past 20 years, cell-penetrating peptides (CPPs) have captured the attention of biomedical researchers, biophysicists, and (bio)organic chemists. These molecules efficiently enter cells and mediate entry of (macro)molecules that by themselves do not cross the plasma membrane. Since their

  18. Alternative Mechanisms for the Interaction of the Cell-Penetrating Peptides Penetratin and the TAT Peptide with Lipid Bilayers

    NARCIS (Netherlands)

    Yesylevskyy, Semen; Marrink, Siewert-Jan; Mark, Alan E.

    2009-01-01

    Cell-penetrating peptides (CPPs) have recently attracted much interest due to their apparent ability to penetrate cell membranes in an energy-independent manner. Here molecular-dynamics simulation techniques were used to study the interaction of two CPPs: penetratin and the TAT peptide with

  19. Sensing lymphoma cells based on a cell-penetrating/apoptosis-inducing/electron-transfer peptide probe

    International Nuclear Information System (INIS)

    Sugawara, Kazuharu; Shinohara, Hiroki; Kadoya, Toshihiko; Kuramitz, Hideki

    2016-01-01

    To electrochemically sense lymphoma cells (U937), we fabricated a multifunctional peptide probe that consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. Electron-transfer peptides derive from cysteine residue combined with the C-terminals of four tyrosine residues (Y_4). A peptide whereby Y_4C is bound to the C-terminals of protegrin 1 (RGGRLCYCRRRFCVCVGR-NH_2) is known to be an apoptosis-inducing agent against U937 cells, and is referred to as a peptide-1 probe. An oxidation response of the peptide-1 probe has been observed due to a phenolic hydroxyl group, and this response is decreased by the uptake of the peptide probe into the cells. To improve the cell membrane permeability against U937 cells, the RGGR at the N-terminals of the peptide-1 probe was replaced by RRRR (peptide-2 probe). In contrast, RNRCKGTDVQAWY_4C (peptide-3 probe), which recognizes ovalbumin, was constructed as a control. Compared with the other probes, the change in the peak current of the peptide-2 probe was the greatest at low concentrations and occurred in a short amount of time. Therefore, the cell membrane permeability of the peptide-2 probe was increased based on the arginine residues and the apoptosis-inducing peptides. The peak current was linear and ranged from 100 to 1000 cells/ml. The relative standard deviation of 600 cells/ml was 5.0% (n = 5). Furthermore, the membrane permeability of the peptide probes was confirmed using fluorescent dye. - Highlights: • We constructed a multifunctional peptide probe for the electrochemical sensing of lymphoma cells. • The peptide probe consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. • The electrode response of the peptide probe changes due to selective uptake into the cells.

  20. Sensing lymphoma cells based on a cell-penetrating/apoptosis-inducing/electron-transfer peptide probe

    Energy Technology Data Exchange (ETDEWEB)

    Sugawara, Kazuharu, E-mail: kzsuga@maebashi-it.ac.jp [Maebashi Institute of Technology, Gunma 371-0816 (Japan); Shinohara, Hiroki; Kadoya, Toshihiko [Maebashi Institute of Technology, Gunma 371-0816 (Japan); Kuramitz, Hideki [Department of Environmental Biology and Chemistry, Graduate School of Science and Engineering for Research, University of Toyama, Toyama 930-8555 (Japan)

    2016-06-14

    To electrochemically sense lymphoma cells (U937), we fabricated a multifunctional peptide probe that consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. Electron-transfer peptides derive from cysteine residue combined with the C-terminals of four tyrosine residues (Y{sub 4}). A peptide whereby Y{sub 4}C is bound to the C-terminals of protegrin 1 (RGGRLCYCRRRFCVCVGR-NH{sub 2}) is known to be an apoptosis-inducing agent against U937 cells, and is referred to as a peptide-1 probe. An oxidation response of the peptide-1 probe has been observed due to a phenolic hydroxyl group, and this response is decreased by the uptake of the peptide probe into the cells. To improve the cell membrane permeability against U937 cells, the RGGR at the N-terminals of the peptide-1 probe was replaced by RRRR (peptide-2 probe). In contrast, RNRCKGTDVQAWY{sub 4}C (peptide-3 probe), which recognizes ovalbumin, was constructed as a control. Compared with the other probes, the change in the peak current of the peptide-2 probe was the greatest at low concentrations and occurred in a short amount of time. Therefore, the cell membrane permeability of the peptide-2 probe was increased based on the arginine residues and the apoptosis-inducing peptides. The peak current was linear and ranged from 100 to 1000 cells/ml. The relative standard deviation of 600 cells/ml was 5.0% (n = 5). Furthermore, the membrane permeability of the peptide probes was confirmed using fluorescent dye. - Highlights: • We constructed a multifunctional peptide probe for the electrochemical sensing of lymphoma cells. • The peptide probe consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. • The electrode response of the peptide probe changes due to selective uptake into the cells.

  1. Conformational analysis of Infectious bursal disease virus (IBDV derived cell penetrating peptide (CPP analogs

    Directory of Open Access Journals (Sweden)

    Vinay G. Joshi

    2013-12-01

    Full Text Available Aim: This study was designed to develop peptide analogs of Infectious Bursal Disease (IBD virus VP5 protein segment having cell penetrating ability to improve their interaction with cargo molecule (Nucleic acid without affecting the backbone conformation. Materials and Methods: IBDV VP5 protein segment designated as RATH peptide were synthesized using solid phase peptide synthesis and their solution conformation was elucidated using CD spectroscopy in polar (water and apolar (TFE solvents. Cell penetrating ability of RATH-CONH2 was observed using FITC labeled peptide internalization in to HeLa cells under fluorescent microscopy. The efficacy of RATH analog interactions with nucleic acids was evaluated using FITC labeled oligonucleotides by fluorescence spectroscopy and plasmid constructs in gel retardation assay. Results: CD spectra of RATH analogs in water and apolar trifluroethanol (TFE helped to compare their secondary structures which were almost similar with dominant beta conformations suggesting successful induction of positive charge in the analogs without affecting back bone conformation of CPP designed. Cell penetrating ability of RATH CONH2 in HeLa cell was more than 90%. The fluorescence spectroscopy and plasmid constructs in gel retardation assay demonstrated successful interaction of amide analogs with nucleic acid. Conclusion: Intentional changes made in IBDV derived peptide RATH COOH to RATH CONH2 did not showed major changes in backbone conformation and such modifications may help to improve the cationic charge in most CPPs to interact with nucleic acid. [Vet World 2013; 6(6.000: 307-312

  2. Cell-Penetrating Ability of Peptide Hormones: Key Role of Glycosaminoglycans Clustering

    Directory of Open Access Journals (Sweden)

    Armelle Tchoumi Neree

    2015-11-01

    Full Text Available Over the last two decades, the potential usage of cell-penetrating peptides (CPPs for the intracellular delivery of various molecules has prompted the identification of novel peptidic identities. However, cytotoxic effects and unpredicted immunological responses have often limited the use of various CPP sequences in the clinic. To overcome these issues, the usage of endogenous peptides appears as an appropriate alternative approach. The hormone pituitary adenylate-cyclase-activating polypeptide (PACAP38 has been recently identified as a novel and very efficient CPP. This 38-residue polycationic peptide is a member of the secretin/glucagon/growth hormone-releasing hormone (GHRH superfamily, with which PACAP38 shares high structural and conformational homologies. In this study, we evaluated the cell-penetrating ability of cationic peptide hormones in the context of the expression of cell surface glycosaminoglycans (GAGs. Our results indicated that among all peptides evaluated, PACAP38 was unique for its potent efficiency of cellular uptake. Interestingly, the abilities of the peptides to reach the intracellular space did not correlate with their binding affinities to sulfated GAGs, but rather to their capacity to clustered heparin in vitro. This study demonstrates that the uptake efficiency of a given cationic CPP does not necessarily correlate with its affinity to sulfated GAGs and that its ability to cluster GAGs should be considered for the identification of novel peptidic sequences with potent cellular penetrating properties.

  3. Polymeric pH nanosensor with extended measurement range bearing octaarginine as cell penetrating peptide

    DEFF Research Database (Denmark)

    Ke, Peng; Sun, Honghao; Liu, Mingxing

    2016-01-01

    A synthetic peptide octaarginine which mimics human immunodeficiency virus-1, Tat protein is used as cell penetrating moiety for new pH nanosensors which demonstrate enhanced cellular uptake and expanded measurement range from pH 3.9 to pH 7.3 by simultaneously incorporating two complemental pH-s......H-sensitive fluorophores in a same nanoparticle. The authors believe that this triple fluorescent pH sensor provides a new tool to pH measurements that can have application in cellular uptake mechanism study and new nanomedicine design.......A synthetic peptide octaarginine which mimics human immunodeficiency virus-1, Tat protein is used as cell penetrating moiety for new pH nanosensors which demonstrate enhanced cellular uptake and expanded measurement range from pH 3.9 to pH 7.3 by simultaneously incorporating two complemental p...

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

    Science.gov (United States)

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

    2005-12-01

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

  5. Effect of a Fusion Peptide by Covalent Conjugation of a Mitochondrial Cell-Penetrating Peptide and a Glutathione Analog Peptide

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    Carmine Pasquale Cerrato

    2017-06-01

    Full Text Available Previously, we designed and synthesized a library of mitochondrial antioxidative cell-penetrating peptides (mtCPPs superior to the parent peptide, SS31, to protect mitochondria from oxidative damage. A library of antioxidative glutathione analogs called glutathione peptides (UPFs, exceptional in hydroxyl radical elimination compared with glutathione, were also designed and synthesized. Here, a follow-up study is described, investigating the effects of the most promising members from both libraries on reactive oxidative species scavenging ability. None of the peptides influenced cell viability at the concentrations used. Fluorescence microscopy studies showed that the fluorescein-mtCPP1-UPF25 (mtgCPP internalized into cells, and spectrofluorometric analysis determined the presence and extent of peptide into different cell compartments. mtgCPP has superior antioxidative activity compared with mtCPP1 and UPF25 against H2O2 insult, preventing ROS formation by 2- and 3-fold, respectively. Moreover, we neither observed effects on mitochondrial membrane potential nor production of ATP. These data indicate that mtgCPP is targeting mitochondria, protecting them from oxidative damage, while also being present in the cytosol. Our hypothesis is based on a synergistic effect resulting from the fused peptide. The mitochondrial peptide segment is targeting mitochondria, whereas the glutathione analog peptide segment is active in the cytosol, resulting in increased scavenging ability.

  6. Potent inhibition of late stages of hepadnavirus replication by a modified cell penetrating peptide

    DEFF Research Database (Denmark)

    Abdul, Fabien; Ndeboko, Bénédicte; Buronfosse, Thierry

    2012-01-01

    Cationic cell-penetrating peptides (CPPs) and their lipid domain-conjugates (CatLip) are agents for the delivery of (uncharged) biologically active molecules into the cell. Using infection and transfection assays we surprisingly discovered that CatLip peptides were able to inhibit replication...... by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)8-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral...

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  8. Selective mono-radioiodination and characterization of a cell-penetrating peptide. L-Tyr-maurocalcine

    Energy Technology Data Exchange (ETDEWEB)

    Ahmadi, Mitra; Bacot, Sandrine; Perret, Pascale; Riou, Laurent; Ghezzi, Catherine [Universite Joseph Fourier, Grenoble (France); INSERM U1039, Grenoble (France). Radiopharmaceutiques Biocliniques; Poillot, Cathy; Cestele, Sandrine [INSERM U836, Grenoble (France). Grenoble Inst. of Neuroscience; Universite Joseph Fourier, Grenoble (France); Desruet, Marie-Dominique [INSERM U1039, Grenoble (France). Radiopharmaceutiques Biocliniques; Couvet, Morgane; Bourgoin, Sandrine; Seve, Michel [CRI-INSERM U823, Grenoble (France). Inst. of Albert Bonniot; Universite Joseph Fourier, Grenoble (France); Waard, Michel de [INSERM U836, Grenoble (France). Grenoble Inst. of Neuroscience; Universite Joseph Fourier, Grenoble (France); Smartox Biotechnologies, Grenoble (France)

    2014-07-01

    Mono-and poly-iodinated peptides form frequently during radioiodination procedures. However, the formation of a single species in its mono-iodinated form is essential for quantitative studies such as determination of tissue concentration or image quantification. Therefore, the aim of the present study was to define the optimal experimental conditions in order to exclusively obtain the mono-iodinated form of L-maurocalcine (L-MCa). L-MCa is an animal venom toxin which was shown to act as a cell-penetrating peptide. In order to apply the current direct radioiodination technique using oxidative agents including chloramine T, Iodo-Gen {sup registered} or lactoperoxidase, an analogue of this peptide containing a tyrosine residue (Tyr-L-MCa) was synthesized and was shown to fold/oxidize properly. The enzymatic approach using lactoperoxidase/H{sub 2}O{sub 2} was found to be the best method for radioiodination of Tyr-L-MCa. MALDI-TOF mass spectrometry analyses were then used for identification of the chromatographic eluting components of the reaction mixtures. We observed that the production of different radioiodinated species depended upon the reaction conditions. Our results successfully described the experimental conditions of peptide radioiodination allowing the exclusive production of the mono-iodinated form with high radiochemical purity and without the need for a purification step. Mono-radioiodination of L-Tyr-MCa will be crucial for future quantitative studies, investigating the mechanism of cell penetration and in vivo biodistribution.

  9. Cell-Penetrating Peptides as Carriers for Oral Delivery of Biopharmaceuticals

    DEFF Research Database (Denmark)

    Kristensen, Mie; Nielsen, Hanne Mørck

    2016-01-01

    Oral delivery of biopharmaceuticals, for example peptides and proteins, constitutes a great challenge in drug delivery due to their low chemical stability and poor permeation across the intestinal mucosa, to a large extent limiting the mode of administration to injections, which is not favouring...... patient compliance. Nevertheless, cell-penetrating peptides (CPPs) have shown promising potential as carriers to overcome the epithelium, and this minireview highlights recent knowledge gained within the field of CPP-mediated transepithelial delivery of therapeutic peptides and proteins from the intestine...... is to be preferred depends on the physicochemical properties of both the specific CPP and the specific cargo. In addition to the physical epithelial barrier, a metabolic barrier must be overcome in order to obtain CPP-mediated delivery of a cargo drug from the intestine, and a number of strategies have been employed...

  10. Cell-penetrating peptides as tools to enhance non-injectable delivery of biopharmaceuticals

    DEFF Research Database (Denmark)

    Kristensen, Mie; Nielsen, Hanne Mørck

    2016-01-01

    Non-injectable delivery of peptide and protein drugs is hampered by their labile nature, hydrophilicity, and large molecular size; thus limiting their permeation across mucosae, which represent major biochemical and physical barriers to drugs administered via e.g. the oral, nasal, and pulmonary...... routes. However, in recent years cell-penetrating peptides (CPP) have emerged as promising tools to enhance mucosal delivery of co-administered or conjugated peptide and protein cargo and more advanced CPP-cargo formulations are emerging. CPPs act as transepithelial delivery vectors, but the mechanism...... understanding, documentation of CPP-mediated delivery in higher animal species than rodent as well as extensive toxicological studies are necessary for CPP-containing non-injectable DDSs to reach the clinic....

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

    KAUST Repository

    Haidar, Malak

    2017-09-08

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

  12. Photochemical internalisation of a macromolecular protein toxin using a cell penetrating peptide-photosensitiser conjugate.

    Science.gov (United States)

    Wang, Julie T-W; Giuntini, Francesca; Eggleston, Ian M; Bown, Stephen G; MacRobert, Alexander J

    2012-01-30

    Photochemical internalisation (PCI) is a site-specific technique for improving cellular delivery of macromolecular drugs. In this study, a cell penetrating peptide, containing the core HIV-1 Tat 48-57 sequence, conjugated with a porphyrin photosensitiser has been shown to be effective for PCI. Herein we report an investigation of the photophysical and photobiological properties of a water soluble bioconjugate of the cationic Tat peptide with a hydrophobic tetraphenylporphyrin derivative. The cellular uptake and localisation of the amphiphilic bioconjugate was examined in the HN5 human head and neck squamous cell carcinoma cell line. Efficient cellular uptake and localisation in endo/lysosomal vesicles was found using fluorescence detection, and light-induced, rupture of the vesicles resulting in a more diffuse intracellular fluorescence distribution was observed. Conjugation of the Tat sequence with a hydrophobic porphyrin thus enables cellular delivery of an amphiphilic photosensitiser which can then localise in endo/lysosomal membranes, as required for effective PCI treatment. PCI efficacy was tested in combination with a protein toxin, saporin, and a significant reduction in cell viability was measured versus saporin or photosensitiser treatment alone. This study demonstrates that the cell penetrating peptide-photosensitiser bioconjugation strategy is a promising and versatile approach for enhancing the therapeutic potential of bioactive agents through photochemical internalisation. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides.

    Science.gov (United States)

    Bolhassani, Azam; Jafarzade, Behnaz Sadat; Mardani, Golnaz

    2017-01-01

    The failure of proteins to penetrate mammalian cells or target tumor cells restricts their value as therapeutic tools in a variety of diseases such as cancers. Recently, protein transduction domains (PTDs) or cell penetrating peptides (CPPs) have been shown to promote the delivery of therapeutic proteins or peptides into live cells. The successful delivery of proteins mainly depends on their physicochemical properties. Although, linear cell penetrating peptides are one of the most effective delivery vehicles; but currently, cyclic CPPs has been developed to potently transport bioactive full-length proteins into cells. Up to now, several small protein transduction domains from viral proteins including Tat or VP22 could be fused to other peptides or proteins to entry them in various cell types at a dose-dependent approach. A major disadvantage of PTD-fusion proteins is primary uptake into endosomal vesicles leading to inefficient release of the fusion proteins into the cytosol. Recently, non-covalent complex formation (Chariot) between proteins and CPPs has attracted a special interest to overcome some delivery limitations (e.g., toxicity). Many preclinical and clinical trials of CPP-based delivery are currently under evaluation. Generally, development of more efficient protein transduction domains would significantly increase the potency of protein therapeutics. Moreover, the synergistic or combined effects of CPPs with other delivery systems for protein/peptide drug delivery would promote their therapeutic effects in cancer and other diseases. In this review, we will describe the functions and implications of CPPs for delivering the therapeutic proteins or peptides in preclinical and clinical studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides

    Institute of Scientific and Technical Information of China (English)

    Tingting Lin; Ergang Liu; Huining He; Meong Cheol Shin; Cheol Moon; Victor C.Yang; Yongzhuo Huang

    2016-01-01

    Brain delivery of macromolecular therapeutics(e.g., proteins) remains an unsolved problem because of the formidable blood–brain barrier(BBB). Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs,new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways(e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion), a low molecular weight protamine(LMWP) cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides(CPP)have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration.The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

  15. Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tingting Lin

    2016-07-01

    Full Text Available Brain delivery of macromolecular therapeutics (e.g., proteins remains an unsolved problem because of the formidable blood–brain barrier (BBB. Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion, a low molecular weight protamine (LMWP cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

  16. A novel chimeric cell-penetrating peptide with membrane-disruptive properties for efficient endosomal escape.

    Science.gov (United States)

    Salomone, Fabrizio; Cardarelli, Francesco; Di Luca, Mariagrazia; Boccardi, Claudia; Nifosì, Riccardo; Bardi, Giuseppe; Di Bari, Lorenzo; Serresi, Michela; Beltram, Fabio

    2012-11-10

    Efficient endocytosis into a wide range of target cells and low toxicity make the arginine-rich Tat peptide (Tat(11): YGRKKRRQRRR, residues 47-57 of HIV-1 Tat protein) an excellent transporter for delivery purposes. Unfortunately, molecules taken up by endocytosis undergo endosomal entrapment and possible metabolic degradation. Escape from the endosome is therefore actively researched. In this context, antimicrobial peptides (AMPs) provide viable templates for the design of new membrane-disruptive motifs. In particular the Cecropin-A and Melittin hybrids (CMs) are among the smallest and most effective peptides with membrane-perturbing abilities. Here we present a novel chimeric peptide in which the Tat(11) motif is fused to the CM(18) hybrid (KWKLFKKIGAVLKVLTTG, residues 1-7 of Cecropin-A and 2-12 of Melittin). When administered to cells, CM(18)-Tat(11) combines the two desired functionalities: efficient uptake and destabilization of endocytotic-vesicle membranes. We show that this chimeric peptide effectively increases cargo-molecule cytoplasm availability and allows the subsequent intracellular localization of diverse membrane-impermeable molecules (i.e. Tat(11)-EGFP fusion protein, calcein, dextrans, and plasmidic DNA) with no detectable cytotoxicity. The present results open the way to the rational engineering of "modular" cell-penetrating peptides (CPPs) that combine (i) efficient translocation from the extracellular milieu into vesicles and (ii) efficient release of molecules from vesicles into the cytoplasm. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease.

    Science.gov (United States)

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T

    2016-02-22

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

  18. Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides

    KAUST Repository

    Rydberg, Hanna A.; Matson, Maria; Å mand, Helene L.; Esbjö rner, Elin K.; Nordé n, Bengt

    2012-01-01

    Cell-penetrating peptides (CPPs) are able to traverse cellular membranes and deliver macromolecular cargo. Uptake occurs through both endocytotic and nonendocytotic pathways, but the molecular requirements for efficient internalization are not fully understood. Here we investigate how the presence of tryptophans and their position within an oligoarginine influence uptake mechanism and efficiency. Flow cytometry and confocal fluorescence imaging are used to estimate uptake efficiency, intracellular distribution and toxicity in Chinese hamster ovarian cells. Further, membrane leakage and lipid membrane affinity are investigated. The peptides contain eight arginine residues and one to four tryptophans, the tryptophans positioned either at the N-terminus, in the middle, or evenly distributed along the amino acid sequence. Our data show that the intracellular distribution varies among peptides with different tryptophan content and backbone spacing. Uptake efficiency is higher for the peptides with four tryptophans in the middle, or evenly distributed along the peptide sequence, than for the peptide with four tryptophans at the N-terminus. All peptides display low cytotoxicity except for the one with four tryptophans at the N-terminus, which was moderately toxic. This finding is consistent with their inability to induce efficient leakage of dye from lipid vesicles. All peptides have comparable affinities for lipid vesicles, showing that lipid binding is not a decisive parameter for uptake. Our results indicate that tryptophan content and backbone spacing can affect both the CPP uptake efficiency and the CPP uptake mechanism. The low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view. © 2012 American Chemical Society.

  19. Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides

    KAUST Repository

    Rydberg, Hanna A.

    2012-07-10

    Cell-penetrating peptides (CPPs) are able to traverse cellular membranes and deliver macromolecular cargo. Uptake occurs through both endocytotic and nonendocytotic pathways, but the molecular requirements for efficient internalization are not fully understood. Here we investigate how the presence of tryptophans and their position within an oligoarginine influence uptake mechanism and efficiency. Flow cytometry and confocal fluorescence imaging are used to estimate uptake efficiency, intracellular distribution and toxicity in Chinese hamster ovarian cells. Further, membrane leakage and lipid membrane affinity are investigated. The peptides contain eight arginine residues and one to four tryptophans, the tryptophans positioned either at the N-terminus, in the middle, or evenly distributed along the amino acid sequence. Our data show that the intracellular distribution varies among peptides with different tryptophan content and backbone spacing. Uptake efficiency is higher for the peptides with four tryptophans in the middle, or evenly distributed along the peptide sequence, than for the peptide with four tryptophans at the N-terminus. All peptides display low cytotoxicity except for the one with four tryptophans at the N-terminus, which was moderately toxic. This finding is consistent with their inability to induce efficient leakage of dye from lipid vesicles. All peptides have comparable affinities for lipid vesicles, showing that lipid binding is not a decisive parameter for uptake. Our results indicate that tryptophan content and backbone spacing can affect both the CPP uptake efficiency and the CPP uptake mechanism. The low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view. © 2012 American Chemical Society.

  20. Characterization of the cell penetrating properties of a human salivary proline-rich peptide.

    Science.gov (United States)

    Radicioni, Giorgia; Stringaro, Annarita; Molinari, Agnese; Nocca, Giuseppina; Longhi, Renato; Pirolli, Davide; Scarano, Emanuele; Iavarone, Federica; Manconi, Barbara; Cabras, Tiziana; Messana, Irene; Castagnola, Massimo; Vitali, Alberto

    2015-11-01

    Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.

  1. Antibacterial Effects of a Cell-Penetrating Peptide Isolated from Kefir.

    Science.gov (United States)

    Miao, Jianyin; Guo, Haoxian; Chen, Feilong; Zhao, Lichao; He, Liping; Ou, Yangwen; Huang, Manman; Zhang, Yi; Guo, Baoyan; Cao, Yong; Huang, Qingrong

    2016-04-27

    Kefir is a traditional fermented milk beverage used throughout the world for centuries. A cell-penetrating peptide, F3, was isolated from kefir by Sephadex G-50 gel filtration, DEAE-52 ion exchange, and reverse-phase high-performance liquid chromatography. F3 was determined to be a low molecular weight peptide containing one leucine and one tyrosine with two phosphate radicals. This peptide displayed antimicrobial activity across a broad spectrum of organisms including several Gram-positive and Gram-negative bacteria as well as fungi, with minimal inhibitory concentration (MIC) values ranging from 125 to 500 μg/mL. Cellular penetration and accumulation of F3 were determined by confocal laser scanning microscopy. The peptide was able to penetrate the cellular membrane of Escherichia coli and Staphylococcus aureus. Changes in cell morphology were observed by scanning electron microscopy (SEM). The results indicate that peptide F3 may be a good candidate for use as an effective biological preservative in agriculture and the food industry.

  2. 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.

  3. Harnessing the capacity of cell-penetrating peptides for drug delivery to the central nervous system.

    Science.gov (United States)

    Kang, Ting; Gao, Xiaoling; Chen, Jun

    2014-01-01

    The existence of blood-brain barrier (BBB) represents the most formidable challenge for drug delivery to the central nervous system (CNS). Modern breakthrough in biology offers multiple choices for overcoming this barrier but yields modest outcomes for clinical application due to various problems such as safety concerns, insufficient delivery efficiency and poor penetration. Cell penetrating peptides (CPPs) possessing powerful transmembrane capacity have been shown to be effective transport vectors for bioactive molecules and an attractive alternative to traditional active targeting approaches. However, the non-specificity of CPPs has hindered them from targeting a desired site of action. Promisingly, design of novel CPP-mediated nanoparticulate delivery systems with specific targeting property may extricate CPPs from the dilemma. In this review, both the traditional and novel applications of CPPs-based strategies for CNS drug delivery will be discussed.

  4. Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides.

    Science.gov (United States)

    Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina

    2016-01-01

    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

  5. Brain delivery of insulin boosted by intranasal coadministration with cell-penetrating peptides.

    Science.gov (United States)

    Kamei, Noriyasu; Takeda-Morishita, Mariko

    2015-01-10

    Intranasal administration is considered as an alternative route to enable effective drug delivery to the central nervous system (CNS) by bypassing the blood-brain barrier. Several reports have proved that macromolecules can be transferred directly from the nasal cavity to the brain. However, strategies to enhance the delivery of macromolecules from the nasal cavity to CNS are needed because of their low delivery efficiencies via this route in general. We hypothesized that the delivery of biopharmaceuticals to the brain parenchyma can be facilitated by increasing the uptake of drugs by the nasal epithelium including supporting and neuronal cells to maximize the potentiality of the intranasal pathway. To test this hypothesis, the CNS-related model peptide insulin was intranasally coadministered with the cell-penetrating peptide (CPP) penetratin to mice. As a result, insulin coadministered with l- or d-penetratin reached the distal regions of the brain from the nasal cavity, including the cerebral cortex, cerebellum, and brain stem. In particular, d-penetratin could intranasally deliver insulin to the brain with a reduced risk of systemic insulin exposure. Thus, the results obtained in this study suggested that CPPs are potential tools for the brain delivery of peptide- and protein-based pharmaceuticals via intranasal administration. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Secondary structure of cell-penetrating peptides during interaction with fungal cells.

    Science.gov (United States)

    Gong, Zifan; Ikonomova, Svetlana P; Karlsson, Amy J

    2018-03-01

    Cell-penetrating peptides (CPPs) are peptides that cross cell membranes, either alone or while carrying molecular cargo. Although their interactions with mammalian cells have been widely studied, much less is known about their interactions with fungal cells, particularly at the biophysical level. We analyzed the interactions of seven CPPs (penetratin, Pep-1, MPG, pVEC, TP-10, MAP, and cecropin B) with the fungal pathogen Candida albicans using experiments and molecular simulations. Circular dichroism (CD) of the peptides revealed a structural transition from a random coil or weak helix to an α-helix occurs for all peptides when the solvent is changed from aqueous to hydrophobic. However, CD performed in the presence of C. albicans cells showed that proximity to the cell membrane is not necessarily sufficient to induce this structural transition, as penetratin, Pep-1, and MPG did not display a structural shift in the presence of cells. Monte Carlo simulations were performed to further probe the molecular-level interaction with the cell membrane, and these simulations suggested that pVEC, TP-10, MAP, and cecropin B strongly penetrate into the hydrophobic domain of the membrane lipid bilayer, inducing a transition to an α-helical conformation. In contrast, penetratin, Pep-1 and MPG remained in the hydrophilic region without a shift in conformation. The experimental data and MC simulations combine to explain how peptide structure affects their interaction with cells and their mechanism of translocation into cells (direct translocation vs. endocytosis). Our work also highlights the utility of combining biophysical experiments, biological experiments, and molecular modeling to understand biological phenomena. © 2017 The Protein Society.

  7. Paramagnetic particles carried by cell-penetrating peptide tracking of bone marrow mesenchymal stem cells, a research in vitro

    International Nuclear Information System (INIS)

    Liu Min; Guo Youmin; Wu Qifei; Yang Junle; Wang Peng; Wang Sicen; Guo Xiaojuan; Qiang Yongqian; Duan Xiaoyi

    2006-01-01

    The ability to track the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging of the mesenchymal stem cells. The mesenchymal stem cells were isolated from rat bone marrow by Percoll and identified by osteogenic differentiation in vitro. The cell-penetrating peptides labeled with fluorescein-5-isothiocyanate and gadolinium were synthesized by a solid-phase peptide synthesis method and the relaxivity of cell-penetrating peptide-gadolinium paramagnetic conjugate on 400 MHz nuclear magnetic resonance was 5.7311 ± 0.0122 mmol -1 s -1 , higher than that of diethylenetriamine pentaacetic acid gadolinium (p < 0.05). Fluorescein imaging confirmed that this new peptide could internalize into the cytoplasm and nucleus. Gadolinium was efficiently internalized into mesenchymal stem cells by the peptide in a time- or concentration-dependent fashion, resulting in intercellular T1 relaxation enhancement, which was obviously detected by 1.5 T magnetic resonance imaging. Cytotoxicity assay and flow cytometric analysis showed the intercellular contrast medium incorporation did not affect cell viability and membrane potential gradient. The research in vitro suggests that the newly constructed peptides could be a vector for tracking mesenchymal stem cells

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

    Science.gov (United States)

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

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

  9. Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides.

    Directory of Open Access Journals (Sweden)

    Caghan Kizil

    Full Text Available Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI, RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

  10. Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine.

    Directory of Open Access Journals (Sweden)

    Divyamani Srinivasan

    2011-03-01

    Full Text Available Cell-penetrating peptides (CPPs can transport macromolecular cargos into live cells. However, the cellular delivery efficiency of these reagents is often suboptimal because CPP-cargo conjugates typically remain trapped inside endosomes. Interestingly, irradiation of fluorescently labeled CPPs with light increases the release of the peptide and its cargos into the cytosol. However, the mechanism of this phenomenon is not clear. Here we investigate the molecular basis of the photo-induced endosomolytic activity of the prototypical CPPs TAT labeled to the fluorophore 5(6-carboxytetramethylrhodamine (TMR.We report that TMR-TAT acts as a photosensitizer that can destroy membranes. TMR-TAT escapes from endosomes after exposure to moderate light doses. However, this is also accompanied by loss of plasma membrane integrity, membrane blebbing, and cell-death. In addition, the peptide causes the destruction of cells when applied extracellularly and also triggers the photohemolysis of red blood cells. These photolytic and photocytotoxic effects were inhibited by hydrophobic singlet oxygen quenchers but not by hydrophilic quenchers.Together, these results suggest that TAT can convert an innocuous fluorophore such as TMR into a potent photolytic agent. This effect involves the targeting of the fluorophore to cellular membranes and the production of singlet oxygen within the hydrophobic environment of the membranes. Our findings may be relevant for the design of reagents with photo-induced endosomolytic activity. The photocytotoxicity exhibited by TMR-TAT also suggests that CPP-chromophore conjugates could aid the development of novel Photodynamic Therapy agents.

  11. Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos

    Directory of Open Access Journals (Sweden)

    Mie Kristensen

    2016-01-01

    Full Text Available The hydrophilic nature of peptides and proteins renders them impermeable to cell membranes. Thus, in order to successfully deliver peptide and protein-based therapeutics across the plasma membrane or epithelial and endothelial barriers, a permeation enhancing strategy must be employed. Cell-penetrating peptides (CPPs constitute a promising tool and have shown applications for peptide and protein delivery into cells as well as across various epithelia and the blood-brain barrier (BBB. CPP-mediated delivery of peptides and proteins may be pursued via covalent conjugation of the CPP to the cargo peptide or protein or via physical complexation obtained by simple bulk-mixing of the CPP with its cargo. Both approaches have their pros and cons, and which is the better choice likely relates to the physicochemical properties of the CPP and its cargo as well as the route of administration, the specific barrier and the target cell. Besides the physical barrier, a metabolic barrier must be taken into consideration when applying peptide-based delivery vectors, such as the CPPs, and stability-enhancing strategies are commonly employed to prolong the CPP half-life. The mechanisms by which CPPs translocate cell membranes are believed to involve both endocytosis and direct translocation, but are still widely investigated and discussed. The fact that multiple factors influence the mechanisms responsible for cellular CPP internalization and the lack of sensitive methods for detection of the CPP, and in some cases the cargo, further complicates the design and conduction of conclusive mechanistic studies.

  12. Recent in vivo advances in cell-penetrating peptide-assisted drug delivery.

    Science.gov (United States)

    Kurrikoff, Kaido; Gestin, Maxime; Langel, Ülo

    2016-01-01

    Delivery of macromolecular drugs is an important field in medical research. However, macromolecules are usually unable to cross the cell membrane without the assistance of a delivery system. Cell penetrating peptides (CPPs) are unique tools to gain access to the cell interior and deliver a bioactive cargo into the cytosol or nucleus. In addition to macromolecular delivery, CPPs have been used to deliver smaller bioactive molecules. Therefore CPPs have become an intensive field of research for medical treatment. In this review, we highlight studies that include CPP in vivo disease models. We review different strategies and approaches that have been used, with specific attention on recent publications. The approaches that have been used include CPP-cargo covalent conjugation strategies and nanoparticle strategies. Various additional strategies have been used to achieve disease targeting, including active targeting, passive targeting, and combined active/passive strategies. As a result, delivery of various types of molecule has been achieved, including small drug molecules, proteins and nucleic acid-based macromolecules (e.g. siRNA, antisense nucleotides and plasmid DNA). Despite recent advances in the field, confusions surrounding CPP internalization mechanisms and intracellular trafficking are hindering the development of new and more efficient vectors. Nevertheless, the recent increase in the number of publications containing in vivo CPP utilization looks promising that the number of clinical trials would also increase in the near future.

  13. Liposomes equipped with cell penetrating peptide BR2 enhances chemotherapeutic effects of cantharidin against hepatocellular carcinoma.

    Science.gov (United States)

    Zhang, Xue; Lin, Congcong; Lu, Aiping; Lin, Ge; Chen, Huoji; Liu, Qiang; Yang, Zhijun; Zhang, Hongqi

    2017-11-01

    A main hurdle for the success of tumor-specific liposomes is their inability to penetrate tumors efficiently. In this study, we incorporated a cell-penetrating peptide BR2 onto the surface of a liposome loaded with the anticancer drug cantharidin (CTD) to create a system targeting hepatocellular carcinoma (HCC) cells more efficiently and effectively. The in vitro cytotoxicity assay comparing the loaded liposomes' effects on hepatocellular cancer HepG2 and the control Miha cells showed that CTD-loaded liposomes had a stronger anticancer effect after BR2 modification. The cellular uptake results of HepG2 and Miha cells further confirmed the superior ability of BR2-modified liposomes to penetrate cancer cells. The colocalization study revealed that BR2-modified liposomes could enter tumor cells and subsequently release drugs. A higher efficiency of delivery by BR2 liposomes as compared to unmodified liposomes was evident by evaluation of the HepG2 tumor spheroids penetration and inhibition. The biodistribution studies and anticancer efficacy results in vivo showed the significant accumulation of BR2-modified liposomes into tumor sites and an enhanced tumor inhibition. In conclusion, BR2-modified liposomes improve the anticancer potency of drugs for HCC.

  14. Cell penetrating peptide-modified poly(lactic-co-glycolic acid) nanoparticles with enhanced cell internalization.

    Science.gov (United States)

    Steinbach, Jill M; Seo, Young-Eun; Saltzman, W Mark

    2016-01-01

    The surface modification of nanoparticles (NPs) can enhance the intracellular delivery of drugs, proteins, and genetic agents. Here we studied the effect of different surface ligands, including cell penetrating peptides (CPPs), on the cell binding and internalization of poly(lactic-co-glycolic) (PLGA) NPs. Relative to unmodified NPs, we observed that surface-modified NPs greatly enhanced cell internalization. Using one CPP, MPG (unabbreviated notation), that achieved the highest degree of internalization at both low and high surface modification densities, we evaluated the effect of two different NP surface chemistries on cell internalization. After 2h, avidin-MPG NPs enhanced cellular internalization by 5 to 26-fold relative to DSPE-MPG NP formulations. Yet, despite a 5-fold increase in MPG density on DSPE compared to Avidin NPs, both formulations resulted in similar internalization levels (48 and 64-fold, respectively) after 24h. Regardless of surface modification, all NPs were internalized through an energy-dependent, clathrin-mediated process, and became dispersed throughout the cell. Overall both Avidin- and DSPE-CPP modified NPs significantly increased internalization and offer promising delivery options for applications in which internalization presents challenges to efficacious delivery. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  15. The uptake of arginine-rich cell-penetrating peptides: putting the puzzle together.

    Science.gov (United States)

    Brock, Roland

    2014-05-21

    Over the past 20 years, cell-penetrating peptides (CPPs) have captured the attention of biomedical researchers, biophysicists, and (bio)organic chemists. These molecules efficiently enter cells and mediate entry of (macro)molecules that by themselves do not cross the plasma membrane. Since their discovery, models on the mechanism by which uptake occurs have seen major revisions. Starting from direct penetration across the plasma membrane, it later became apparent that for large molecular weight cargos in particular, endocytosis plays a role in uptake and furthermore that the route of uptake is a function of CPP, cell-type, cargo, and concentration. For the class of arginine-rich CPPs, this dependence on conditions has been elucidated in particular. As I will discuss here for this class of CPPs, a downside of this multitude of possibilities has been a lack of attention for commonalities in the observation of apparently distinct phenomena. At the same time, differences of apparently similar observations were not appreciated sufficiently. In addition, there has been insufficient acknowledgment of observations that are incompatible with the proposed models. Nevertheless, a considerable amount of data can be assembled into a quite coherent picture and the data that is left creates the basis for concrete future lines of research to resolve the questions that remain. Moreover, any uptake mechanism has its distinct structure-activity relationship for uptake giving room for the molecular design of molecules to preferentially direct uptake to either of them.

  16. 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

    We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting......-PNA (cPNA1(7)-(D-Arg)8) and hexamer carrier decanoyl-CPP-PNA (Deca-cPNA1(6)-(D-Arg)8), respectively, without showing significant additional cellular toxicity. Most interestingly, the activity reached the same level obtained by enhancement with endosomolytic chloroquine (CQ) treatment, suggesting...... 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...

  17. Translocation of cell penetrating peptides and calcium-induced membrane fusion share same mechanism

    Czech Academy of Sciences Publication Activity Database

    Magarkar, Aniket; Allolio, Christoph; Jurkiewicz, Piotr; Baxová, Katarína; Šachl, Radek; Horinek, D.; Heinz, V.; Rachel, R.; Ziegler, C.; Jungwirth, Pavel

    2017-01-01

    Roč. 46, Suppl 1 (2017), S386 ISSN 0175-7571. [IUPAB congress /19./ and EBSA congress /11./. 16.07.2017-20.07.2017, Edinburgh] Institutional support: RVO:61388963 ; RVO:61388955 Keywords : membrane interactions * membrane fusion * cell penetration Subject RIV: BO - Biophysics

  18. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Science.gov (United States)

    Suh, Jin Sook; Lee, Jue Yeon; Choi, Yoon Jung; You, Hyung Keun; Hong, Seong-Doo; Chung, Chong Pyoung; Park, Yoon Jeong

    2014-01-01

    Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue

  19. Enhanced Cellular Uptake of Albumin-Based Lyophilisomes when Functionalized with Cell-Penetrating Peptide TAT in HeLa Cells

    NARCIS (Netherlands)

    van Bracht, Etienne; Versteegden, Luuk R. M.; Stolle, Sarah; Verdurmen, Wouter P. R.; Woestenenk, Rob; Raave, Rene; Hafmans, Theo; Oosterwijk, Egbert; Brock, Roland; van Kuppevelt, Toin H.; Daamen, Willeke F.

    2014-01-01

    Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various

  20. Molecular imaging of a cell-penetrating peptide labeled fluorescein-5-isothiocyanate and MR contrast agents: gadopentetate dimeglumine

    International Nuclear Information System (INIS)

    Liu Min; Guo Youmin; Duan Xiaoyi; Guo Xiaojuan; Yang Junle; Xu Min

    2006-01-01

    Objective: To study the value of a new intracellular contrast agent--cell penetrating peptide labeled Fluorescein-5-isothiocyanate (FITC) and MRI contrast agent, Gadopentetate dimeglumine in molecular imaging. Methods: A new cell penetration peptides (CPPs)sequence LAGRRRRRRRRRK were synthesized in solid phase on the base of arginine (9) and were labelled with FITC (CPP 13 -FITC) and Gd - DTPA (CPP 13 -DTPA-Gd). Hepatic carcinoma cell line-HEPG 2 and mouse bone marrow stem cell was respectively stained by CPP 13 -FITC for different time intervals for observing the uptake and intracellular distribution. HEPG 2 in three l00 mm 2 culture plates was respectively incubated with CPP 13 -DTPA-Gd, Gd- DTPA and Dulbecco minimum essential medium for 30 min and imaged by 1.5 T MRI for studying the intracellular uptake and T 1 WI signal characteristics. Results: The peptide was synthesized by the manual solid-phase method successfully. The calculated molecular weight was 1792.78 and the chemical purity was over 95%. By inverted fluorescence microscope, HEPG 2 and mouse stem cell could transport CPP-FITC in cytoplasm and nuclear in 10 min. By MR imaging, CPP-DTPA-Gd could be uptake by HEPG 2 in 30 min and had a short T 1 short T 2 signal, furthermore. T 1 WI signal intensity ratio between in-tube (Ii) and out-tube (Io) in three groups of three scan slices were shown below: Iil/Io of group 1 (Group 1 was the cell incubated by CPP 13 -DTPA-Gd ) respectively was 2.84, 2.60, 2. 48; Iil/Io of group 2 (Group 2 was the cell incubated by DTPA-Gd) respectively is 1.15, 1.11, 1.12; Iil/Io of group 3 (Group 3 was the controled cell ) respectively was 1.15, 1.11, 1.11. By ANVOA analysis, the signal intensity among group 1, group 2 and group 3 had significant difference(F (1,2) = 201.88 P (1,3) =206.37 P (2,3) =0.529 P=0.507). Conclusion: The new constructed cell penetration peptide on the base of the polyargnine can translocate cell by carting FITC and MRI contrast agent-DTPA-Gd and the

  1. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    Energy Technology Data Exchange (ETDEWEB)

    Amand, Helene L., E-mail: helene.amand@chalmers.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden); Norden, Bengt, E-mail: norden@chalmers.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden); Fant, Kristina, E-mail: kristina.fant@sp.se [Chalmers University of Technology, Department of Chemical and Biological Engineering/Physical Chemistry, SE-412 96 Gothenburg (Sweden)

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ('peptiplexes') enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the 'chelate effect' and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide

  2. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    International Nuclear Information System (INIS)

    Åmand, Helene L.; Nordén, Bengt; Fant, Kristina

    2012-01-01

    Highlights: ► Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. ► Dimer formation enhances peptiplex stability, resulting in increased transfection. ► By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes (“peptiplexes”) enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the “chelate effect” and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from its stronger binding to DNA.

  3. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

    Directory of Open Access Journals (Sweden)

    Andrea-Anneliese Keller

    2013-02-01

    Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

  4. Hemocompatible poly(NIPAm-MBA-AMPS) colloidal nanoparticles as carriers of anti-inflammatory cell penetrating peptides.

    Science.gov (United States)

    Bartlett, Rush L; Medow, Matthew R; Panitch, Alyssa; Seal, Brandon

    2012-04-09

    Anionic copolymer systems containing sulfated monomers have great potential for delivery of cationic therapeutics, but N-isopropylacrylamide (NIPAm) 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) copolymer nanoparticles have seen limited characterization to date with regard to physical properties relevant to loading and release of therapeutics. Characterization of polymeric nanoparticles incorporating AMPS showed an increased size and decreased thermodynamic swelling ratios of AMPS containing particles as compared to NIPAm nanoparticles lacking AMPS. Particles with increasing AMPS addition showed an increased propensity for uniformity, intraparticle colloidal stability, and drug loading capacity. Peptide encapsulated in particles was shielded from peptide degradation in serum. Particles were shown not impede blood coagulation or to cause hemolysis. This study has demonstrated that AMPS incorporation into traditional NIPAm nanoparticles presents a tunable parameter for changing particle LCST, size, swelling ratio, ζ potential, and cationic peptide loading potential. This one-pot synthesis results in a thermosensitive anionic nanoparticle system that is a potentially useful platform to deliver cationic cell penetrating peptides.

  5. Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

    Science.gov (United States)

    Upadhya, Archana; Sangave, Preeti C

    2016-10-01

    Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R 8 ) and CHK 6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH 3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  6. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells.

    Science.gov (United States)

    Kaitsuka, Taku; Tomizawa, Kazuhito

    2015-11-06

    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

  7. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Taku Kaitsuka

    2015-11-01

    Full Text Available Protein transduction using cell-penetrating peptides (CPPs is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

  8. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Directory of Open Access Journals (Sweden)

    Suh JS

    2014-03-01

    Full Text Available Jin Sook Suh,1,* Jue Yeon Lee,2,* Yoon Jung Choi,1 Hyung Keun You,3 Seong-Doo Hong,4 Chong Pyoung Chung,2 Yoon Jeong Park1,2 1Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 2Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Seoul, 3Department of Periodontology, College of Dentistry, Wonkwang University, Iksan, 4Department of Oral Pathology, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP, and a transcriptional coactivator with a PDZ-binding motif (TAZ protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in

  9. Synthesis and in vitro evaluation of PNA-peptide-DETA conjugates as potential cell penetrating artificial ribonucleases.

    Science.gov (United States)

    Petersen, Lene; de Koning, Martijn C; van Kuik-Romeijn, Petra; Weterings, Jimmy; Pol, Christine J; Platenburg, Gerard; Overhand, Mark; van der Marel, Gijsbert A; van Boom, Jacques H

    2004-01-01

    We report the synthesis of novel artificial ribonucleases with potentially improved cellular uptake. The design of trifunctional conjugates 1a and 1b is based on the specific RNA-recognizing properties of PNA, the RNA-cleaving abilities of diethylenetriamine (DETA), and the peptide (KFF)(3)K for potential uptake into E. coli. The conjugates were assembled in a convergent synthetic route involving native chemical ligation of a PNA, containing an N-terminal cysteine, with the C-terminal thioester of the cell-penetrating (KFF)(3)K peptide to give 12a and 12b. These hybrids contained a free cysteine side-chain, which was further functionalized with an RNA-hydrolyzing diethylenetriamine (DETA) moiety. The trifunctional conjugates (1a, 1b) were evaluated for RNA-cleaving properties in vitro and showed efficient degradation of the target RNA at two major cleavage sites. It was also established that the cleavage efficiency strongly depended on the type of spacer connecting the PNA and the peptide.

  10. Vesicles mimicking normal and cancer cell membranes exhibit differential responses to the cell-penetrating peptide Pep-1.

    Science.gov (United States)

    Almarwani, Bashiyar; Phambu, Esther Nzuzi; Alexander, Christopher; Nguyen, Ha Aimee T; Phambu, Nsoki; Sunda-Meya, Anderson

    2018-06-01

    The cell-penetrating peptide (CPP) Pep-1 presents a great potential in drug delivery due to its intrinsic property to cross plasma membrane. However, its mechanism of entry into the cell remains unresolved. In this study, we compare the selectivity of Pep-1 towards vesicles mimicking normal and cancer cell membranes. The interaction was performed in a wide range of peptide-to-lipid molar ratios using infrared (IR), fluorescence, scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) techniques. At low peptide concentration, fluorescence experiments show that lipid-phosphatidylserine (PS) seems to enable Pep-1 translocation into cancer cell membrane as evidenced by the blue shift of its maximal emission wavelength. DSC data show that Pep-1 induces segregation of lipids. At high peptide concentration, IR data indicate that the interaction of Pep-1 is relatively stronger with normal cell membrane than with cancer cell membrane through the phosphate groups, while the interaction is weaker with normal cell membrane than with cancer cell membrane through the carbonyl groups. TGA and DSC data reveal that vesicles of normal cell membrane are thermally more stable than vesicles of cancer cell membrane. This suggests that the additional lipid PS included in cancer cell membrane has a destabilizing effect on the membrane structure. SEM images reveal that Pep-1 form superstructures including spherical particles and fibrils in the presence of both model membranes. PS seems to enhance peptide transport across cellular membranes. The biophysical techniques in this study provide valuable insights into the properties of CPPs in drug delivery systems. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery

    DEFF Research Database (Denmark)

    Tuttolomondo, Martina; Casella, Cinzia; Hansen, Pernille Lund

    2017-01-01

    tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using......-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10-800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and β-helix conformation. We...

  12. [Cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy].

    Science.gov (United States)

    Tan, Jiao; Wang, Ya-Ping; Wang, Hui-Xin; Liang, Jian-Ming; Zhang, Meng; Sun, Xun; Huang, Yong-Zhuo

    2014-12-01

    To develop a cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy, we prepared the AVPI-LMWP/pTRAIL self-assembled complexes containing a therapeutic combination of peptide drug AVPI and DNA drug TRAIL. The chimeric apoptotic peptide AVPI-LMWP was synthesized using the standard solid-phase synthesis. The cationic AVPI-LMWP could condense pTRAIL by electrostatic interaction. The physical-chemical properties of the AVPI-LMWP/pTRAIL complexes were characterized. The cellular uptake efficiency and the inhibitory activity of the AVPI-LMWP/pTRAIL complexes on tumor cell were also performed. The results showed that the AVPI-LMWP/pTRAIL complexes were successfully prepared by co-incubation. With the increase of mass ratio (AVPI-LMWP/DNA), the particle size was decreased and the zeta potential had few change. Agarose gel electrophoresis showed that AVPI-LMWP could fully bind and condense pTRAIL at a mass ratio above 15:1. Cellular uptake efficiency was improved along with the increased ratio of W(AVPI-LMWP)/WpTRAIL. The in vitro cytotoxicity experiments demonstrated that the AVPI-LMWP/pTRAIL (W:W = 20:1) complexes was significantly more effective than the pTRAIL, AVPI-LMWP alone or LMWP/pTRAIL complexes on inhibition of HeLa cell growth. Our studies indicated that the AVPI-LMWP/pTRAIL co-delivery system could deliver plasmid into HeLa cell and induce tumor cell apoptosis efficiently, which showed its potential in cancer therapy using combination of apoptoic peptide and gene drugs.

  13. Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

    DEFF Research Database (Denmark)

    Ndeboko, Bénédicte; Lemamy, Guy Joseph; Nielsen, Peter E

    2015-01-01

    , such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck......-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics...

  14. The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.

    Science.gov (United States)

    Luo, Zhao; Cao, Xue-Wei; Li, Chen; Wu, Miao-Dan; Yang, Xu-Zhong; Zhao, Jian; Wang, Fu-Jun

    2016-11-01

    Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  15. Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide.

    Science.gov (United States)

    Brezden, Anna; Mohamed, Mohamed F; Nepal, Manish; Harwood, John S; Kuriakose, Jerrin; Seleem, Mohamed N; Chmielewski, Jean

    2016-08-31

    Bacterial infection caused by intracellular pathogens, such as Mycobacterium, Salmonella, and Brucella, is a burgeoning global health epidemic that necessitates urgent action. However, the therapeutic value of a number of antibiotics, including aminoglycosides, against intracellular pathogenic bacteria is compromised due to their inability to traverse eukaryotic membranes. For this significant problem to be addressed, a cleavable conjugate of the antibiotic kanamycin and a nonmembrane lytic, broad-spectrum antimicrobial peptide with efficient mammalian cell penetration, P14LRR, was prepared. This approach allows kanamycin to enter mammalian cells as a conjugate linked via a tether that breaks down in the reducing environment within cells. Potent antimicrobial activity of the P14KanS conjugate was demonstrated in vitro, and this reducible conjugate effectively cleared intracellular pathogenic bacteria within macrophages more potently than that of a conjugate lacking the disulfide moiety. Notably, successful clearance of Mycobacterium tuberculosis within macrophages was observed with the dual antibiotic conjugate, and Salmonella levels were significantly reduced in an in vivo Caenorhabditis elegans model.

  16. Cell-penetrating peptide-driven Cre recombination in porcine primary cells and generation of marker-free pigs.

    Science.gov (United States)

    Kang, Qianqian; Sun, Zhaolin; Zou, Zhiyuan; Wang, Ming; Li, Qiuyan; Hu, Xiaoxiang; Li, Ning

    2018-01-01

    Cell-penetrating peptides (CPPs) have been increasingly used to deliver various molecules, both in vitro and in vivo. However, there are no reports of CPPs being used in porcine fetal fibroblasts (PFFs). The increased use of transgenic pigs for basic research and biomedical applications depends on the availability of technologies for efficient genetic-modification of PFFs. Here, we report that three CPPs (CPP5, TAT, and R9) can efficiently deliver active Cre recombinase protein into PFFs via an energy-dependent endocytosis pathway. The three CPP-Cre proteins can enter PFFs and subsequently perform recombination with different efficiencies. The recombination efficacy of CPP5-Cre was found to be nearly 90%. The rate-limiting step for CPP-Cre-mediated recombination was the step of endosome escape. HA2 and chloroquine were found to improve the recombination efficiency of TAT-Cre. Furthermore, we successfully obtained marker-free transgenic pigs using TAT-Cre and CPP5-Cre. We provide a framework for the development of CPP-based farm animal transgenic technologies that would be beneficial to agriculture and biomedicine.

  17. Formulation of Stable and Homogeneous Cell-Penetrating Peptide NF55 Nanoparticles for Efficient Gene Delivery In Vivo

    Directory of Open Access Journals (Sweden)

    Krista Freimann

    2018-03-01

    Full Text Available Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo.

  18. Current Understanding of Physicochemical Mechanisms for Cell Membrane Penetration of Arginine-rich Cell Penetrating Peptides: Role of Glycosaminoglycan Interactions.

    Science.gov (United States)

    Takechi-Haraya, Yuki; Saito, Hiroyuki

    2018-01-01

    Arginine-rich cell penetrating peptides (CPPs) are very promising drug carriers to deliver membrane-impermeable pharmaceuticals, such as siRNA, bioactive peptides and proteins. CPPs directly penetrate into cells across cell membranes via a spontaneous energy-independent process, in which CPPs appear to interact with acidic lipids in the outer leaflet of the cell membrane. However, acidic lipids represent only 10 to 20% of the total membrane lipid content and in mammalian cell membranes they are predominantly located in the inner leaflet. Alternatively, CPPs favorably bind in a charge density- dependent manner to negatively charged, sulfated glycosaminoglycans (GAGs), such as heparan sulfate and chondroitin sulfate, which are abundant on the cell surface and are involved in many biological functions. We have recently demonstrated that the interaction of CPPs with sulfated GAGs plays a critical role in their direct cell membrane penetration: the favorable enthalpy contribution drives the high-affinity binding of arginine-rich CPPs to sulfated GAGs, initiating an efficient cell membrane penetration. The favorable enthalpy gain is presumably mainly derived from a unique property of the guanidino group of arginine residues forming multidentate hydrogen bonding with sulfate and carboxylate groups in GAGs. Such interactions can be accompanied with charge neutralization of arginine-rich CPPs, promoting their partition into cell membranes. This review summarizes the current understanding of the physicochemical mechanism for lipid membrane penetration of CPPs, and discusses the role of the GAG interactions on the cell membrane penetration of CPPs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. A cancer specific cell-penetrating peptide, BR2, for the efficient delivery of an scFv into cancer cells.

    Directory of Open Access Journals (Sweden)

    Ki Jung Lim

    Full Text Available Cell-penetrating peptides (CPPs have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2 was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49-57. The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv directed toward a mutated K-ras (G12V. BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.

  20. A cancer specific cell-penetrating peptide, BR2, for the efficient delivery of an scFv into cancer cells.

    Science.gov (United States)

    Lim, Ki Jung; Sung, Bong Hyun; Shin, Ju Ri; Lee, Young Woong; Kim, Da Jung; Yang, Kyung Seok; Kim, Sun Chang

    2013-01-01

    Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49-57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.

  1. Cell Penetrating Capacity and Internalization Mechanisms Used by the Synthetic Peptide CIGB-552 and Its Relationship with Tumor Cell Line Sensitivity.

    Science.gov (United States)

    Astrada, Soledad; Fernández Massó, Julio Raúl; Vallespí, Maribel G; Bollati-Fogolín, Mariela

    2018-03-30

    CIGB-552 is a twenty-amino-acid novel synthetic peptide that has proven to be effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Such capability is conferred by its cell-penetrating peptide character, which allows it to enter cells and elicit a pro-apoptotic effect through its major mediator, COMMD1 protein. Cell-penetrating peptides are able to use different internalization mechanisms, such as endocytosis or direct transduction through the plasma membrane. Although CIGB-552 cytotoxicity has been evaluated in several non-tumor- and tumor-derived cell lines, no data regarding the relationship between cell line sensitivity, cell penetrating capacity, the internalization mechanisms involved, COMMD1 expression levels, or its subcellular localization has yet been produced. Here, we present the results obtained from a comparative analysis of CIGB-552 sensitivity, internalization capacity and the mechanisms involved in three human tumor-derived cell lines from different origins: mammary gland, colon and lung (MCF-7, HT-29 and H460, respectively). Furthermore, cell surface markers relevant for internalization processes such as phosphatidylserine, as well as CIGB-552 target COMMD1 expression/localization, were also evaluated. We found that both endocytosis and transduction are involved in CIGB-552 internalization in the three cell lines evaluated. However, CIGB-552 incorporation efficiency and contribution of each mechanism is cell-line dependent. Finally, sensitivity was directly correlated with high internalization capacity in those cell lines where endocytosis had a major contribution on CIGB-552 internalization.

  2. Topical Delivery of Anti-VEGF Drugs to the Ocular Posterior Segment Using Cell-Penetrating Peptides.

    Science.gov (United States)

    de Cogan, Felicity; Hill, Lisa J; Lynch, Aisling; Morgan-Warren, Peter J; Lechner, Judith; Berwick, Matthew R; Peacock, Anna F A; Chen, Mei; Scott, Robert A H; Xu, Heping; Logan, Ann

    2017-05-01

    To evaluate the efficacy of anti-VEGF agents for treating choroidal neovascularization (CNV) when delivered topically using novel cell-penetrating peptides (CPPs) compared with delivery by intravitreal (ivit) injection. CPP toxicity was investigated in cell cultures. Ivit concentrations of ranibizumab and bevacizumab after topical administration were measured using ELISA. The biological efficacy of topical anti-VEGF + CPP complexes was compared with ivit anti-VEGF injections using an established model of CNV. CPPs were nontoxic in vitro. In vivo, after topical eye drop delivery, CPPs were present in the rat anterior chamber within 6 minutes. A single application of CPP + bevacizumab eye drop delivered clinically relevant concentrations of bevacizumab to the posterior chamber of the rat eye in vivo. Similarly, clinically relevant levels of CPP + ranibizumab and CPP + bevacizumab were detected in the porcine vitreous and retina ex vivo. In an established model of CNV, mice treated with either a single ivit injection of anti-VEGF, twice daily CPP + anti-VEGF eye drops or daily dexamethasone gavage for 10 days all had significantly reduced areas of CNV when compared with lasered eyes without treatment. CPPs are nontoxic to ocular cells and can be used to deliver therapeutically relevant doses of ranibizumab and bevacizumab by eye drop to the posterior segment of mouse, rat, and pig eyes. The CPP + anti-VEGF drug complexes were cleared from the retina within 24 hours, suggesting a daily eye drop dosing regimen. Daily, topically delivered anti-VEGF with CPP was as efficacious as a single ivit injection of anti-VEGF in reducing areas of CNV in vivo.

  3. Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1

    International Nuclear Information System (INIS)

    Zhu, W.L.; Lan Hongliang; Park, Il-Seon; Kim, Jae Il; Jin, H.Z.; Hahm, Kyung-Soo; Shin, S.Y.

    2006-01-01

    Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 μM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 μM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 μM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein

  4. The feasibility of a targeted ultrasound contrast agent carrying genes and cell-penetrating peptides to hypoxic HUVEC

    International Nuclear Information System (INIS)

    Tian Ju; Wang Zhigang; Ren Jianli; Zhang Qingfeng; Liu Li

    2012-01-01

    Objective: To prepare an anti-P-selectin targeted ultrasound contrast agent carrying genes and cell-penetrating peptides (CPP) and to investigate its feasibility of delivery to hypoxic human umbilical vein endothelial cells (HUVEC). Methods: Anti-P-selectin targeted ultrasound contrast agent carrying a green fluorescent protein gene (pEGFP-N1) and CPP was prepared by mechanical vibration and carbodiimide techniques. The appearance, distribution, concentration and diameter of the ultrasound contrast agent were measured. The gene and CPP distribution on the agent was investigated using confocal laser scanning microscopy (CLSM). The efficiency of the ultrasound contrast agent to carry the gene and CPP was investigated by fluorospectrophotometry. HUVEC were cultured in vitro and hypoxic HUVEC were prepared using hydrogen peroxide (H 2 O 2 ). Hypoxic HUVEC were randomly assigned targeted ultrasound contrast agents and non-targeted ultrasound contrast agents for transfection. The transfection effect of green fluorescent protein in the two groups was observed using fluorescence microscopy and flow cytometry. T-test and linear correlation analysis were used for statistical analysis. Results: The average diameter of anti-P-selectin targeted ultrasound contrast agents carrying gene and CPP was (2.15 ±0.36) μm and the concentration was (1.58 ± 0.23) × 10 7 /ml.The results of CLSM showed that gene and CPP were distributed on the shell of the agent. The gene encapsulation efficiency was 28% (y=0.932x-0.09, r=0.993, P<0.05), and the CPP encapsulation efficiency was 25% (y=5.875x-0.81, r=0.987, P<0.05). EGFP expression was observed using fluorescence microscopy in targeted ultrasound contrast agents and non-targeted ultrasound contrast agents. The average transfection efficiencies of targeted ultrasound contrast agents and non-targeted ultrasound contrast agents were (18.74 ± 0.47) % and (15.34 ± 0.22) % after 24 h (t=10.923, P<0.001). Conclusions: The in vitro studies

  5. A novel strategy to improve antigen presentation for active immunotherapy in cancer. Fusion of the human papillomavirus type 16 E7 antigen to a cell penetrating peptide

    International Nuclear Information System (INIS)

    Granadillo, Milaid; Torrens, Isis; Guerra, Maribel

    2012-01-01

    Facilitating the delivery of exogenous antigens to antigen-presenting cells, ensuing processing and presentation via the major histocompatibility complex class I and induction of an effective immune response are fundamental for an effective therapeutic cancer vaccine. In this regard, we propose the use of cell-penetrating peptides fused to a tumor antigen. To demonstrate this concept we designed a fusion protein comprising a novel cell-penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus anti-lipopolysaccharide factor protein (LALF 32-51 ) linked to human papillomavirus 16 E7 antigen (LALF 32-51 -E7). In this work, we demonstrated that the immunization with LALF 32-51 -E7 using the TC-1 mouse model induces a potent and long-lasting anti-tumor response supported on an effective E7-specific CD8 +T -cell response. The finding that therapeutic immunization with LALF 32-51 or E7 alone, or an admixture of LALF32-51 and E7, does not induce significant tumor reduction indicates that covalent linkage between LALF 32-51 and E7 is required for the anti-tumor effect. These results support the use of this novel cell-penetrating peptide as an efficient means for delivering therapeutic targets into cellular compartments with the induction of a cytotoxic CD8 +T lymphocyte immune response. This approach is promissory for the treatment of tumors associated with the human papillomavirus 16, which is responsible for the 50% of cervical cancer cases worldwide and other malignancies. Furthermore, protein-based vaccines can circumvent the major histocompatibility complex specificity limitation associated with peptide vaccines providing a greater extent in their application

  6. 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

  7. A+-Helix of Protein C Inhibitor (PCI) Is a Cell-penetrating Peptide That Mediates Cell Membrane Permeation of PCI*

    Science.gov (United States)

    Yang, Hanjiang; Wahlmüller, Felix Christof; Sarg, Bettina; Furtmüller, Margareta; Geiger, Margarethe

    2015-01-01

    Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His1–Arg11) and mPCI (Arg1–Ala18) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI. PMID:25488662

  8. The potent antimicrobial properties of cell penetrating peptide-conjugated silver nanoparticles with excellent selectivity for gram-positive bacteria over erythrocytes.

    Science.gov (United States)

    Liu, Lihong; Yang, Jun; Xie, Jianping; Luo, Zhentao; Jiang, Jiang; Yang, Yi Yan; Liu, Shaomin

    2013-05-07

    Silver nanoparticles are of great interest for use as antimicrobial agents. Studies aimed at producing potent nano-silver biocides have focused on manipulation of particle size, shape, composition and surface charge. Here, we report the cell penetrating peptide catalyzed formation of antimicrobial silver nanoparticles in N,N-dimethylformamide. The novel nano-composite demonstrated a distinctly enhanced biocidal effect toward bacteria (gram-positive Bacillus subtilis, gram-negative Escherichia coli) and pathogenic yeast (Candida albicans), as compared to triangular and extremely small silver nanoparticles. In addition, a satisfactory biocompatibility was verified by a haemolysis test. Our results provide a paradigm in developing strategies that can maximize the silver nanoparticle application potentials while minimizing the toxic effects.

  9. Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport across Phospholipid Membranes Induced by a Proton Gradient

    Directory of Open Access Journals (Sweden)

    Fatemeh Madani

    2011-01-01

    Full Text Available Detergent-mediated reconstitution of bacteriorhodopsin (BR into large unilamellar vesicles (LUVs was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

  10. Visualization and Quantitative Assessment of the Brain Distribution of Insulin through Nose-to-Brain Delivery Based on the Cell-Penetrating Peptide Noncovalent Strategy.

    Science.gov (United States)

    Kamei, Noriyasu; Shingaki, Tomotaka; Kanayama, Yousuke; Tanaka, Misa; Zochi, Riyo; Hasegawa, Koki; Watanabe, Yasuyoshi; Takeda-Morishita, Mariko

    2016-03-07

    Our recent work suggested that intranasal coadministration with the cell-penetrating peptide (CPP) penetratin increased the brain distribution of the peptide drug insulin. The present study aimed to distinctly certify the ability of penetratin to facilitate the nose-to-brain delivery of insulin by quantitatively evaluating the distribution characteristics in brain using radioactive (64)Cu-NODAGA-insulin. Autoradiography and analysis using a gamma counter of brain areas demonstrated that the accumulation of radioactivity was greatest in the olfactory bulb, the anterior part of the brain closest to the administration site, at 15 min after intranasal administration of (64)Cu-NODAGA-insulin with l- or d-penetratin. The brain accumulation of (64)Cu-NODAGA-insulin with penetratin was confirmed by ELISA using unlabeled insulin in which intact insulin was delivered to the brain after intranasal coadministration with l- or d-penetratin. By contrast, quantification of cerebrospinal fluid (CSF) samples showed increased insulin concentration in only the anterior portion of the CSF at 15 min after intranasal coadministration with l-penetratin. This study gives the first concrete proof that penetratin can accelerate the direct transport of insulin from the nasal cavity to the brain parenchyma. Further optimization of intranasal administration with CPP may increase the efficacy of delivery of biopharmaceuticals to the brain while reducing the risk of systemic drug exposure.

  11. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    Directory of Open Access Journals (Sweden)

    Betty R Liu

    Full Text Available Cell-penetrating peptides (CPPs have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW from bovine lactoferricin (LFcin, stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

  12. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    Science.gov (United States)

    Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2016-01-01

    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

  13. Modulation of mitochondrial activity in HaCaT keratinocytes by the cell penetrating peptide Z-Gly-RGD(DPhe)-mitoparan.

    Science.gov (United States)

    Richardson, Adam; Muir, Lewis; Mousdell, Sasha; Sexton, Darren; Jones, Sarah; Howl, John; Ross, Kehinde

    2018-01-30

    Biologically active cell penetrating peptides (CPPs) are an emerging class of therapeutic agent. The wasp venom peptide mastoparan is an established CPP that modulates mitochondrial activity and triggers caspase-dependent apoptosis in cancer cells, as does the mastoparan analogue mitoparan (mitP). Mitochondrial depolarisation and activation of the caspase cascade also underpins the action of dithranol, a topical agent for treatment of psoriasis. The effects of a potent mitP analogue on mitochondrial activity were therefore examined to assess its potential as a novel approach for targeting mitochondria for the treatment of psoriasis. In HaCaT keratinocytes treated with the mitP analogue Z-Gly-RGD(DPhe)-mitP for 24 h, a dose-dependent loss of mitochondrial activity was observed using the methyl-thiazolyl-tetrazolium (MTT) assay. At 10 μmol L -1 , MTT activity was less than 30% that observed in untreated cells. Staining with the cationic dye JC-1 suggested that Z-Gly-RGD(DPhe)-mitP also dissipated the mitochondrial membrane potential, with a threefold increase in mitochondrial depolarisation levels. However, caspase activity appeared to be reduced by 24 h exposure to Z-Gly-RGD(DPhe)-mitP treatment. Furthermore, Z-Gly-RGD(DPhe)-mitP treatment had little effect on overall cell viability. Our findings suggest Z-Gly-RGD(DPhe)-mitP promotes the loss of mitochondrial activity but does not appear to evoke apoptosis in HaCaT keratinocytes.

  14. Synthesis, characterization and applications of carboxylated and polyethylene-glycolated bifunctionalized InP/ZnS quantum dots in cellular internalization mediated by cell-penetrating peptides.

    Science.gov (United States)

    Liu, Betty R; Winiarz, Jeffrey G; Moon, Jong-Sik; Lo, Shih-Yen; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2013-11-01

    Semiconductor nanoparticles, also known as quantum dots (QDs), are widely used in biomedical imaging studies and pharmaceutical research. Cell-penetrating peptides (CPPs) are a group of small peptides that are able to traverse cell membrane and deliver a variety of cargoes into living cells. CPPs deliver QDs into cells with minimal nonspecific absorption and toxic effect. In this study, water-soluble, monodisperse, carboxyl-functionalized indium phosphide (InP)/zinc sulfide (ZnS) QDs coated with polyethylene glycol lipids (designated QInP) were synthesized for the first time. The physicochemical properties (optical absorption, fluorescence and charging state) and cellular internalization of QInP and CPP/QInP complexes were characterized. CPPs noncovalently interact with QInP in vitro to form stable CPP/QInP complexes, which can then efficiently deliver QInP into human A549 cells. The introduction of 500nM of CPP/QInP complexes and QInP at concentrations of less than 1μM did not reduce cell viability. These results indicate that carboxylated and polyethylene-glycolylated (PEGylated) bifunctionalized QInP are biocompatible nanoparticles with potential for use in biomedical imaging studies and drug delivery applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Interference with RUNX1/ETO Leukemogenic Function by Cell-Penetrating Peptides Targeting the NHR2 Oligomerization Domain

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    Yvonne Bartel

    2013-01-01

    Full Text Available The leukemia-associated fusion protein RUNX1/ETO is generated by the chromosomal translocation t(8;21 which appears in about 12% of all de novo acute myeloid leukemias (AMLs. Essential for the oncogenic potential of RUNX1/ETO is the oligomerization of the chimeric fusion protein through the nervy homology region 2 (NHR2 within ETO. In previous studies, we have shown that the intracellular expression of peptides containing the NHR2 domain inhibits RUNX1/ETO oligomerization, thereby preventing cell proliferation and inducing differentiation of RUNX1/ETO transformed cells. Here, we show that introduction of a recombinant TAT-NHR2 fusion polypeptide into the RUNX1/ETO growth-dependent myeloid cell line Kasumi-1 results in decreased cell proliferation and increased numbers of apoptotic cells. This effect was highly specific and mediated by binding the TAT-NHR2 peptide to ETO sequences, as TAT-polypeptides containing the oligomerization domain of BCR did not affect cell proliferation or apoptosis in Kasumi-1 cells. Thus, the selective interference with NHR2-mediated oligomerization by peptides represents a challenging but promising strategy for the inhibition of the leukemogenic potential of RUNX1/ETO in t(8;21-positive leukemia.

  16. An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery

    Directory of Open Access Journals (Sweden)

    Ding Y

    2015-10-01

    Full Text Available Yuan Ding,1,* Dan Sun,1,* Gui-Ling Wang,1 Hong-Ge Yang,1 Hai-Feng Xu,1 Jian-Hua Chen,2 Ying Xie,1,3 Zhi-Qiang Wang4 1Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing, 2School of Medicine, Jianghan University, Wuhan, 3State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, People’s Republic of China; 4Department of Chemistry and Biochemistry, Kent State University Geauga, Burton, OH, USA *These authors contributed equally to this work Abstract: Cell-penetrating peptides (CPPs as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into

  17. A novel nanoemulsion-based method to produce ultrasmall, water-dispersible nanoparticles from chitosan, surface modified with cell-penetrating peptide for oral delivery of proteins and peptides

    Directory of Open Access Journals (Sweden)

    Barbari GR

    2017-05-01

    Full Text Available Ghullam Reza Barbari,1 Farid Abedin Dorkoosh,1 Mohsen Amini,2 Mohammad Sharifzadeh,3 Fateme Atyabi,1 Saeed Balalaie,4 Niyousha Rafiee Tehrani,5 Morteza Rafiee Tehrani1 1Department of Pharmaceutics, 2Department of Medicinal Chemistry, 3Department of Pharmacology, School of Pharmacy, Tehran University of Medical Sciences, 4Department of Chemistry, Khaje Nasiroddin University, 5Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Abstract: A simple and reproducible water-in-oil (W/O nanoemulsion technique for making ultrasmall (<15 nm, monodispersed and water-dispersible nanoparticles (NPs from chitosan (CS is reported. The nano-sized (50 nm water pools of the W/O nanoemulsion serve as “nano-containers and nano-reactors”. The entrapped polymer chains of CS inside these “nano-reactors” are covalently cross-linked with the chains of polyethylene glycol (PEG, leading to rigidification and formation of NPs. These NPs possess excessive swelling properties in aqueous medium and preserve integrity in all pH ranges due to chemical cross-linking with PEG. A potent and newly developed cell-penetrating peptide (CPP is further chemically conjugated to the surface of the NPs, leading to development of a novel peptide-conjugated derivative of CS with profound tight-junction opening properties. The CPP-conjugated NPs can easily be loaded with almost all kinds of proteins, peptides and nucleotides for oral delivery applications. Feasibility of this nanoparticulate system for oral delivery of a model peptide (insulin is investigated in Caco-2 cell line. The cell culture results for translocation of insulin across the cell monolayer are very promising (15%–19% increase, and animal studies are actively under progress and will be published separately. Keywords: ultrasmall, cell-penetrating peptide, chitosan, oral insulin, nanoemulsion, Caco-2 cell

  18. Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides.

    Science.gov (United States)

    Dixon, James E; Osman, Gizem; Morris, Gavin E; Markides, Hareklea; Rotherham, Michael; Bayoussef, Zahia; El Haj, Alicia J; Denning, Chris; Shakesheff, Kevin M

    2016-01-19

    Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application.

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

    Science.gov (United States)

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

    2017-07-13

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

  20. Solid-Phase Synthesis of Difficult Purine-Rich PNAs through Selective Hmb Incorporation: Application to the Total Synthesis of Cell Penetrating Peptide-PNAs

    Directory of Open Access Journals (Sweden)

    Julien Tailhades

    2017-10-01

    Full Text Available Antisense oligonucleotide (ASO-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino and Spinraza (2′-O-methoxyethyl-phosphorothioate in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl and Dmb (2,4-dimethoxybenzyl to ameliorate difficult couplings and reduce “on-resin” aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.

  1. Solid-phase synthesis of difficult purine-rich PNAs through selective Hmb incorporation: Application to the total synthesis of cell penetrating peptide-PNAs

    Science.gov (United States)

    Tailhades, Julien; Takizawa, Hotake; Gait, Michael J.; Wellings, Don A.; Wade, John D.; Aoki, Yoshitsugu; Shabanpoor, Fazel

    2017-10-01

    Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2’-O-methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce “on-resin” aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.

  2. Identification of a cell-penetrating peptide domain from human beta-defensin 3 and characterization of its anti-inflammatory activity

    Directory of Open Access Journals (Sweden)

    Lee JY

    2015-08-01

    sites, such as the cytoplasm or nucleus, as hBD3-3 has the ability to be used as a carrier, and suggest a potential approach to effectively treat inflammatory diseases. Keywords: human beta-defensin 3, cell-penetrating peptide, anti-inflammatory activity, lipopolysaccharide, NF-κB canonical pathway

  3. Improved proteolytic stability and potent activity against Leishmania infantum trypanothione reductase of α/β-peptide foldamers conjugated to cell-penetrating peptides.

    Science.gov (United States)

    de Lucio, Héctor; Gamo, Ana María; Ruiz-Santaquiteria, Marta; de Castro, Sonia; Sánchez-Murcia, Pedro A; Toro, Miguel A; Gutiérrez, Kilian Jesús; Gago, Federico; Jiménez-Ruiz, Antonio; Camarasa, María-José; Velázquez, Sonsoles

    2017-11-10

    The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β 3 -peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β 3 -peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo

    International Nuclear Information System (INIS)

    Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung; Park, Yoon-Jeong; Chung, Chong-Pyoung

    2012-01-01

    Highlights: ► HBP sequence identified from HB-EGF has cell penetration activity. ► HBP inhibits the NF-κB dependent inflammatory responses. ► HBP directly blocks phosphorylation and degradation of IκBα. ► HBP inhibits nuclear translocation of NF-κB p65 subunit. -- Abstract: A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-α and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-α and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-κB-dependent inflammatory responses by directly blocking the phosphorylation and degradation of IκBα and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-κB. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.

  5. 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

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

    Science.gov (United States)

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

    2014-03-01

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

  7. Biophysical and biological properties of small linear peptides derived from crotamine, a cationic antimicrobial/antitumoral toxin with cell penetrating and cargo delivery abilities.

    Science.gov (United States)

    Dal Mas, C; Pinheiro, D A; Campeiro, J D; Mattei, B; Oliveira, V; Oliveira, E B; Miranda, A; Perez, K R; Hayashi, M A F

    2017-12-01

    Crotamine is a natural polypeptide from snake venom which delivers nucleic acid molecules into cells, besides having pronounced affinity for negatively charged membranes and antifungal activity. We previously demonstrated that crotamine derived short linear peptides were not very effective as antifungal, although the non-structured recombinant crotamine was overridingly more potent compared to the native structured crotamine. Aiming to identify the features necessary for the antifungal activity of crotamine, two linear short peptides, each comprising half of the total positively charged amino acid residues of the full-length crotamine were evaluated here to show that these linear peptides keep the ability to interact with lipid membrane model systems with different phospholipid compositions, even after forming complexes with DNA. Interestingly, the presence of cysteine residues in the structure of these linear peptides highly influenced the antifungal activity, which was not associated to the lipid membrane lytic activity. In addition to the importance of the positive charges, the crucial role of cysteine residues was noticed for these linear analogs of crotamine, although the tridimensional structure and lipid membrane lytic activity observed only for native crotamine was not essential for the antifungal activity. As these peptides still keep the ability to form complexes with DNA molecules with no prejudice to their ability to bind to lipid membranes, they may be potentially advantageous as membrane translocation vector, as they do not show lipid membrane lytic activity and may harbor or not antifungal activity, by keeping or not the semi-essential amino acid cysteine in their sequence. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Alberto Malerba

    2012-01-01

    Full Text Available The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD. In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO. Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD.

  9. [The construction of cell-penetrating peptide R8 and pH sensitive cleavable polyethylene glycols co-modified liposomes].

    Science.gov (United States)

    Zhang, Li; Wang, Yang; Gao, Hui-le; He, Qin

    2015-06-01

    The purpose of the study is to construct R8 peptide (RRRRRRRR) and pH sensitive polyethylene glycols (PEG) co-modified liposomes (Cl-Lip) and utilize them in breast cancer treatment. The co-modified liposomes were prepared with soybean phospholipid, cholesterol, DSPE-PEG2K-R8 and PEG5K-Hz-PE (pH sensitive PEG). The size and zeta potential of Cl-Lip were also characterized. The in vitro experiment demonstrated that the Cl-Lip had high serum stability in 50% fetal bovine serum. The cellular uptake of Cl-Lip under different pre-incubated conditions was evaluated on 4T1 cells. And the endocytosis pathway, lysosome escape ability and tumor spheroid penetration ability were also evaluated. The results showed the particle size of the Cl-Lip was (110.4 ± 5.2) nm, PDI of the Cl-Lip was 0.207 ± 0.039 and zeta potential of the Cl-Lip was (-3.46 ± 0.05) mV. The cellular uptake of Cl-Lip on 4T1 cells was pH sensitive, as the cellular uptake of Cl-Lip pre-incubated in pH 6.0 was higher than that of pH 7.4 under each time point. The main endocytosis pathways of Cl-Lip under pH 6.0 were micropinocytosis and energy-dependent pathway. At the same time, the Cl-Lip with pre-incubation in pH 6.0 had high lysosome escape ability and high tumor spheroid penetration ability. All the above results demonstrated that the Cl-Lip we constructed had high pH sensitivity and is a promising drug delivery system.

  10. Thrombin-Activatable Microbubbles as Potential Ultrasound Contrast Agents for the Detection of Acute Thrombosis.

    Science.gov (United States)

    Lux, Jacques; Vezeridis, Alexander M; Hoyt, Kenneth; Adams, Stephen R; Armstrong, Amanda M; Sirsi, Shashank R; Mattrey, Robert F

    2017-11-01

    Acute deep vein thrombosis (DVT) is the formation of a blood clot in the deep veins of the body that can lead to fatal pulmonary embolism. Acute DVT is difficult to distinguish from chronic DVT by ultrasound (US), the imaging modality of choice, and is therefore treated aggressively with anticoagulants, which can lead to internal bleeding. Here we demonstrate that conjugating perfluorobutane-filled (PFB-filled) microbubbles (MBs) with thrombin-sensitive activatable cell-penetrating peptides (ACPPs) could lead to the development of contrast agents that detect acute thrombosis with US imaging. Successful conjugation of ACPP to PFB-filled MBs was confirmed by fluorescence microscopy and flow cytometry. Fluorescein-labeled ACPP was used to evaluate the efficiency of thrombin-triggered cleavage by measuring the mean fluorescence intensity of ACPP-labeled MBs (ACPP-MBs) before and after incubation at 37 °C with thrombin. Lastly, control MBs and ACPP-MBs were infused through a tube containing a clot, and US contrast enhancement was measured with or without the presence of a thrombin inhibitor after washing the clot with saline. With thrombin activity, 91.7 ± 14.2% of the signal was retained after ACPP-MB infusion and washing, whereas only 16.7 ± 4% of the signal was retained when infusing ACPP-MBs in the presence of hirudin, a potent thrombin inhibitor.

  11. In vivo study of doxorubicin-loaded cell-penetrating peptide-modified pH-sensitive liposomes: biocompatibility, bio-distribution, and pharmacodynamics in BALB/c nude mice bearing human breast tumors

    Directory of Open Access Journals (Sweden)

    Ding Y

    2017-10-01

    Full Text Available Yuan Ding,1,* Wei Cui,2,* Dan Sun,1 Gui-Ling Wang,1 Yu Hei,1 Shuai Meng,1 Jian-Hua Chen,3 Ying Xie,1 Zhi-Qiang Wang4 1Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, 2School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing, 3School of Medicine, Jianghan University, Wuhan, People’s Republic of China; 4Department of Chemistry and Biochemistry, Kent State University Geauga, Burton, OH, USA *These authors contributed equally to this work Abstract: In vivo evaluation of drug delivery vectors is essential for clinical translation. In BALB/c nude mice bearing human breast cancer tumors, we investigated the biocompatibility, pharmacokinetics, and pharmacodynamics of doxorubicin (DOX-loaded novel cell-penetrating peptide (CPP-modified pH-sensitive liposomes (CPPL (referred to as CPPL(DOX with an optimal CPP density of 4%. In CPPL, a polyethylene glycol (PEG derivative formed by conjugating PEG with stearate via acid-degradable hydrazone bond (PEG2000-Hz-stearate was inserted into the surface of liposomes, and CPP was directly attached to liposome surfaces via coupling with stearate to simultaneously achieve long circulation time in blood and improve the selectivity and efficacy of CPP for tumor targeting. Compared to PEGylated liposomes, CPPL enhanced DOX accumulation in tumors up to 1.9-fold (p<0.01 and resulted in more cell apoptosis as a result of DNA disruption as well as a relatively lower tumor growth ratio (T/C%. Histological examination did not show any signs of necrosis or inflammation in normal tissues, but large cell dissolving areas were found in tumors following the treatment of animals with CPPL(DOX. Our findings provide important and detailed information regarding the distribution of CPPL(DOX in vivo and reveal their abilities of tumor penetration and potential for the treatment of

  12. Antimicrobial and cell-penetrating properties of penetratin analogs

    DEFF Research Database (Denmark)

    Bahnsen, Jesper Søborg; Franzyk, Henrik; Sandberg-Schaal, Anne

    2013-01-01

    Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well as octaar......Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well...... as octaarginine and different Tat sequences. The effects of peptide length, guanidinium content, and sequence of non-cationic residues were assessed in mammalian and bacterial cells. The arginine (Arg) content in the penetratin analogs was found to influence eukaryotic cell uptake efficiency, antimicrobial...... was similar, the eukaryotic cellular uptake of the shuffled analogs was noticeably lower than for native penetratin. Moreover, a point substitution of Met to Leu in native penetratin had no influence on eukaryotic cellular uptake and antimicrobial effect, and only a minor effect on cytotoxicity, in contrast...

  13. Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)

    DEFF Research Database (Denmark)

    Valnickova, Zuzana; Thaysen-Andersen, Morten; Højrup, Peter

    2009-01-01

    -activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. RESULTS: The four N-linked glycosylation sequons within the activation peptide were all occupied...

  14. Molecular Imaging with Activatable Reporter Systems

    Directory of Open Access Journals (Sweden)

    Gang Niu, Xiaoyuan Chen

    2012-01-01

    Full Text Available Molecular imaging is a newly emerged multiple disciplinary field that aims to visualize, characterize and quantitatively measure biological processes at cellular and molecular levels in humans and other living systems. A reporter gene is a piece of DNA encoding reporter protein, which presents as a readily measurable phenotype that can be distinguished easily from the background of endogenous protein. After being transferred into cells of organ systems (transgenes, the reporter gene can be utilized to visualize transcriptional and posttranscriptional regulation of gene expression, protein-protein interactions, or trafficking of proteins or cells in living subjects. Herein, we review previous classification of reporter genes and regroup the reporter gene based imaging as basic, inducible and activatable, based on the regulation of reporter gene transcription and post-translational modification of reporter proteins. We then focus on activatable reporters, in which the signal can be activated at the posttranslational level for visualizing protein-protein interactions, protein phosphorylation or tertiary structure changes. The applications of several types of activatable reporters will also be summarized. We conclude that activatable reporter imaging can benefit both basic biomedical research and drug development.

  15. Cellular Reprogramming Using Protein and Cell-Penetrating Peptides

    Directory of Open Access Journals (Sweden)

    Bong Jong Seo

    2017-03-01

    Full Text Available Recently, stem cells have been suggested as invaluable tools for cell therapy because of their self-renewal and multilineage differentiation potential. Thus, scientists have developed a variety of methods to generate pluripotent stem cells, from nuclear transfer technology to direct reprogramming using defined factors, or induced pluripotent stem cells (iPSCs. Considering the ethical issues and efficiency, iPSCs are thought to be one of the most promising stem cells for cell therapy. Induced pluripotent stem cells can be generated by transduction with a virus, plasmid, RNA, or protein. Herein, we provide an overview of the current technology for iPSC generation and describe protein-based transduction technology in detail.

  16. Tunable protease-activatable virus nanonodes.

    Science.gov (United States)

    Judd, Justin; Ho, Michelle L; Tiwari, Abhinav; Gomez, Eric J; Dempsey, Christopher; Van Vliet, Kim; Igoshin, Oleg A; Silberg, Jonathan J; Agbandje-McKenna, Mavis; Suh, Junghae

    2014-05-27

    We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

  17. 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

    (antisense activity) is still limited by endocytotic entrapment. We have shown that this low bioavailability can be greatly improved by combining CPP-PNA conjugate administration with a photochemical internalization technique using photosensitizers such as aluminum phthalocyanine (AlPcS(2a...

  18.   Cell Penetrating Peptoids (CPPos: Synthesis of a Small Combinatorial Library by Using IRORI MiniKans

    Directory of Open Access Journals (Sweden)

    Dominik K. Kölmel

    2012-11-01

    Full Text Available Cell penetrating peptoids (CPPos are potent mimics of the corresponding cell penetrating peptides (CPPs. The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used to either target different cellular organelles or even different tissues and organs. In this study we established the submonomer-based solid phase synthesis of a “proof of principle” peptoid library in IRORI MiniKans to expand the amount for phenotypic high throughput screens of CPPos. The library consisting of tetrameric peptoids [oligo(N-alkylglycines] was established on Rink amide resin in a split and mix approach with hydrophilic and hydrophobic peptoid side chains. All CPPos of the presented library were labeled with rhodamine B to allow for the monitoring of cellular uptake by fluorescent confocal microscopy. Eventually, all the purified peptoids were subjected to live cell imaging to screen for CPPos with organelle specificity. While highly charged CPPos enter the cells by endocytosis with subsequent endosomal release, critical levels of lipophilicity allow other CPPos to specifically localize to mitochondria once a certain lipophilicity threshold is reached.

  19. Visualization of Protease Activity In Vivo Using an Activatable Photo-Acoustic Imaging Probe Based on CuS Nanoparticles

    Science.gov (United States)

    Yang, Kai; Zhu, Lei; Nie, Liming; Sun, Xiaolian; Cheng, Liang; Wu, Chenxi; Niu, Gang; Chen, Xiaoyuan; Liu, Zhuang

    2014-01-01

    Herein, we for the first time report a novel activatable photoacoustic (PA) imaging nano-probe for in vivo detection of cancer-related matrix metalloproteinases (MMPs). A black hole quencher 3 (BHQ3) which absorbs red light is conjugated to near-infrared (NIR)-absorbing copper sulfide (CuS) nanoparticles via a MMP-cleavable peptide linker. The obtained CuS-peptide-BHQ3 (CPQ) nano-probe exhibits two distinctive absorption peaks at 630 nm and 930 nm. Inside the tumor microenviorment where MMPs present, the MMP-sensitive peptide would be cleaved, releasing BHQ3 from the CuS nanoparticles, the former of which as a small molecule is then rapidly cleared out from the tumor, whereas the latter of which as large nanoparticles would retain inside the tumor for a much longer period of time. As the result, the PA signal at 680 nm which is contributed by BHQ3 would be quickly diminished while that at 930 nm would be largely retained. The PA signal ratio of 680 nm / 930 nm could thus serve as an in vivo indicator of MMPs activity inside the tumor. Our work presents a novel strategy of in vivo sensing of MMPs based on PA imaging, which should offer remarkably improved detection depth compared with traditional optical imaging techniques. PMID:24465271

  20. Improved cellular uptake of antisense Peptide nucleic acids by conjugation to a cell-penetrating Peptide and a lipid domain

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2011-01-01

    based on a splicing correction of a mutated luciferase gene in HeLa pLuc705 cells by targeting antisense oligonucleotides to a cryptic splice site. Further improvement in the delivery of CatLip-PNA conjugates is achieved by using auxiliary agents/treatments (e.g., chloroquine, calcium ions......Unaided cellular uptake of RNA interference agents such as antisense oligonucleotides and siRNA is extremely poor, and in vivo bioavailability is also limited. Thus, effective delivery strategies for such potential drugs are in high demand. Recently, a novel approach using a class of short cationic....... We have found, however, that this low -bioavailability can be significantly improved by chemical conjugation to a lipid domain ("Lip," such as a fatty acid), thereby creating "CatLip"-conjugates. The cellular uptake of these conjugates is conveniently evaluated using a sensitive cellular assay system...

  1. Thrombin activatable fibrinolysis inhibitor (TAFI in cord blood.

    Directory of Open Access Journals (Sweden)

    Krzysztof Góralczyk

    2007-03-01

    Full Text Available Thrombin activatable fibrinolysis inhibitor (TAFI is a plasma zymogene (procarboxypeptidase B which can decrease fibrinolysis and thus act as a haemostatic factor. TAFI is now extensively studied in many complications as well as in physiological and complicated pregnancy. The question we posed in the present study was whether TAFI antigen is present in cord blood plasma. The study group consisted of 38 parturient women, 26 primiparous and 12 multiparous with normal course of pregnancy and delivery. The cord blood was sampled from the cord vein, and the mother's blood from the antecubital vein. 3.2% sodium citrate was used as an anticoagulant. TAFIa/ai antigen was measured by ELISA method. TAFIa/ai antigen was identified in all samples of cord blood plasma. Its level was 91.50 ng/ml (range: 71.76 - 160.77 ng/ml vs. 55.46 ng/ml (range: 39.77 - 68.54 ng/ml in the mother's blood, which means that the level of TAFIa/ai antigen was significantly higher in fetal blood than in maternal blood (p<0.00001. TAFIa/ai antigen is an integral component of cord blood plasma. The concentration of TAFIa/ai antigen is about two times higher in fetal blood than in maternal blood.

  2. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

    OpenAIRE

    HaiFang Yin; Prisca Boisguerin; Hong M Moulton; Corinne Betts; Yiqi Seow; Jordan Boutilier; Qingsong Wang; Anthony Walsh; Bernard Lebleu; Matthew JA Wood

    2013-01-01

    We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was ...

  3. Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans

    KAUST Repository

    Å mand, Helene L.; Rydberg, Hanna A.; Fornander, Louise H.; Lincoln, Per; Nordé n, Bengt; Esbjö rner, Elin K.

    2012-01-01

    Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved

  4. A functional single nucleotide polymorphism in the thrombin-activatable fibrinolysis inhibitor (TAFI) gene associates with outcome of meningococcal disease

    NARCIS (Netherlands)

    Kremer Hovinga, J. A.; Franco, R. F.; Zago, M. A.; ten Cate, Hugo; Westendorp, R. G. J.; Reitsma, P. H.

    2004-01-01

    In meningococcal sepsis, disseminated intravascular coagulation with deposition of fibrin and formation of microthrombi occurs in various organs and enhanced inhibition of fibrinolysis is associated with adverse outcome. Recently, TAFI (thrombin-activatable fibrinolysis inhibitor) was identified as

  5. Cascading of molecular logic gates for advanced functions: a self-reporting, activatable photosensitizer.

    Science.gov (United States)

    Erbas-Cakmak, Sundus; Akkaya, Engin U

    2013-10-18

    Logical progress: Independent molecular logic gates have been designed and characterized. Then, the individual molecular logic gates were coerced to work together within a micelle. Information relay between the two logic gates was achieved through the intermediacy of singlet oxygen. Working together, these concatenated logic gates result in a self-reporting and activatable photosensitizer. GSH=glutathione. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Cell-penetrating superoxide dismutase attenuates oxidative stress-induced senescence by regulating the p53-p21Cip1 pathway and restores osteoblastic differentiation in human dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Park YJ

    2012-09-01

    Full Text Available Yoon Jung Choi,1,* Jue Yeon Lee,2,* Chong Pyoung Chung,2 Yoon Jeong Park,1,21Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea; 2Research Institute, Nano Intelligent Biomedical Engineering, Seoul, Republic of Korea*These authors contributed equally to this workBackground: Human dental pulp stem cells (DPSCs have potential applications in tissue regeneration because of their convenient cell harvesting procedures and multipotent capacity. However, the tissue regenerative potential of DPSCs is known to be negatively regulated by aging in long-term culture and under oxidative stress. With an aim of reducing cellular senescence and oxidative stress in DPSCs, an intracellular delivery system for superoxide dismutase 1 (SOD1 was developed. We conjugated SOD1 with a cell-penetrating peptide known as low-molecular weight protamine (LMWP, and investigated the effect of LMWP-SOD1 conjugates on hydrogen peroxide-induced cellular senescence and osteoblastic differentiation.Results: LMWP-SOD1 significantly attenuated enlarged and flattened cell morphology and increased senescence-associated β-galactosidase activity. Under the same conditions, LMWP-SOD1 abolished activation of the cell cycle regulator proteins, p53 and p21Cip1, induced by hydrogen peroxide. In addition, LMWP-SOD1 reversed the inhibition of osteoblastic differentiation and downregulation of osteogenic gene markers induced by hydrogen peroxide. However, LMWP-SOD1 could not reverse the decrease in odontogenesis caused by hydrogen peroxide.Conclusion: Overall, cell-penetrating LMWP-SOD1 conjugates are effective for attenuation of cellular senescence and reversal of osteoblastic differentiation of DPSCs caused by oxidative stress inhibition. This result suggests potential application in the field of antiaging and tissue engineering to overcome the limitations of senescent stem cells.Keywords: superoxide

  7. Interactions of Bio-Inspired Membranes with Peptides and Peptide-Mimetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Michael Sebastiano

    2015-08-01

    Full Text Available Via Dissipative Particle Dynamics (DPD and implicit solvent coarse-grained (CG Molecular Dynamics (MD we examine the interaction of an amphiphilic cell-penetrating peptide PMLKE and its synthetic counterpart with a bio-inspired membrane. We use the DPD technique to investigate the interaction of peptide-mimetic nanoparticles, or nanopins, with a three-component membrane. The CG MD approach is used to investigate the interaction of a cell-penetrating peptide PMLKE with single-component membrane. We observe the spontaneous binding and subsequent insertion of peptide and nanopin in the membrane by using CG MD and DPD approaches, respectively. In addition, we find that the insertion of peptide and nanopins is mainly driven by the favorable enthalpic interactions between the hydrophobic components of the peptide, or nanopin, and the membrane. Our study provides insights into the mechanism underlying the interactions of amphiphilic peptide and peptide-mimetic nanoparticles with a membrane. The result of this study can be used to guide the functional integration of peptide and peptide-mimetic nanoparticles with a cell membrane.

  8. Membrane adsorption and binding, cellular uptake and cytotoxicity of cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone

    DEFF Research Database (Denmark)

    Jing, Xiaona; Yang, Mingjun; Kasimova, Marina Robertovna

    2012-01-01

    to evaluate the effect of α-chirality in the β-peptoid residues and the presence of guanidinium groups in the α-amino acid residues on membrane interaction. The molecular properties of the peptidomimetics in solution (surface and intramolecular hydrogen bonding, aqueous diffusion rate and molecular size) were...... studied along with their adsorption to lipid bilayers, cellular uptake, and toxicity. The surface hydrogen bonding ability of the peptidomimetics reflected their adsorbed amounts onto lipid bilayers as well as with their cellular uptake, indicating the importance of hydrogen bonding for their membrane...

  9. Conjugation of a cell-penetrating peptide to parathyroid hormone affects its structure, potency, and transepithelial permeation

    DEFF Research Database (Denmark)

    Kristensen, Mie; de Groot, Anne Marit; Berthelsen, Jens

    2015-01-01

    hormone, i.e. PTH(1-34), and to evaluate the effect with regards to secondary structure, potency in Saos-2 cells, immunogenicity, safety as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1-34) as an alternative...

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

    Science.gov (United States)

    Ahmed, Marya

    2017-10-24

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

  11. [Reversion of overweight and obesity in Vilafranca del Penedès child population: ACTIVA'T Program (2012)].

    Science.gov (United States)

    Bibiloni, Maria Del Mar; Fernández-Blanco, Jordi; Pujol-Plana, Noemí; Surià Sonet, Sònia; Pujol-Puyané, Maria Cèlia; Mercadé Fuentes, Sílvia; Ojer Fernández de Soto, Laura; Tur, Josep A

    2017-11-20

    To assess a 6-month nutritional and physical activity intervention program on the nutritional status of overweight or obese and not very active 8-14 years old children by means of a controlled pre-post design (ACTIVA'T program). Pre-post study in 8-14 years old overweight or obese and low active children from Vilafranca del Penedès (Barcelona, Spain) randomized in control group (n = 51, 47.1% girls, nutritional intervention and ≤3h/wk physical activity) and ACTIVA'T group (n = 45, 37.8% girls, nutritional and physical activity ≥5h/wk intervention). Body mass index, waist/height index, and diet quality by means of KIDMED test at the beginning and at the end of the program were assessed. During the intervention, each participant was accompanied by a relative (father or mother) who performed the same activities as the children. Dietary recommendations have positively changed the habits of both ACTIVA'T and control group. The reversion in the prevalence of overweight and obesity was 93.8% and 58.6%, respectively, in the ACTIVA'T group, compared to 25.0% and 35.8% in the control group. Abdominal obesity was decreased from 42.2% to 17.8% in the ACTIVA'T group and from 47.1% to 27.5% in the control group. The program ACTIVA'T (nutritional education and physical activity promotion) improves the quality of diet and reverses the prevalence of overweight and obesity in the underactive child population. Copyright © 2017 SESPAS. Publicado por Elsevier España, S.L.U. All rights reserved.

  12. Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery

    International Nuclear Information System (INIS)

    Gaspar, V M; Marques, J G; Sousa, F; Queiroz, J A; Correia, I J; Louro, R O

    2013-01-01

    Bridging the gap between nanoparticulate delivery systems and translational gene therapy is a long sought after requirement in nanomedicine-based applications. However, recent developments regarding nanoparticle functionalization have brought forward the ability to synthesize materials with biofunctional moieties that mimic the evolved features of viral particles. Herein we report the versatile conjugation of both cell penetrating arginine and pH-responsive histidine moieties into the chitosan polymeric backbone, to improve the physicochemical characteristics of the native material. Amino acid coupling was confirmed by 2D TOCSY NMR and Fourier transform infrared spectroscopy. The synthesized chitosan–histidine–arginine (CH–H–R) polymer complexed plasmid DNA biopharmaceuticals, and spontaneously assembled into stable 105 nm nanoparticles with spherical morphology and positive surface charge. The functionalized delivery systems were efficiently internalized into the intracellular compartment, and exhibited remarkably higher transfection efficiency than unmodified chitosan without causing any cytotoxic effect. Additional findings regarding intracellular trafficking events reveal their preferential escape from degradative lysosomal pathways and nuclear localization. Overall, this assembly of nanocarriers with bioinspired moieties provides the foundations for the design of efficient and customizable materials for cancer gene therapy. (paper)

  13. Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.

    Directory of Open Access Journals (Sweden)

    Yoshinori Ohno

    Full Text Available Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.

  14. Antimicrobial Peptide-PNA Conjugates Selectively Targeting Bacterial Genes

    Science.gov (United States)

    2013-07-22

    antibacterial therapy. Initial publications suggest that conjugates of cell penetrating peptides and PNA’s can overcome the barrier in transporting ...Zhou, Y., Hou, Z., Meng, J., and Luo, X. Targeting RNA polymerase primary σ70 as a therapeutic strategy against methicillin - resistant ... Staphylococcus aureus by antisense peptide nucleic acid. PLoS One. 2012; 7(1):e29886. 2. Good, L., Sandberg, R., Larsson, O., Nielsen, P.E., and Wahlestedt, C

  15. Anticancer activities of bovine and human lactoferricin-derived peptides.

    Science.gov (United States)

    Arias, Mauricio; Hilchie, Ashley L; Haney, Evan F; Bolscher, Jan G M; Hyndman, M Eric; Hancock, Robert E W; Vogel, Hans J

    2017-02-01

    Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits broad-spectrum anticancer activity, while a similar peptide derived from human LF (hLF) is not as active. In this work, several peptides derived from the N-terminal regions of bLF and hLF were studied for their anticancer activities against leukemia and breast-cancer cells, as well as normal peripheral blood mononuclear cells. The cyclized LFcinB-CLICK peptide, which possesses a stable triazole linkage, showed improved anticancer activity, while short peptides hLF11 and bLF10 were not cytotoxic to cancer cells. Interestingly, hLF11 can act as a cell-penetrating peptide; when combined with the antimicrobial core sequence of LFcinB (RRWQWR) through either a Pro or Gly-Gly linker, toxicity to Jurkat cells increased. Together, our work extends the library of LF-derived peptides tested for anticancer activity, and identified new chimeric peptides with high cytotoxicity towards cancerous cells. Additionally, these results support the notion that short cell-penetrating peptides and antimicrobial peptides can be combined to create new adducts with increased potency.

  16. Sensitive and selective tumor imaging with novel and highly activatable fluorescence probes

    International Nuclear Information System (INIS)

    Urano, Yasuteru

    2008-01-01

    Selective and sensitive tumor imaging in vivo is one of the most requested methodologies in medical sciences. Although several imaging modalities have been developed including positron emission tomography (PET) and magnetic resonance (MR) imaging for the detection of tumors, none of these modalities can activate the signals upon being accumulated or uptaken to tumor sites. Among these modalities, only optical fluorescence imaging has a marked advantage, that is, their signals can be dramatically increased upon detecting some biological features. In this short review, I will introduce some recent strategies for activatable optical fluorescence imaging of tumors, and discuss their advantages over other modalities. (author)

  17. Thrombin-activatable fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis.

    Science.gov (United States)

    Valls Serón, M; Haiko, J; DE Groot, P G; Korhonen, T K; Meijers, J C M

    2010-10-01

     Pathogenic bacteria modulate the host coagulation system to evade immune responses or to facilitate dissemination through extravascular tissues. In particular, the important bacterial pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic system. Thrombin-activatable fibrinolysis inhibitor (TAFI) has anti-fibrinolytic properties as the active enzyme (TAFIa) removes C-terminal lysine residues from fibrin, thereby attenuating accelerated plasmin formation.  Here, we demonstrate inactivation and cleavage of TAFI by homologous surface proteases, the omptins Pla of Y. pestis and PgtE of S. enterica. We show that omptin-expressing bacteria decrease TAFI activatability by thrombin-thrombomodulin and that the anti-fibrinolytic potential of TAFIa was reduced by recombinant Escherichia coli expressing Pla or PgtE. The functional impairment resulted from C-terminal cleavage of TAFI by the omptins.  Our results indicate that TAFI is degraded directly by the omptins PgtE of S. enterica and Pla of Y. pestis. This may contribute to the ability of PgtE and Pla to damage tissue barriers, such as fibrin, and thereby to enhance spread of S. enterica and Y. pestis during infection. © 2010 International Society on Thrombosis and Haemostasis.

  18. Factor XI dependent and independent activation of thrombin activatable fibrinolysis inhibitor (TAFI) in plasma associated with clot formation

    NARCIS (Netherlands)

    Bouma, B. N.; Mosnier, L. O.; Meijers, J. C.; Griffin, J. H.

    1999-01-01

    Thrombin Activatable Fibrinolysis Inhibitor (TAFI) also known as plasma procarboxypeptidase B is activated by relatively high concentrations of thrombin in a reaction stimulated by thrombomodulin. In plasma an intact factor XI-dependent feed back loop via the intrinsic pathway is necessary to

  19. An eco-friendly in situ activatable antibiotic via cucurbit[8]uril-mediated supramolecular crosslinking of branched polyethylenimine.

    Science.gov (United States)

    Li, Shengke; Jiang, Nan; Zhao, Wenxuan; Ding, Yuan-Fu; Zheng, Ying; Wang, Lian-Hui; Zheng, Jun; Wang, Ruibing

    2017-05-30

    We report an unprecedented, eco-friendly, in situ activatable model antibiotic, phenylalanyl-polyethylenimine (PhePEI), to potentially diminish antibiotic pollution of the environment and associated antibiotic resistance. The inactive PhePEI can be reversibly activated upon supramolecular crosslinking by cucurbit[8]uril, conferring potent antibacterial activity.

  20. Binding characteristics of thrombin-activatable fibrinolysis inhibitor to streptococcal surface collagen-like proteins A and B

    NARCIS (Netherlands)

    Seron, Mercedes Valls; Plug, Tom; Marquart, J. Arnoud; Marx, Pauline F.; Herwald, Heiko; de Groot, Philip G.; Meijers, Joost C. M.

    2011-01-01

    Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins ScIA and ScIB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic

  1. DNA-interactive properties of crotamine, a cell-penetrating polypeptide and a potential drug carrier.

    Directory of Open Access Journals (Sweden)

    Pei-Chun Chen

    Full Text Available Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss or (ds double stranded molecules. The affinities of the protein for ss- vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of ∼3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more

  2. Preconcentration method using an activatable yield tracer for neutron activation analysis

    International Nuclear Information System (INIS)

    Tsukada, Masamichi; Yamamoto, Daijiro; Endo, Kazutoyo; Nakahara, Hiromichi

    1991-01-01

    Previously, the use of an enriched stable isotope as an activatable yield tracer in preconcentration steps has been tested by use of commercially available enriched 116 Cd and 156 Dy for biological standard reference materials. In the present work, this method has been further applied to the determination of lanthanoid contents in various kinds of samples: one coal fly-ash, three Japanese standard rocks, and eight standard soils. Samples were decomposed by alkali fusion in the preconcentration step. Thirteen elements were determined for coal fly-ash and soil samples, and 14 elements for rocks. The data obtained for coal fly-ash and standard rocks are compared with the data reported in literature. The data for soil samples have been newly determined in the present work. The ordinary instrumental neutron activation analysis and radiochemical neutron activation analysis were also performed to confirm the accuracy and usefulness of the present method. (author) 10 refs.; 4 tabs

  3. Thrombin-activatable fibrinolysis inhibitor activity in healthy and diseased dogs

    DEFF Research Database (Denmark)

    Jessen, Lisbeth Rem; Wiinberg, Bo; Kjelgaard-Hansen, Mads

    2010-01-01

    Background: In people, increased thrombin-activatable fibrinolysis inhibitor (TAFI) antigen has been associated with increased risk of thrombosis, and decreased TAFI may contribute to bleeding diathesis. TAFI activity in dogs has been described in experimental models, but not in dogs...... with spontaneous disease. Objective: The aim of this study was to compare TAFI activity in healthy dogs with TAFI activity in dogs with spontaneous disease. Methods: Plasma samples from 20 clinically healthy Beagles and from 35 dogs with various diseases were analyzed using a commercial chromogenic assay...... that measured TAFI activity relative to activity in standardized pooled human plasma. Results: Median TAFI activity for the 20 Beagles was 46.1% (range 32.2-70.8%) compared with 62.6% (29.1-250%) for the 35 diseased dogs, and 14/35 (40%) had TAFI activities >the upper limit for controls. The highest individual...

  4. Use of radioactive and neutron-activatable tracers to determine effective hydrogeologic parameters

    International Nuclear Information System (INIS)

    Yu, C.; Jester, W.A.; Jarrett, A.R.

    1985-01-01

    The migration of nuclides in a geologic medium is controlled by the hydrogeologic parameters of the medium such as the dispersion coefficient, pore water velocity, retardation factor, degradation rate, mass transfer coefficient, water content, and fraction of dead-end pores. The breakthrough curve (BTC), which is the graphical relationship between the tracer concentration and the elapsed time since introduction, is an integrated picture of the hydrogeologic parameters that produced the BTC. Both radioactive and neutron-activatable tracers have been used to generate BTCs. The BTC of a 92 Br radioactive tracer generated under saturated conditions in a nonhomogeneous (fractured) soil column is shown. From BTCs, the effective hydrogeologic parameters can be identified if appropriate techniques are applied

  5. Gadolinium-Encapsulating Iron Oxide Nanoprobe as Activatable NMR/MRI Contrast Agent

    Science.gov (United States)

    Santra, Santimukul; Jativa, Samuel D.; Kaittanis, Charalambos; Normand, Guillaume; Grimm, Jan; Perez, J. Manuel

    2012-01-01

    Herein we report a novel gadolinium-encapsulating iron oxide nanoparticle-based activatable NMR/MRI nanoprobe. In our design, Gd-DTPA is encapsulated within the polyacrylic acid (PAA) polymer coating of a superparamagnetic iron oxide nanoparticle (IO-PAA) yielding a composite magnetic nanoprobe (IO-PAA-Gd-DTPA) with quenched longitudinal spin-lattice magnetic relaxation (T1). Upon release of the Gd-DTPA complex from the nanoprobe's polymeric coating in acidic media, an increase in the T1 relaxation rate (1/T1) of the composite magnetic nanoprobe was observed, indicating a dequenching of the nanoprobe with a corresponding increase in the T1-weighted MRI signal. When a folate-conjugated nanoprobe was incubated in HeLa cells, a cancer cell line overexpressing folate receptors, an increase in the 1/T1 signal was observed. This result suggests that upon receptor-mediated internalization, the composite magnetic nanoprobe degraded within the cell's lysosome acidic (pH = 5.0) environment, resulting in an intracellular release of Gd-DTPA complex with subsequent T1 activation. No change in T1 was observed when the Gd-DTPA complex was chemically conjugated on the surface of the nanoparticle's polymeric coating or when encapsulated in the polymeric coating of a non-magnetic nanoparticle. These results confirmed that the observed (T1) quenching of the composite magnetic nanoprobe is due to the encapsulation and close proximity of the Gd ion to the nanoparticles superparamagnetic iron oxide (IO) core. In addition, when an anticancer drug (Taxol) was co-encapsulated with the Gd-DTPA within the folate receptor targeting composite magnetic nanoprobe, the T1 activation of the probe coincide with the rate of drug release and corresponding cytotoxic effect in cell culture studies. Taken together, these results suggest that our activatable T1 nanoagent could be of great importance for the detection of acidic tumors and assessment of drug targeting and release by MRI. PMID:22809405

  6. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis

    International Nuclear Information System (INIS)

    Bazzi, Zainab A.; Lanoue, Danielle; El-Youssef, Mouhanned; Romagnuolo, Rocco; Tubman, Janice; Cavallo-Medved, Dora; Porter, Lisa A.; Boffa, Michael B.

    2016-01-01

    Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis. To assess the role of TAFIa in breast cancer metastasis, in vitro migration and invasion assays, live cell proteolysis and cell proliferation using MDA-MB-231 and SUM149 cells were carried out in the presence of a TAFIa inhibitor, recombinant TAFI variants, or soluble TM. Inhibition of TAFIa with potato tuber carboxypeptidase inhibitor increased cell invasion, migration and proteolysis of both cell lines, whereas addition of TM resulted in a decrease in all these parameters. A stable variant of TAFIa, TAFIa-CIIYQ, showed enhanced inhibitory effects on cell invasion, migration and proteolysis. Furthermore, pericellular plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa. Taken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis in the breast cancer microenvironment. Enhancement of TAFI activation in this microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells

  7. Force-activatable biosensor enables single platelet force mapping directly by fluorescence imaging.

    Science.gov (United States)

    Wang, Yongliang; LeVine, Dana N; Gannon, Margaret; Zhao, Yuanchang; Sarkar, Anwesha; Hoch, Bailey; Wang, Xuefeng

    2018-02-15

    Integrin-transmitted cellular forces are critical for platelet adhesion, activation, aggregation and contraction during hemostasis and thrombosis. Measuring and mapping single platelet forces are desired in both research and clinical applications. Conventional force-to-strain based cell traction force microscopies have low resolution which is not ideal for cellular force mapping in small platelets. To enable platelet force mapping with submicron resolution, we developed a force-activatable biosensor named integrative tension sensor (ITS) which directly converts molecular tensions to fluorescent signals, therefore enabling cellular force mapping directly by fluorescence imaging. With ITS, we mapped cellular forces in single platelets at 0.4µm resolution. We found that platelet force distribution has strong polarization which is sensitive to treatment with the anti-platelet drug tirofiban, suggesting that the ITS force map can report anti-platelet drug efficacy. The ITS also calibrated integrin molecular tensions in platelets and revealed two distinct tension levels: 12-54 piconewton (nominal values) tensions generated during platelet adhesion and tensions above 54 piconewton generated during platelet contraction. Overall, the ITS is a powerful biosensor for the study of platelet mechanobiology, and holds great potential in antithrombotic drug development and assessing platelet activity in health and disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Design of ultrasonically-activatable nanoparticles using low boiling point perfluorocarbons.

    Science.gov (United States)

    Sheeran, Paul S; Luois, Samantha H; Mullin, Lee B; Matsunaga, Terry O; Dayton, Paul A

    2012-04-01

    Recently, an interest has developed in designing biomaterials for medical ultrasonics that can provide the acoustic activity of microbubbles, but with improved stability in vivo and a smaller size distribution for extravascular interrogation. One proposed alternative is the phase-change contrast agent. Phase-change contrast agents (PCCAs) consist of perfluorocarbons (PFCs) that are initially in liquid form, but can then be vaporized with acoustic energy. Crucial parameters for PCCAs include their sensitivity to acoustic energy, their size distribution, and their stability, and this manuscript provides insight into the custom design of PCCAs for balancing these parameters. Specifically, the relationship between size, thermal stability and sensitivity to ultrasound as a function of PFC boiling point and ambient temperature is illustrated. Emulsion stability and sensitivity can be 'tuned' by mixing PFCs in the gaseous state prior to condensation. Novel observations illustrate that stable droplets can be generated from PFCs with extremely low boiling points, such as octafluoropropane (b.p. -36.7 °C), which can be vaporized with acoustic parameters lower than previously observed. Results demonstrate the potential for low boiling point PFCs as a useful new class of compounds for activatable agents, which can be tailored to the desired application. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction.

    Science.gov (United States)

    Evans, Brian C; Hocking, Kyle M; Kilchrist, Kameron V; Wise, Eric S; Brophy, Colleen M; Duvall, Craig L

    2015-06-23

    A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.

  10. Selenium as an alternative peptide label - comparison to fluorophore-labelled penetratin

    DEFF Research Database (Denmark)

    Hyrup Møller, Laura; Bahnsen, Jesper Søborg; Nielsen, Hanne Mørck

    2015-01-01

    lysates, primarily the intact peptide (PenMSe, TAMRA-PenMSe or TAMRA-Pen) was observed. Selenium labelling caused minimal alteration of the physicochemical properties of the peptide and allowed for absolute quantitative determination of cellular uptake by inductively coupled plasma mass spectrometry......In the present study, the impact on peptide properties of labelling peptides with the fluorophore TAMRA or the selenium (Se) containing amino acid SeMet was evaluated. Three differently labelled variants of the cell-penetrating peptide (CPP) penetratin (Pen) were synthesized, PenMSe, TAMRA....... Selenium is thus proposed as a promising alternative label for quantification of peptides in general, altering the properties of the peptide to a minor extent as compared to commonly used peptide labels....

  11. Membrane interaction and secondary structure of de novo designed arginine-and tryptophan peptides with dual function

    KAUST Repository

    Rydberg, Hanna A.

    2012-10-01

    Cell-penetrating peptides and antimicrobial peptides are two classes of positively charged membrane active peptides with several properties in common. The challenge is to combine knowledge about the membrane interaction mechanisms and structural properties of the two classes to design peptides with membrane-specific actions, useful either as transporters of cargo or as antibacterial substances. Membrane active peptides are commonly rich in arginine and tryptophan. We have previously designed a series of arg/trp peptides and investigated how the position and number of tryptophans affect cellular uptake. Here we explore the antimicrobial properties and the interaction with lipid model membranes of these peptides, using minimal inhibitory concentrations assay (MIC), circular dichroism (CD) and linear dichroism (LD). The results show that the arg/trp peptides inhibit the growth of the two gram positive strains Staphylococcus aureus and Staphylococcus pyogenes, with some individual variations depending on the position of the tryptophans. No inhibition of the gram negative strains Proteus mirabilis or Pseudomonas aeruginosa was noticed. CD indicated that when bound to lipid vesicles one of the peptides forms an α-helical like structure, whereas the other five exhibited rather random coiled structures. LD indicated that all six peptides were somehow aligned parallel with the membrane surface. Our results do not reveal any obvious connection between membrane interaction and antimicrobial effect for the studied peptides. By contrast cell-penetrating properties can be coupled to both the secondary structure and the degree of order of the peptides. © 2012 Elsevier Inc.

  12. A novel chimeric peptide with antimicrobial activity.

    Science.gov (United States)

    Alaybeyoglu, Begum; Akbulut, Berna Sariyar; Ozkirimli, Elif

    2015-04-01

    Beta-lactamase-mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co-administration of beta-lactam type antibiotics and beta-lactamase inhibitors. Antimicrobial peptides are promising broad-spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46-Tyr51 beta-hairpin loop of beta-lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta-lactamase. Here, our goal was to modify this peptide for improved beta-lactamase inhibition and cellular uptake. Motivated by the cell-penetrating pVEC sequence, which includes a hydrophobic stretch at its N-terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N-terminus to facilitate uptake. Activity measurements of the peptide based on the 45-53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta-lactamase inhibitor with a K(i) value of 58 μM. Incubation of beta-lactam-resistant cells with peptide decreased the number of viable cells, while it had no effect on beta-lactamase-free cells, indicating that this peptide had antimicrobial activity via beta-lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N-terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta-lactamase but also for other intracellular targets. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  14. INTERNALIZATION OF ANTIMICROBIAL PEPTIDE ACIPENSIN 1 INTO HUMAN TUMOR CELLS

    Directory of Open Access Journals (Sweden)

    E. S. Umnyakova

    2016-01-01

    Full Text Available Search for new compounds providing delivery of drugs into infected or neoplastic cells, is an important direction of biomedical research. Cell-penetrating peptides are among those compounds, due to their ability to translocate through membranes of eukaryotic cells, serving as potential carriers of various therapeutic agents to the target cells. The aim of present work was to investigate the ability of acipensin 1, an antimicrobial peptide of innate immune system, for in vitro penetration into human tumor cells. Acipensin 1 is a cationic peptide that we have previously isolated from leukocytes of the Russian sturgeon, Acipenser gueldenstaedtii. Capability of acipensin 1 to enter the human erytroleukemia K-562 cells has been investigated for the first time. A biotechnological procedure for producing a recombinant acipensin 1 peptide has been developed. The obtained peptide was conjugated with a fluorescent probe BODIPY FL. By means of confocal microscopy, we have shown that the tagged acipensin 1 rapidly enters into K-562 cells and can be detected in the intracellular space within 5 min after its addition to the cell culture. Using flow cytometry technique, penetration kinetics of the labeled peptide into K-562 cells (at nontoxic micromolar concentrations has been studied. We have observed a rapid internalization of the peptide to the target cells, thus confirming the results of microscopic analysis, i.e, the labeled acipensin was detectable in K-562 cells as soon as wihin 2-3 seconds after its addition to the incubation medium. The maximum of fluorescence was reached within a period of approx. 45 seconds, with further “plateau” at the terms of >100 seconds following cell stimulation with the test compound. These data support the concept, that the antimicrobial peptides of innate immunity system possess the features of cell-penetrating peptides, and allow us to consider the studied sturgeon peptide a promising template for development of new

  15. Theranostic Niosomes for Efficient siRNA/microRNA Delivery and Activatable Near-Infrared Fluorescent Tracking of Stem Cells

    DEFF Research Database (Denmark)

    Yang, Chuanxu; Shan, Gao; Song, Ping

    2018-01-01

    RNA interference (RNAi) mediated gene regulation in stem cells offers great potential in regenerative medicine. In this study, we developed a theranostic platform for efficient delivery of small RNAs (siRNA/miRNA) to human mesenchymal stem cells (hMSCs) to promote differentiation, and meanwhile...... OFF/ON activatable fluorescence upon cellular internalization, resulting in efficient NIR labeling and the capability to dynamically monitor stem cells in mice. In addition, iSPN/siRNA achieved simultaneous long-term cell tracking and in vivo gene silencing after implantation in mice. These results...

  16. New Potent Membrane-Targeting Antibacterial Peptides from Viral Capsid Proteins

    Science.gov (United States)

    Dias, Susana A.; Freire, João M.; Pérez-Peinado, Clara; Domingues, Marco M.; Gaspar, Diana; Vale, Nuno; Gomes, Paula; Andreu, David; Henriques, Sónia T.; Castanho, Miguel A. R. B.; Veiga, Ana S.

    2017-01-01

    The increasing prevalence of multidrug-resistant bacteria urges the development of new antibacterial agents. With a broad spectrum activity, antimicrobial peptides have been considered potential antibacterial drug leads. Using bioinformatic tools we have previously shown that viral structural proteins are a rich source for new bioactive peptide sequences, namely antimicrobial and cell-penetrating peptides. Here, we test the efficacy and mechanism of action of the most promising peptides among those previously identified against both Gram-positive and Gram-negative bacteria. Two cell-penetrating peptides, vCPP 0769 and vCPP 2319, have high antibacterial activity against Staphylococcus aureus, MRSA, Escherichia coli, and Pseudomonas aeruginosa, being thus multifunctional. The antibacterial mechanism of action of the two most active viral protein-derived peptides, vAMP 059 and vCPP 2319, was studied in detail. Both peptides act on both Gram-positive S. aureus and Gram-negative P. aeruginosa, with bacterial cell death occurring within minutes. Also, these peptides cause bacterial membrane permeabilization and damage of the bacterial envelope of P. aeruginosa cells. Overall, the results show that structural viral proteins are an abundant source for membrane-active peptides sequences with strong antibacterial properties. PMID:28522994

  17. Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins[S

    Science.gov (United States)

    Subra, Caroline; Grand, David; Laulagnier, Karine; Stella, Alexandre; Lambeau, Gérard; Paillasse, Michael; De Medina, Philippe; Monsarrat, Bernard; Perret, Bertrand; Silvente-Poirot, Sandrine; Poirot, Marc; Record, Michel

    2010-01-01

    Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA2-IVA, the calcium-independent iPLA2-VIA, and the secreted sPLA2-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPγS triggered activation of phospholipase A2 (PLA2)and PLD2. A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandinJ2 (15-d PGJ2), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell. PMID:20424270

  18. A Cancer Cell-Activatable Aptamer-Reporter System for One-Step Assay of Circulating Tumor Cells

    Directory of Open Access Journals (Sweden)

    Zihua Zeng

    2014-01-01

    Full Text Available The current antibody-mediated numeration assays of circulating tumor cells (CTCs require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

  19. Smart activatable and traceable dual-prodrug for image-guided combination photodynamic and chemo-therapy.

    Science.gov (United States)

    Hu, Fang; Yuan, Youyong; Mao, Duo; Wu, Wenbo; Liu, Bin

    2017-11-01

    Activatable photosensitizers (PSs) and chemo-prodrugs are highly desirable for anti-cancer therapy to reduce systemic toxicity. However, it is difficult to integrate both together into a molecular probe for combination therapy due to the complexity of introducing PS, singlet oxygen quencher, chemo-drug, chemo-drug inhibitor and active linker at the same time. To realize activatable PS and chemo-prodrug combination therapy, we develop a smart therapeutic platform in which the chemo-prodrug serves as the singlet oxygen quencher for the PS. Specifically, the photosensitizing activity and fluorescence of the PS (TPEPY-SH) are blocked by the chemo-prodrug (Mitomycin C, MMC) in the probe. Meanwhile, the cytotoxicity of MMC is also inhibited by the electron-withdrawing acyl at the nitrogen position next to the linker. Upon glutathione activation, TPEPY-S-MMC can simultaneously release active PS and MMC for combination therapy. The restored fluorescence of TPEPY-SH is also used to report the activation for both PS and MMC as well as to guide the photodynamic therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent.

    Science.gov (United States)

    Schwenck, Johannes; Maier, Florian C; Kneilling, Manfred; Wiehr, Stefan; Fuchs, Kerstin

    2017-05-08

    This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models. Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

  1. Two-photon imaging of formaldehyde in live cells and animals utilizing a lysosome-targetable and acidic pH-activatable fluorescent probe.

    Science.gov (United States)

    Xie, Xilei; Tang, Fuyan; Shangguan, Xiaoyan; Che, Shiyi; Niu, Jinye; Xiao, Yongsheng; Wang, Xu; Tang, Bo

    2017-06-13

    Lyso-TPFP presents lysosomal targetability and an acidic pH-activatable response toward formaldehyde. Thus, it exclusively visualizes lysosomal formaldehyde and is immune against it in neutral cytosol and other organelles. In addition, two-photon fluorescence imaging endows Lyso-TPFP with the capability of in situ tracking formaldehyde in live cells and animals.

  2. Quantification of pharmaceutical peptides using selenium as an elemental detection label

    DEFF Research Database (Denmark)

    Møller, Laura Hyrup; Gabel-Jensen, Charlotte; Franzyk, Henrik

    2014-01-01

    analysis of cell samples by LC-ICP-MS showed mainly uptake of the intact peptides, while the amount of intact peptides in cell lysates was semi-quantitatively determined. The selenium-containing penetratin analogues were to some extent degraded in pure cell medium, while an extensive degradation......The aim of the present work was to demonstrate how selenium labelling of a synthetic cell-penetrating peptide may be employed in evaluation of stability and quantitative estimation of cellular uptake by inductively coupled plasma mass spectrometry (ICP-MS). Two analogues of the cell...

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

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Shiraishi, Takehiko; Zachar, Vladimir

    2008-01-01

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

  4. Characterisation of the membrane affinity of an isoniazide peptide conjugate by tensiometry, atomic force microscopy and sum-frequency vibrational spectroscopy, using a phospholipid Langmuir monolayer model.

    Science.gov (United States)

    Hill, Katalin; Pénzes, Csanád Botond; Schnöller, Donát; Horváti, Kata; Bosze, Szilvia; Hudecz, Ferenc; Keszthelyi, Tamás; Kiss, Eva

    2010-10-07

    Tensiometry, sum-frequency vibrational spectroscopy, and atomic force microscopy were employed to assess the cell penetration ability of a peptide conjugate of the antituberculotic agent isoniazide. Isoniazide was conjugated to peptide (91)SEFAYGSFVRTVSLPV(106), a functional T-cell epitope of the immunodominant 16 kDa protein of Mycobacterium tuberculosis. As a simple but versatile model of the cell membrane a phospholipid Langmuir monolayer at the liquid/air interface was used. Changes induced in the structure of the phospholipid monolayer by injection of the peptide conjugate into the subphase were followed by tensiometry and sum-frequency vibrational spectroscopy. The drug penetrated lipid films were transferred to a solid support by the Langmuir-Blodgett technique, and their structures were characterized by atomic force microscopy. Peptide conjugation was found to strongly enhance the cell penetration ability of isoniazide.

  5. Current status of multiple antigen-presenting peptide vaccine systems: Application of organic and inorganic nanoparticles

    Directory of Open Access Journals (Sweden)

    Taguchi Hiroaki

    2011-08-01

    Full Text Available Abstract Many studies are currently investigating the development of safe and effective vaccines to prevent various infectious diseases. Multiple antigen-presenting peptide vaccine systems have been developed to avoid the adverse effects associated with conventional vaccines (i.e., live-attenuated, killed or inactivated pathogens, carrier proteins and cytotoxic adjuvants. Recently, two main approaches have been used to develop multiple antigen-presenting peptide vaccine systems: (1 the addition of functional components, e.g., T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2 synthetic approaches using size-defined nanomaterials, e.g., self-assembling peptides, non-peptidic dendrimers, and gold nanoparticles, as antigen-displaying platforms. This review summarizes the recent experimental studies directed to the development of multiple antigen-presenting peptide vaccine systems.

  6. A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

    Science.gov (United States)

    Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

    2015-01-01

    The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores.

    Science.gov (United States)

    Chen, Zhixing; Wei, Lu; Zhu, Xinxin; Min, Wei

    2012-08-13

    It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

  8. The effect of a beta-lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study.

    Science.gov (United States)

    Alaybeyoglu, Begum; Uluocak, Bilge Gedik; Akbulut, Berna Sariyar; Ozkirimli, Elif

    2017-05-01

    Co-administration of beta-lactam antibiotics and beta-lactamase inhibitors has been a favored treatment strategy against beta-lactamase-mediated bacterial antibiotic resistance, but the emergence of beta-lactamases resistant to current inhibitors necessitates the discovery of novel non-beta-lactam inhibitors. Peptides derived from the Ala46-Tyr51 region of the beta-lactamase inhibitor protein are considered as potent inhibitors of beta-lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell-penetrating peptides could guide the design of beta-lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta-lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell-penetrating peptide pVEC, our approach involved the addition of the N-terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta-lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta-lactam antibiotic ampicillin, and the beta-lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N-terminus of the beta-lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N-terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European

  9. Stepwise-activable multifunctional peptide-guided prodrug micelles for cancerous cells intracellular drug release

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jing, E-mail: zhangjing@zjut.edu.cn; Li, Mengfei [Zhejiang University of Technology, College of Materials Science and Engineering (China); Yuan, Zhefan [Zhejiang University, Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering (China); Wu, Dan; Chen, Jia-da; Feng, Jie, E-mail: fengjie@zjut.edu.cn [Zhejiang University of Technology, College of Materials Science and Engineering (China)

    2016-10-15

    A novel type of stepwise-activable multifunctional peptide-guided prodrug micelles (MPPM) was fabricated for cancerous cells intracellular drug release. Deca-lysine sequence (K{sub 10}), a type of cell-penetrating peptide, was synthesized and terminated with azido-glycine. Then a new kind of molecule, alkyne modified doxorubicin (DOX) connecting through disulfide bond (DOX-SS-alkyne), was synthesized. After coupling via Cu-catalyzed azide–alkyne cycloaddition (CuAAC) click chemistry reaction, reduction-sensitive peptide-guided prodrug was obtained. Due to the amphiphilic property of the prodrug, it can assemble to form micelles. To prevent the nanocarriers from unspecific cellular uptake, the prodrug micelles were subsequently modified with 2,3-dimethyl maleic anhydride to obtain MPPM with a negatively charged outer shell. In vitro studies showed that MPPM could be shielded from cells under psychological environment. However, when arriving at mild acidic tumor site, the cell-penetrating capacity of MPPM would be activated by charge reversal of the micelles via hydrolysis of acid-labile β-carboxylic amides and regeneration of K{sub 10}, which enabled efficient internalization of MPPM by tumor cells as well as following glutathione- and protease-induced drug release inside the cancerous cells. Furthermore, since the guide peptide sequences can be accurately designed and synthesized, it can be easily changed for various functions, such as targeting peptide, apoptotic peptide, even aptamers, only need to be terminated with azido-glycine. This method can be used as a template for reduction-sensitive peptide-guided prodrug for cancer therapy.Graphical abstractA novel type of stepwise-activable multifunctional peptide-guided prodrug micelles (MPPM) was fabricated for selective drug delivery in cancerous cells. MPPM could be shielded from cells under psychological environment. However, when arriving at mild acidic tumor site, the cell-penetrating capacity of MPPM would

  10. Peptide dendrimers

    Czech Academy of Sciences Publication Activity Database

    Niederhafner, Petr; Šebestík, Jaroslav; Ježek, Jan

    2005-01-01

    Roč. 11, - (2005), 757-788 ISSN 1075-2617 R&D Projects: GA ČR(CZ) GA203/03/1362 Institutional research plan: CEZ:AV0Z40550506 Keywords : multiple antigen peptides * peptide dendrimers * synthetic vaccine * multipleantigenic peptides Subject RIV: CC - Organic Chemistry Impact factor: 1.803, year: 2005

  11. Effective modification of cell death-inducing intracellular peptides by means of a photo-cleavable peptide array-based screening system.

    Science.gov (United States)

    Kozaki, Ikko; Shimizu, Kazunori; Honda, Hiroyuki

    2017-08-01

    Intracellular functional peptides that play a significant role inside cells have been receiving a lot of attention as regulators of cellular activity. Previously, we proposed a novel screening system for intracellular functional peptides; it combined a photo-cleavable peptide array system with cell-penetrating peptides (CPPs). Various peptides can be delivered into cells and intracellular functions of the peptides can be assayed by means of our system. The aim of the present study was to demonstrate that the proposed screening system can be used for assessing the intracellular activity of peptides. The cell death-inducing peptide (LNLISKLF) identified in a mitochondria-targeting domain (MTD) of the Noxa protein served as an original peptide sequence for screening of peptides with higher activity via modification of the peptide sequence. We obtained 4 peptides with higher activity, in which we substituted serine (S) at the fifth position with phenylalanine (F), valine (V), tryptophan (W), or tyrosine (Y). During analysis of the mechanism of action, the modified peptides induced an increase in intracellular calcium concentration, which was caused by the treatment with the original peptide. Higher capacity for cell death induction by the modified peptides may be caused by increased hydrophobicity or an increased number of aromatic residues. Thus, the present work suggests that the intracellular activity of peptides can be assessed using the proposed screening system. It could be used for identifying intracellular functional peptides with higher activity through comprehensive screening. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Peptides Displayed as High Density Brush Polymers Resist Proteolysis and Retain Bioactivity

    Science.gov (United States)

    2015-01-01

    We describe a strategy for rendering peptides resistant to proteolysis by formulating them as high-density brush polymers. The utility of this approach is demonstrated by polymerizing well-established cell-penetrating peptides (CPPs) and showing that the resulting polymers are not only resistant to proteolysis but also maintain their ability to enter cells. The scope of this design concept is explored by studying the proteolytic resistance of brush polymers composed of peptides that are substrates for either thrombin or a metalloprotease. Finally, we demonstrate that the proteolytic susceptibility of peptide brush polymers can be tuned by adjusting the density of the polymer brush and offer in silico models to rationalize this finding. We contend that this strategy offers a plausible method of preparing peptides for in vivo use, where rapid digestion by proteases has traditionally restricted their utility. PMID:25314576

  13. DNA-histone complexes as ligands amplify cell penetration and nuclear targeting of anti-DNA antibodies via energy-independent mechanisms.

    Science.gov (United States)

    Zannikou, Markella; Bellou, Sofia; Eliades, Petros; Hatzioannou, Aikaterini; Mantzaris, Michael D; Carayanniotis, George; Avrameas, Stratis; Lymberi, Peggy

    2016-01-01

    We have generated three monoclonal cell-penetrating antibodies (CPAbs) from a non-immunized lupus-prone (NZB × NZW)F1 mouse that exhibited high anti-DNA serum titres. These CPAbs are polyreactive because they bind to DNA and other cellular components, and localize mainly in the nucleus of HeLa cells, albeit with a distinct nuclear labelling profile. Herein, we have examined whether DNA-histone complexes (DHC) binding to CPAbs, before cell entry, could modify the cell penetration of CPAbs or their nuclear staining properties. By applying confocal microscopy and image analysis, we found that extracellular binding of purified CPAbs to DHC significantly enhanced their subsequent cell-entry, both in terms of percentages of positively labelled cells and fluorescence intensity (internalized CPAb amount), whereas there was a variable effect on their nuclear staining profile. Internalization of CPAbs, either alone or bound to DHC, remained unaltered after the addition of endocytosis-specific inhibitors at 37° or assay performance at 4°, suggesting the involvement of energy-independent mechanisms in the internalization process. These findings assign to CPAbs a more complex pathogenetic role in systemic lupus erythematosus where both CPAbs and nuclear components are abundant. © 2015 John Wiley & Sons Ltd.

  14. In vivo imaging of membrane type-1 matrix metalloproteinase with a novel activatable near-infrared fluorescence probe.

    Science.gov (United States)

    Shimizu, Yoichi; Temma, Takashi; Hara, Isao; Makino, Akira; Kondo, Naoya; Ozeki, Ei-Ichi; Ono, Masahiro; Saji, Hideo

    2014-08-01

    Membrane type-1 matrix metalloproteinase (MT1-MMP) is a protease activating MMP-2 that mediates cleavage of extracellular matrix components and plays pivotal roles in tumor migration, invasion and metastasis. Because in vivo noninvasive imaging of MT1-MMP would be useful for tumor diagnosis, we developed a novel near-infrared (NIR) fluorescence probe that can be activated following interaction with MT1-MMP in vivo. MT1-hIC7L is an activatable fluorescence probe comprised of anti-MT1-MMP monoclonal antibodies conjugated to self-assembling polymer micelles that encapsulate NIR dyes (IC7-1, λem : 858 nm) at concentrations sufficient to cause fluorescence self-quenching. In aqueous buffer, MT1-hIC7L fluorescence was suppressed to background levels and increased approximately 35.5-fold in the presence of detergent. Cellular uptake experiments revealed that in MT1-MMP positive C6 glioma cells, MT1-hIC7L showed significantly higher fluorescence that increased with time as compared to hIC7L, a negative control probe lacking the anti-MT1-MMP monoclonal antibody. In MT1-MMP negative MCF-7 breast adenocarcinoma cells, both MT1-hIC7L and hIC7L showed no obvious fluorescence. In addition, the fluorescence intensity of C6 cells treated with MT1-hIC7L was suppressed by pre-treatment with an MT1-MMP endocytosis inhibitor (P imaging using probes intravenously administered to tumor-bearing mice showed that MT1-hIC7L specifically visualized C6 tumors (tumor-to-background ratios: 3.8 ± 0.3 [MT1-hIC7L] vs 3.1 ± 0.2 [hIC7L] 48 h after administration, P fluorescence in MCF-7 tumors. Together, these results show that MT1-hIC7L would be a potential activatable NIR probe for specifically detecting MT1-MMP-expressing tumors. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  15. Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

    Directory of Open Access Journals (Sweden)

    Park Sung

    2008-12-01

    Full Text Available Abstract Background Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli have been unsuccessful. Results Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM of the precursor proteins by two orders of magnitude. Conclusion A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.

  16. Thrombin activatable fibrinolysis inhibitor and clot lysis time in pregnant patients with antiphospholipid syndrome: relationship with pregnancy outcome and thrombosis.

    Science.gov (United States)

    Martinez-Zamora, Maria Angeles; Tassies, Dolors; Carmona, Francisco; Espinosa, Gerard; Cervera, Ricard; Reverter, Juan Carlos; Balasch, Juan

    2009-12-01

    Antiphospholipid syndrome (APS) pregnancies are associated with thrombotic obstetric complications, despite treatment. This study evaluated Thrombin Activatable Fibrinolysis Inhibitor (TAFI) levels, TAFI gene polymorphisms and Clot Lysis Time (CLT) in pregnant patients with APS in relation to pregnancy outcome and thrombosis. Group 1 consisted of 67 pregnant patients with APS. Group 2 included 66 pregnant patients with uneventful term pregnancies and delivery. Patients were sampled during each trimester and at baseline. TAFI antigen and CLT and two polymorphisms of the TAFI gene, Ala147Thr and +1542C/G, were determined. Significantly prolonged CLT was found at baseline in Group 1. Allele distribution of the TAFI gene polymorphisms was similar in both groups. Basal TAFI and CLT in patients with APS having an adverse or a good obstetrical outcome were similar. Comparison of TAFI and CLT baseline levels in patients with APS with or without previous thrombosis showed no statistical differences. Patients with APS have impairment in fibrinolysis evidenced by prolonged CLT at baseline. TAFI and CLT do not seem to be useful as markers of obstetric outcome or risk of thrombosis in patients with APS.

  17. Thrombin activatable fibrinolysis inhibitor (TAFI) - A possible link between coagulation and complement activation in the antiphospholipid syndrome (APS).

    Science.gov (United States)

    Grosso, Giorgia; Vikerfors, Anna; Woodhams, Barry; Adam, Mariette; Bremme, Katarina; Holmström, Margareta; Ågren, Anna; Eelde, Anna; Bruzelius, Maria; Svenungsson, Elisabet; Antovic, Aleksandra

    2017-10-01

    Thrombosis and complement activation are pathogenic features of antiphospholipid syndrome (APS). Their molecular link is Plasma carboxypeptidase-B, also known as thrombin activatable fibrinolysis inhibitor (TAFIa), which plays a dual role: anti-fibrinolytic, by cleaving carboxyl-terminal lysine residues from partially degraded fibrin, and anti-inflammatory, by downregulating complement anaphylatoxins C3a and C5a. To investigate the levels of TAFI (proenzyme) and TAFIa (active enzyme) in relation to complement activation, fibrin clot permeability and fibrinolytic function in clinical and immunological subsets of 52 APS patients and 15 controls. TAFI (pAPS patients compared to controls. Furthermore, TAFIa was increased (pAPS patients affected by arterial thrombosis compared to other APS-phenotypes. Positive associations were found between TAFI and age, fibrinogen and C5a, and between TAFIa and age, fibrinogen and thrombomodulin. TAFI and TAFIa levels were increased in patients with APS as a potential response to complement activation. Interestingly, TAFI activation was associated with arterial thrombotic APS manifestations. Thus, TAFIa may be considered a novel biomarker for arterial thrombosis in APS. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency.

    Science.gov (United States)

    Yin, Haifang; Boisguerin, Prisca; Moulton, Hong M; Betts, Corinne; Seow, Yiqi; Boutilier, Jordan; Wang, Qingsong; Walsh, Anthony; Lebleu, Bernard; Wood, Matthew Ja

    2013-09-24

    We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.Molecular Therapy-Nucleic Acids (2013) 2, e124; doi:10.1038/mtna.2013

  19. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

    Directory of Open Access Journals (Sweden)

    HaiFang Yin

    2013-01-01

    Full Text Available We have recently reported that cell-penetrating peptides (CPPs and novel chimeric peptides containing CPP (referred as B peptide and muscle-targeting peptide (referred as MSP motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO and control peptide 3 (B-3-PMO and 3-B-PMO were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO, further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG, indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.

  20. Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers

    Science.gov (United States)

    Chuah, Jo-Ann; Yoshizumi, Takeshi; Kodama, Yutaka; Numata, Keiji

    2015-01-01

    Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

  1. Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis

    Energy Technology Data Exchange (ETDEWEB)

    Nair, Baiju G.; Fukuda, Takahiro; Mizuki, Toru; Hanajiri, Tatsuro [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan); Maekawa, Toru, E-mail: maekawa@toyo.jp [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan)

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer We study the intracellular localisation of TAT-SPIONs using 3-D electron tomography. Black-Right-Pointing-Pointer 3-D images of TAT-SPIONs in a cell are clearly shown. Black-Right-Pointing-Pointer Release of TAT-SPIONs from endocytic vesicles into the cytoplasm is clearly shown. -- Abstract: Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to various drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to investigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be utilised for further investigations of the behaviours of nanoparticles in cells and eventually for the development of nano drug delivery systems.

  2. Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis

    International Nuclear Information System (INIS)

    Nair, Baiju G.; Fukuda, Takahiro; Mizuki, Toru; Hanajiri, Tatsuro; Maekawa, Toru

    2012-01-01

    Highlights: ► We study the intracellular localisation of TAT-SPIONs using 3-D electron tomography. ► 3-D images of TAT-SPIONs in a cell are clearly shown. ► Release of TAT-SPIONs from endocytic vesicles into the cytoplasm is clearly shown. -- Abstract: Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to various drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to investigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be utilised for further investigations of the behaviours of nanoparticles in cells and eventually for the development of nano drug delivery systems.

  3. A lysosome-locating and acidic pH-activatable fluorescent probe for visualizing endogenous H2O2 in lysosomes.

    Science.gov (United States)

    Liu, Jun; Zhou, Shunqing; Ren, Jing; Wu, Chuanliu; Zhao, Yibing

    2017-11-20

    There is increasing evidence indicating that lysosomal H 2 O 2 is closely related to autophagy and apoptotic pathways under both physiological and pathological conditions. Therefore, fluorescent probes that can be exploited to visualize H 2 O 2 in lysosomes are potential tools for exploring diverse roles of H 2 O 2 in cells. However, functional exploration of lysosomal H 2 O 2 is limited by the lack of fluorescent probes capable of compatibly sensing H 2 O 2 under weak acidic conditions (pH = 4.5) of lysosomes. Lower spatial resolution of the fluorescent visualization of lysosomal H 2 O 2 might be caused by the interference of signals from cytosolic and mitochondrial H 2 O 2 , as well as the non-specific distribution of the probes in cells. In this work, we developed a lysosome-locating and acidic-pH-activatable fluorescent probe for the detection and visualization of H 2 O 2 in lysosomes, which consists of a H 2 O 2 -responsive boronate unit, a lysosome-locating morpholine group, and a pH-activatable benzorhodol fluorophore. The response of the fluorescent probe to H 2 O 2 is significantly more pronounced under acidic pH conditions than that under neutral pH conditions. Notably, the present probe enables the fluorescence sensing of endogenous lysosomal H 2 O 2 in living cells without external stimulations, with signal interference from the cytoplasm and other intracellular organelles being negligible.

  4. Quantitative modeling of the dynamics and intracellular trafficking of far-red light-activatable prodrugs: implications in stimuli-responsive drug delivery system.

    Science.gov (United States)

    Li, Mengjie; Thapa, Pritam; Rajaputra, Pallavi; Bio, Moses; Peer, Cody J; Figg, William D; You, Youngjae; Woo, Sukyung

    2017-12-01

    The combination of photodynamic therapy (PDT) with anti-tumor agents is a complimentary strategy to treat local cancers. We developed a unique photosensitizer (PS)-conjugated paclitaxel (PTX) prodrug in which a PS is excited by near-infrared wavelength light to site-specifically release PTX while generating singlet oxygen (SO) to effectively kill cancer cells with both PTX and SO. The aim of the present study was to identify the determinants influencing the combined efficacy of this light-activatable prodrug, especially the bystander killing effects from released PTX. Using PS-conjugated PTX as a model system, we developed a quantitative mathematical model describing the intracellular trafficking. Dynamics of the prodrug and the model predictions were verified with experimental data using human cancer cells in vitro. The sensitivity analysis suggested that parameters related to extracellular concentration of released PTX, prodrug uptake, target engagement, and target abundance are critical in determining the combined killing efficacy of the prodrug. We found that released PTX cytotoxicity was most sensitive to the retention time of the drug in extracellular space. Modulating drug internalization and conjugating the agents targeted to abundant receptors may provide a new strategy for maximizing the killing capacity of the far-red light-activatable prodrug system. These results provide guidance for the design of the PDT combination study in vivo and have implications for other stimuli-responsive drug delivery systems.

  5. Inhibiting α-synuclein oligomerization by stable cell-penetrating β-synuclein fragments recovers phenotype of Parkinson's disease model flies.

    Directory of Open Access Journals (Sweden)

    Ronit Shaltiel-Karyo

    Full Text Available The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we identified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease.

  6. Comparative analysis of internalisation, haemolytic, cytotoxic and antibacterial effect of membrane-active cationic peptides: aspects of experimental setup.

    Science.gov (United States)

    Horváti, Kata; Bacsa, Bernadett; Mlinkó, Tamás; Szabó, Nóra; Hudecz, Ferenc; Zsila, Ferenc; Bősze, Szilvia

    2017-06-01

    Cationic peptides proved fundamental importance as pharmaceutical agents and/or drug carrier moieties functioning in cellular processes. The comparison of the in vitro activity of these peptides is an experimental challenge and a combination of different methods, such as cytotoxicity, internalisation rate, haemolytic and antibacterial effect, is necessary. At the same time, several issues need to be addressed as the assay conditions have a great influence on the measured biological effects and the experimental setup needs to be optimised. Therefore, critical comparison of results from different assays using representative examples of cell penetrating and antimicrobial peptides was performed and optimal test conditions were suggested. Our main goal was to identify carrier peptides for drug delivery systems of antimicrobial drug candidates. Based on the results of internalisation, haemolytic, cytotoxic and antibacterial activity assays, a classification of cationic peptides is advocated. We found eight promising carrier peptides with good penetration ability of which Penetratin, Tat, Buforin and Dhvar4 peptides showed low adverse haemolytic effect. Penetratin, Transportan, Dhvar4 and the hybrid CM15 peptide had the most potent antibacterial activity on Streptococcus pneumoniae (MIC lower than 1.2 μM) and Transportan was effective against Mycobacterium tuberculosis as well. The most selective peptide was the Penetratin, where the effective antimicrobial concentration on pneumococcus was more than 250 times lower than the HC 50 value. Therefore, these peptides and their analogues will be further investigated as drug delivery systems for antimicrobial agents.

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

    Science.gov (United States)

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

    2017-08-15

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

  8. Chimeric peptides as modulators of CK2-dependent signaling: Mechanism of action and off-target effects.

    Science.gov (United States)

    Zanin, Sofia; Sandre, Michele; Cozza, Giorgio; Ottaviani, Daniele; Marin, Oriano; Pinna, Lorenzo A; Ruzzene, Maria

    2015-10-01

    Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (α/α') and two regulatory (β) subunits. It has a global prosurvival function, especially in cancer, and represents an attractive therapeutic target. Most CK2 inhibitors available so far are ATP-competitive compounds; however, the possibility to block only the phosphorylation of few substrates has been recently explored, and a compound composed of a Tat cell-penetrating peptide and an active cyclic peptide, selected for its ability to bind to the CK2 substrate E7 protein of human papilloma virus, has been developed [Perea et al., Cancer Res. 2004; 64:7127-7129]. By using a similar chimeric peptide (CK2 modulatory chimeric peptide, CK2-MCP), we performed a study to dissect its molecular mechanism of action and the signaling pathways that it affects in cells. We found that it directly interacts with CK2 itself, counteracting the regulatory and stabilizing functions of the β subunit. Cell treatment with CK2-MCP induces a rapid decrease of the amount of CK2 subunits, as well as of other signaling proteins. Concomitant cell death is observed, more pronounced in tumor cells and not accompanied by apoptotic events. CK2 relocalizes to lysosomes, whose proteases are activated, while the proteasome machinery is inhibited. Several sequence variants of the chimeric peptide have been also synthesized, and their effects compared to those of the parental peptide. Intriguingly, the Tat moiety is essential not only for cell penetration but also for the in vitro efficacy of the peptide. We conclude that this class of chimeric peptides, in addition to altering some properties of CK2 holoenzyme, affects several other cellular targets, causing profound perturbations of cell biology. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Cell penetration to nanofibrous scaffolds

    Czech Academy of Sciences Publication Activity Database

    Rampichová, Michala; Buzgo, Matej; Chvojka, J.; Prosecká, Eva; Kofroňová, Olga; Amler, Evžen

    2014-01-01

    Roč. 8, č. 1 (2014), s. 36-41 ISSN 1933-6918 Grant - others:GA UK(CZ) 384311; GA UK(CZ) 626012; GA UK(CZ) 270513; GA UK(CZ) 330611; GA UK(CZ) 648112; GA MZd(CZ) NT12156; GA MŠk(CZ) project IPv6 Institutional support: RVO:68378041 ; RVO:61388971 Keywords : fibrous scaffold * mesenchymal stem cells * Forcespinning (R) Subject RIV: FP - Other Medical Disciplines Impact factor: 4.505, year: 2014

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

    Science.gov (United States)

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

    2018-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Mohamed F Mohamed

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

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

    Science.gov (United States)

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

    2018-02-01

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

  13. Action of the multifunctional peptide BP100 on native biomembranes examined by solid-state NMR

    Energy Technology Data Exchange (ETDEWEB)

    Misiewicz, Julia [Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry (Germany); Afonin, Sergii; Grage, Stephan L.; Berg, Jonas van den; Strandberg, Erik; Wadhwani, Parvesh [Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2) (Germany); Ulrich, Anne S., E-mail: anne.ulrich@kit.edu [Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry (Germany)

    2015-04-15

    Membrane composition is a key factor that regulates the destructive activity of antimicrobial peptides and the non-leaky permeation of cell penetrating peptides in vivo. Hence, the choice of model membrane is a crucial aspect in NMR studies and should reflect the biological situation as closely as possible. Here, we explore the structure and dynamics of the short multifunctional peptide BP100 using a multinuclear solid-state NMR approach. The membrane alignment and mobility of this 11 amino acid peptide was studied in various synthetic lipid bilayers with different net charge, fluidity, and thickness, as well as in native biomembranes harvested from prokaryotic and eukaryotic cells. {sup 19}F-NMR provided the high sensitivity and lack of natural abundance background that are necessary to observe a labelled peptide even in protoplast membranes from Micrococcus luteus and in erythrocyte ghosts. Six selectively {sup 19}F-labeled BP100 analogues gave remarkably similar spectra in all of the macroscopically oriented membrane systems, which were studied under quasi-native conditions of ambient temperature and full hydration. This similarity suggests that BP100 has the same surface-bound helical structure and high mobility in the different biomembranes and model membranes alike, independent of charge, thickness or cholesterol content of the system. {sup 31}P-NMR spectra of the phospholipid components did not indicate any bilayer perturbation, so the formation of toroidal wormholes or micellarization can be excluded as a mechanism of its antimicrobial or cell penetrating action. However, {sup 2}H-NMR analysis of the acyl chain order parameter profiles showed that BP100 leads to considerable membrane thinning and thereby local destabilization.

  14. The homeodomain derived peptide Penetratin induces curvature of fluid membrane domains.

    Directory of Open Access Journals (Sweden)

    Antonin Lamazière

    Full Text Available BACKGROUND: Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, "physical endocytosis". METHODOLOGY/PRINCIPAL FINDINGS: Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in "raft" microdomains versus disordered fluid "non-raft" domains in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like membrane domains. CONCLUSIONS/SIGNIFICANCE: The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we

  15. Cell Penetrating Polymers Containing Guanidinium Trigger Apoptosis in Human Hepatocellular Carcinoma Cells unless Conjugated to a Targeting N-Acetyl-Galactosamine Block.

    Science.gov (United States)

    Tan, Zhe; Dhande, Yogesh K; Reineke, Theresa M

    2017-12-20

    delivery vehicles may be limited by their high cytotoxicity to certain cell types. Thus, the use of cell penetrating structures in polyplex formulations should be used with caution and carefully tailored toward individual cell/tissue types.

  16. Recent Developments in Peptide-Based Nucleic Acid Delivery

    Directory of Open Access Journals (Sweden)

    Tobias Restle

    2008-07-01

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

  17. Peptide aptamers: The versatile role of specific protein function inhibitors in plant biotechnology.

    Science.gov (United States)

    Colombo, Monica; Mizzotti, Chiara; Masiero, Simona; Kater, Martin M; Pesaresi, Paolo

    2015-11-01

    In recent years, peptide aptamers have emerged as novel molecular tools that have attracted the attention of researchers in various fields of basic and applied science, ranging from medicine to analytical chemistry. These artificial short peptides are able to specifically bind, track, and inhibit a given target molecule with high affinity, even molecules with poor immunogenicity or high toxicity, and represent a remarkable alternative to antibodies in many different applications. Their use is on the rise, driven mainly by the medical and pharmaceutical sector. Here we discuss the enormous potential of peptide aptamers in both basic and applied aspects of plant biotechnology and food safety. The different peptide aptamer selection methods available both in vivo and in vitro are introduced, and the most important possible applications in plant biotechnology are illustrated. In particular, we discuss the generation of broad-based virus resistance in crops, "reverse genetics" and aptasensors in bioassays for detecting contaminations in food and feed. Furthermore, we suggest an alternative to the transfer of peptide aptamers into plant cells via genetic transformation, based on the use of cell-penetrating peptides that overcome the limits imposed by both crop transformation and Genetically Modified Organism commercialization. © 2015 Institute of Botany, Chinese Academy of Sciences.

  18. Structure and dynamics of cationic membrane peptides and proteins: Insights from solid-state NMR

    Science.gov (United States)

    Hong, Mei; Su, Yongchao

    2011-01-01

    Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein–lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides. PMID:21344534

  19. Human peptide transporters

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Brodin, Birger; Jørgensen, Flemming Steen

    2002-01-01

    Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2, wh...

  20. Delivery of siRNA Complexed with Palmitoylated α-Peptide/β-Peptoid Cell-Penetrating Peptidomimetics: Membrane Interaction and Structural Characterization of a Lipid-Based Nanocarrier System

    DEFF Research Database (Denmark)

    Jing, Xiaona; Foged, Camilla; Martin-Bertelsen, Birte

    2016-01-01

    . Cryo-transmission electron microscopy (cryo-TEM) revealed multilamellar, onion-like spherical vesicles, and small-angle X-ray scattering (SAXS) analysis confirmed that the majority of the lipids in the nanocarriers were organized in lamellar structures, yet coexisted with a hexagonal phase, which...

  1. Designing of peptides with desired half-life in intestine-like environment

    KAUST Repository

    Sharma, Arun

    2014-08-20

    Background: In past, a number of peptides have been reported to possess highly diverse properties ranging from cell penetrating, tumor homing, anticancer, anti-hypertensive, antiviral to antimicrobials. Owing to their excellent specificity, low-toxicity, rich chemical diversity and availability from natural sources, FDA has successfully approved a number of peptide-based drugs and several are in various stages of drug development. Though peptides are proven good drug candidates, their usage is still hindered mainly because of their high susceptibility towards proteases degradation. We have developed an in silico method to predict the half-life of peptides in intestine-like environment and to design better peptides having optimized physicochemical properties and half-life.Results: In this study, we have used 10mer (HL10) and 16mer (HL16) peptides dataset to develop prediction models for peptide half-life in intestine-like environment. First, SVM based models were developed on HL10 dataset which achieved maximum correlation R/R2 of 0.57/0.32, 0.68/0.46, and 0.69/0.47 using amino acid, dipeptide and tripeptide composition, respectively. Secondly, models developed on HL16 dataset showed maximum R/R2 of 0.91/0.82, 0.90/0.39, and 0.90/0.31 using amino acid, dipeptide and tripeptide composition, respectively. Furthermore, models that were developed on selected features, achieved a correlation (R) of 0.70 and 0.98 on HL10 and HL16 dataset, respectively. Preliminary analysis suggests the role of charged residue and amino acid size in peptide half-life/stability. Based on above models, we have developed a web server named HLP (Half Life Prediction), for predicting and designing peptides with desired half-life. The web server provides three facilities; i) half-life prediction, ii) physicochemical properties calculation and iii) designing mutant peptides.Conclusion: In summary, this study describes a web server \\'HLP\\' that has been developed for assisting scientific

  2. Antiviral activity of α-helical stapled peptides designed from the HIV-1 capsid dimerization domain

    Directory of Open Access Journals (Sweden)

    Cowburn David

    2011-05-01

    Full Text Available Abstract Background The C-terminal domain (CTD of HIV-1 capsid (CA, like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. Mutations of the residues at the CTD dimer interface impair virus assembly and render the virus non-infectious. Therefore, the CTD represents a potential target for designing anti-HIV-1 drugs. Results Due to the pivotal role of the dimer interface, we reasoned that peptides from the α-helical region of the dimer interface might be effective as decoys to prevent CTD dimer formation. However, these small peptides do not have any structure in solution and they do not penetrate cells. Therefore, we used the hydrocarbon stapling technique to stabilize the α-helical structure and confirmed by confocal microscopy that this modification also made these peptides cell-penetrating. We also confirmed by using isothermal titration calorimetry (ITC, sedimentation equilibrium and NMR that these peptides indeed disrupt dimer formation. In in vitro assembly assays, the peptides inhibited mature-like virus particle formation and specifically inhibited HIV-1 production in cell-based assays. These peptides also showed potent antiviral activity against a large panel of laboratory-adapted and primary isolates, including viral strains resistant to inhibitors of reverse transcriptase and protease. Conclusions These preliminary data serve as the foundation for designing small, stable, α-helical peptides and small-molecule inhibitors targeted against the CTD dimer interface. The observation that relatively weak CA binders, such as NYAD-201 and NYAD-202, showed specificity and are able to disrupt the CTD dimer is encouraging for further exploration of a much broader class of antiviral compounds targeting CA. We cannot exclude the possibility that the CA-based peptides described here could elicit additional effects on virus replication not directly linked to their ability to bind

  3. PeptideAtlas

    Data.gov (United States)

    U.S. Department of Health & Human Services — PeptideAtlas is a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments. Mass...

  4. Sensing pH via p-cyanophenylalanine fluorescence: Application to determine peptide pKa and membrane penetration kinetics.

    Science.gov (United States)

    Pazos, Ileana M; Ahmed, Ismail A; Berríos, Mariana I León; Gai, Feng

    2015-08-15

    We expand the spectroscopic utility of a well-known infrared and fluorescence probe, p-cyanophenylalanine, by showing that it can also serve as a pH sensor. This new application is based on the notion that the fluorescence quantum yield of this unnatural amino acid, when placed at or near the N-terminal end of a polypeptide, depends on the protonation status of the N-terminal amino group of the peptide. Using this pH sensor, we are able to determine the N-terminal pKa values of nine tripeptides and also the membrane penetration kinetics of a cell-penetrating peptide. Taken together, these examples demonstrate the applicability of using this unnatural amino acid fluorophore to study pH-dependent biological processes or events that accompany a pH change. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  6. 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.

  7. 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....

  8. 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....

  9. 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....

  10. 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....

  11. 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.

  12. A New Noncanonical Anionic Peptide That Translocates a Cellular Blood–Brain Barrier Model

    Directory of Open Access Journals (Sweden)

    Sara Neves-Coelho

    2017-10-01

    Full Text Available The capacity to transport therapeutic molecules across the blood–brain barrier (BBB represents a breakthrough in the development of tools for the treatment of many central nervous system (CNS-associated diseases. The BBB, while being protective against infectious agents, hinders the brain uptake of many drugs. Hence, finding safe shuttles able to overcome the BBB is of utmost importance. Herein, we identify a new BBB-translocating peptide with unique properties. For years it was thought that cationic sequences were mandatory for a cell-penetrating peptide (CPP to achieve cellular internalization. Despite being anionic at physiological pH, PepNeg (sequence (SGTQEEY is an efficient BBB translocator that is able to carry a large cargo (27 kDa, while maintaining BBB integrity. In addition, PepNeg is able to use two distinct methods of translocation, energy-dependent and -independent, suggesting that direct penetration might occur when low concentrations of peptide are presented to cells. The discovery of this new anionic trans-BBB peptide allows the development of new delivery systems to the CNS and contributes to the need to rethink the role of electrostatic attraction in BBB-translocation.

  13. Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting.

    Science.gov (United States)

    Lin, Ran; Zhang, Pengcheng; Cheetham, Andrew G; Walston, Jeremy; Abadir, Peter; Cui, Honggang

    2015-01-21

    Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.

  14. Probing intra-cellular drug release event using activatable (OFF/ON) CdS:Mn/ZnS quantum dots (Qdots): spectroscopic studies to investigate interaction of Qdots with quencher

    Science.gov (United States)

    Tharkur, Jeremy; Teblum, Andrew; Basumallick, Srijita; Shah, Rikhav; Cantarero, Karishma; Maity, Niharika; Rifai, Sara; Doshi, Mona; Gesquiere, Andre J.; Santra, Swadeshmukul

    2013-02-01

    In recent years, activatable Quantum Dots (AQdots) are gaining popularity in a number of chemical and biological sensing applications. A basic design of AQdot probes involves a suitable quencher which is capable of altering optical properties of the Qdots. In our previous studies we have shown that CdS:Mn/ZnS fluorescence can be effectively quenched using small molecule quenchers (such as dopamine, chemotherapeutic drug) as well as iron oxide nanoparticle via electron/energy transfer process. We have also shown that the quenched Qdot fluorescence can be restored when the Qdots are separated from the quencher. Using Qdot based activatable probes, we detected intracellular drug release event. Qdot fluorescence was restored upon interaction with the intracellular glutathione (GSH). In this paper, we report a GSH induced quenching of water-soluble N-Acetyl Cysteine (NAC) surface-conjugated Cds:Mn/ZnS Qdots. Quenching of NAC-Qdots was due to aggregation of Qdots in solution. This aggregation induced fluorescence quenching phenomenon resembles with the self-quenching phenomenon of traditional organic fluorescence dyes at high concentrations. UV-VIS and fluorescence emission spectroscopy data support the interaction and binding of GSH with the NAC-Qdots. Increase in particle size due to GSH induced aggregation of NAC-Qdots was confirmed by the Dynamic Light Scattering (DLS) data.

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

    Science.gov (United States)

    Ostrowski, Maciej; Kowalczyk, Stanisław

    2015-01-01

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

  16. Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

    DEFF Research Database (Denmark)

    Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E

    2012-01-01

    have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA......Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...

  17. Peptide-micelle hybrids containing fasudil for targeted delivery to the pulmonary arteries and arterioles to treat pulmonary arterial hypertension.

    Science.gov (United States)

    Gupta, Nilesh; Ibrahim, Hany M; Ahsan, Fakhrul

    2014-11-01

    This study investigates the respirability and efficacy of peptide-micelle hybrid nanoparticles as carriers for inhalational therapy of pulmonary arterial hypertension (PAH). CARSKNKDC (CAR), a cell-penetrating and lung-homing peptide, conjugated polyethylene glycol-distearoyl-phosphoethanolamine micelles containing fasudil, an investigational anti-PAH drug, were prepared by solvent evaporation method and characterized for various physicochemical properties. The pharmacokinetics and pharmacological efficacy of hybrid particles containing fasudil were evaluated in healthy rats and monocrotaline-induced PAH rats. CAR micelles containing fasudil had an entrapment efficiency of approximately 58%, showed controlled release of the drug, and were monodispersed with an average size of approximately 14 nm. Nuclear magnetic resonance scan confirmed the drug's presence in the core of peptide-micelle hybrid particles. Compared with plain micelles, CAR peptide increased the cellular uptake by approximately 1.7-fold and extended the drug half-life by approximately fivefold. The formulations were more prone to accumulate in the pulmonary vasculature than in the peripheral blood, which is evident from the ratio of the extent of reduction of pulmonary and systemic arterial pressures. On the whole, this study demonstrates that peptide-polymer hybrid micelles can serve as inhalational carriers for PAH therapy. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  18. The targeted transduction of MMP-overexpressing tumor cells by ACPP-HPMA copolymer-coated adenovirus conjugates.

    Directory of Open Access Journals (Sweden)

    Shuhua Li

    Full Text Available We have designed and tested a new way to selectively deliver HPMA polymer-coated adenovirus type 5 (Ad5 particles into matrix metalloproteinase (MMP-overexpressing tumor cells. An activatable cell penetrating peptide (ACPP was designed and attached to the reactive 4-nitrophenoxy groups of HPMA polymers by the C-terminal amino acid (asparagine, N. ACPPs are activatable cell penetrating peptides (CPPs with a linker between polycationic and polyanionic domains, and MMP-mediated cleavage releases the CPP portion and its attached cargo to enable cell entry. Our data indicate that the transport of these HPMA polymer conjugates by a single ACPP molecule to the cytoplasm occurs via a nonendocytotic and concentration-independent process. The uptake was observed to finish within 20 minutes by inverted fluorescence microscopy. In contrast, HPMA polymer-coated Ad5 without ACPPs was internalized solely by endocytosis. The optimal formulation was not affected by the presence of Ad5 neutralizing antibodies during transduction, and ACPP/polymer-coated Ad5 also retained high targeting capability to several MMP-overexpressing tumor cell types. For the first time, ACPP-mediated cytoplasmic delivery of polymer-bound Ad5 to MMP-overexpressing tumor cells was demonstrated. These findings are significant, as they demonstrate the use of a polymer-based system for the targeted delivery into MMP-overexpressing solid tumors and highlight how to overcome major cellular obstacles to achieve intracellular macromolecular delivery.

  19. The interplay of T1- and T2-relaxation on T1-weighted MRI of hMSCs induced by Gd-DOTA-peptides.

    Science.gov (United States)

    Cao, Limin; Li, Binbin; Yi, Peiwei; Zhang, Hailu; Dai, Jianwu; Tan, Bo; Deng, Zongwu

    2014-04-01

    Three Gd-DOTA-peptide complexes with different peptide sequence are synthesized and used as T1 contrast agent to label human mesenchymal stem cells (hMSCs) for magnetic resonance imaging study. The peptides include a universal cell penetrating peptide TAT, a linear MSC-specific peptide EM7, and a cyclic MSC-specific peptide CC9. A significant difference in labeling efficacy is observed between the Gd-DOTA-peptides as well as a control Dotarem. All Gd-DOTA-peptides as well as Dotarem induce significant increase in T1 relaxation rate which is in favor of T1-weighted MR imaging. Gd-DOTA-CC9 yields the maximum labeling efficacy but poor T1 contrast enhancement. Gd-DOTA-EM7 yields the minimum labeling efficacy but better T1 contrast enhancement. Gd-DOTA-TAT yields a similar labeling efficacy as Gd-DOTA-CC9 and similar T1 contrast enhancement as Gd-DOTA-EM7. The underlying mechanism that governs T1 contrast enhancement effect is discussed. Our results suggest that T1 contrast enhancement induced by Gd-DOTA-peptides depends not only on the introduced cellular Gd content, but more importantly on the effect that Gd-DOTA-peptides exert on the T1-relaxation and T2-relaxation processes/rates. Both T1 and particularly T2 relaxation rate have to be taken into account to interpret T1 contrast enhancement. In addition, the interpretation has to be based on cellular instead of aqueous longitudinal and transverse relaxivities of Gd-DOTA-peptides. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Potent and Selective Peptide-based Inhibition of the G Protein Gαq*

    Science.gov (United States)

    Charpentier, Thomas H.; Waldo, Gary L.; Lowery-Gionta, Emily G.; Krajewski, Krzysztof; Strahl, Brian D.; Kash, Thomas L.; Harden, T. Kendall; Sondek, John

    2016-01-01

    In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq. A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2. In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells. PMID:27742837

  1. Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

    Science.gov (United States)

    Charpentier, Thomas H; Waldo, Gary L; Lowery-Gionta, Emily G; Krajewski, Krzysztof; Strahl, Brian D; Kash, Thomas L; Harden, T Kendall; Sondek, John

    2016-12-02

    In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gα q binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gα q within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gα q in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gα q A representative peptide was specific for active Gα q because it did not bind inactive Gα q or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ 1 γ 2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gα q ; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gα q in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gα q -dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gα q in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gα q in cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Peptides in melanoma therapy.

    Science.gov (United States)

    Mocellin, Simone

    2012-01-01

    Peptides derived from tumor associated antigens can be utilized to elicit a therapeutically effective immune response against melanoma in experimental models. However, patient vaccination with peptides - although it is often followed by the induction of melanoma- specific T lymphocytes - is rarely associated with tumor response of clinical relevance. In this review I summarize the principles of peptide design as well as the results so far obtained in the clinical setting while treating cutaneous melanoma by means of this active immunotherapy strategy. I also discuss some immunological and methodological issues that might be helpful for the successful development of peptide-based vaccines.

  3. Antimicrobial Peptides in Reptiles

    Science.gov (United States)

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  4. Insulin C-peptide test

    Science.gov (United States)

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin the body produces and insulin someone injects ...

  5. Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO.

    Science.gov (United States)

    Yin, Haifang; Moulton, Hong M; Betts, Corinne; Merritt, Thomas; Seow, Yiqi; Ashraf, Shirin; Wang, Qingsong; Boutilier, Jordan; Wood, Matthew Ja

    2010-10-01

    Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

  6. Cre Fused with RVG Peptide Mediates Targeted Genome Editing in Mouse Brain Cells In Vivo.

    Science.gov (United States)

    Zou, Zhiyuan; Sun, Zhaolin; Li, Pan; Feng, Tao; Wu, Sen

    2016-12-14

    Cell penetrating peptides (CPPs) are short peptides that can pass through cell membranes. CPPs can facilitate the cellular entry of proteins, macromolecules, nanoparticles and drugs. RVG peptide (RVG hereinafter) is a 29-amino-acid CPP derived from a rabies virus glycoprotein that can cross the blood-brain barrier (BBB) and enter brain cells. However, whether RVG can be used for genome editing in the brain has not been reported. In this work, we combined RVG with Cre recombinase for bacterial expression. The purified RVG-Cre protein cut plasmids in vitro and traversed cell membranes in cultured Neuro2a cells. By tail vein-injecting RVG-Cre into Cre reporter mouse lines mTmG and Rosa26 lacZ , we demonstrated that RVG-Cre could target brain cells and achieve targeted somatic genome editing in adult mice. This direct delivery of the gene-editing enzyme protein into mouse brains with RVG is much safer than plasmid- or viral-based methods, holding promise for further applications in the treatment of various brain diseases.

  7. N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis

    International Nuclear Information System (INIS)

    Magzoub, Mazin; Sandgren, Staffan; Lundberg, Pontus; Oglecka, Kamila; Lilja, Johanna; Wittrup, Anders; Goeran Eriksson, L.E.; Langel, Ulo; Belting, Mattias; Graeslund, Astrid

    2006-01-01

    A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases

  8. Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity.

    Science.gov (United States)

    Valente, Pierluigi; Fernández-Carvajal, Asia; Camprubí-Robles, María; Gomis, Ana; Quirce, Susana; Viana, Félix; Fernández-Ballester, Gregorio; González-Ros, José M; Belmonte, Carlos; Planells-Cases, Rosa; Ferrer-Montiel, Antonio

    2011-05-01

    The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC(50)100 μM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.

  9. A non-covalent peptide-based strategy for ex vivo and in vivo oligonucleotide delivery.

    Science.gov (United States)

    Crombez, Laurence; Morris, May C; Heitz, Frederic; Divita, Gilles

    2011-01-01

    The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models.

  10. Peptide Vaccines for Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Rory C. F. De Brito

    2018-05-01

    Full Text Available Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.

  11. Peptide Vaccines for Leishmaniasis.

    Science.gov (United States)

    De Brito, Rory C F; Cardoso, Jamille M De O; Reis, Levi E S; Vieira, Joao F; Mathias, Fernando A S; Roatt, Bruno M; Aguiar-Soares, Rodrigo Dian D O; Ruiz, Jeronimo C; Resende, Daniela de M; Reis, Alexandre B

    2018-01-01

    Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.

  12. 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...

  13. Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide.

    Science.gov (United States)

    Dhar, Amlanjyoti; Mallick, Shampa; Ghosh, Piya; Maiti, Atanu; Ahmed, Israr; Bhattacharya, Seemana; Mandal, Tapashi; Manna, Asit; Roy, Koushik; Singh, Sandeep; Nayak, Dipak Kumar; Wilder, Paul T; Markowitz, Joseph; Weber, David; Ghosh, Mrinal K; Chattopadhyay, Samit; Guha, Rajdeep; Konar, Aditya; Bandyopadhyay, Santu; Roy, Siddhartha

    2014-07-01

    Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of α-amino isobutyric acid--an amino acid having high helical propensity--in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development. © 2014 Wiley Periodicals, Inc.

  14. Intelligent "Peptide-Gathering Mechanical Arm" Tames Wild "Trojan-Horse" Peptides for the Controlled Delivery of Cancer Nanotherapeutics.

    Science.gov (United States)

    Shi, Nian-Qiu; Li, Yan; Zhang, Yong; Shen, Nan; Qi, Ling; Wang, Shu-Ran; Qi, Xian-Rong

    2017-12-06

    Cell-penetrating peptides (CPPs), also called "Trojan-Horse" peptides, have been used for facilitating intracellular delivery of numerous diverse cargoes and even nanocarriers. However, the lack of targeting specificity ("wildness" or nonselectivity) of CPP-nanocarriers remains an intractable challenge for many in vivo applications. In this work, we used an intelligent "peptide-gathering mechanical arm" (Int PMA) to curb CPPs' wildness and enhance the selectivity of R 9 -liposome-based cargo delivery for tumor targeting. The peptide NGR, serving as a cell-targeting peptide for anchoring, and peptide PLGLAG, serving as a substrate peptide for deanchoring, were embedded in the Int PMA motif. The Int PMA construct was designed to be sensitive to tumor microenvironmental stimuli, including aminopeptidase N (CD13) and matrix metalloproteinases (MMP-2/9). Moreover, Int PMA could be specifically recognized by tumor tissues via CD13-mediated anchoring and released for cell entry by MMP-2/9-mediated deanchoring. To test the Int PMA design, a series of experiments were conducted in vitro and in vivo. Functional conjugates Int PMA-R 9 -poly(ethylene glycol) (PEG) 2000 -distearoylphosphatidyl-ethanolamine (DSPE) and R 9 -PEG 2000 -DSPE were synthesized by Michael addition reaction and were characterized by thin-layer chromatography and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The Int PMA-R 9 -modified doxorubicin-loaded liposomes (Int PMA-R 9 -Lip-DOX) exhibited a proper particle diameter (approximately 155 nm) with in vitro sustained release characteristics. Cleavage assay showed that Int PMA-R 9 peptide molecules could be cleaved by MMP-2/9 for completion of deanchoring. Flow cytometry and confocal microscopy studies indicated that Int PMA-R 9 -Lip-DOX can respond to both endogenous and exogenous stimuli in the presence/absence of excess MMP-2/9 and MMP-2/9 inhibitor (GM6001) and effectively function under competitive receptor

  15. Diversity-oriented peptide stapling

    DEFF Research Database (Denmark)

    Tran, Thu Phuong; Larsen, Christian Ørnbøl; Røndbjerg, Tobias

    2017-01-01

    as a powerful method for peptide stapling. However, to date CuAAC stapling has not provided a simple method for obtaining peptides that are easily diversified further. In the present study, we report a new diversity-oriented peptide stapling (DOPS) methodology based on CuAAC chemistry. Stapling of peptides...

  16. PNA Peptide chimerae

    DEFF Research Database (Denmark)

    Koch, T.; Næsby, M.; Wittung, P.

    1995-01-01

    Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields....

  17. Tumor penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tambet eTeesalu

    2013-08-01

    Full Text Available Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC, contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is

  18. Peptide aldehyde inhibitors of bacterial peptide deformylases.

    Science.gov (United States)

    Durand, D J; Gordon Green, B; O'Connell, J F; Grant, S K

    1999-07-15

    Bacterial peptide deformylases (PDF, EC 3.5.1.27) are metalloenzymes that cleave the N-formyl groups from N-blocked methionine polypeptides. Peptide aldehydes containing a methional or norleucinal inhibited recombinant peptide deformylase from gram-negative Escherichia coli and gram-positive Bacillus subtilis. The most potent inhibitor was calpeptin, N-CBZ-Leu-norleucinal, which was a competitive inhibitor of the zinc-containing metalloenzymes, E. coli and B. subtilis PDF with Ki values of 26.0 and 55.6 microM, respectively. Cobalt-substituted E. coli and B. subtilis deformylases were also inhibited by these aldehydes with Ki values for calpeptin of 9.5 and 12.4 microM, respectively. Distinct spectral changes were observed upon binding of calpeptin to the Co(II)-deformylases, consistent with the noncovalent binding of the inhibitor rather than the formation of a covalent complex. In contrast, the chelator 1,10-phenanthroline caused the time-dependent inhibition of B. subtilis Co(II)-PDF activity with the loss of the active site metal. The fact that calpeptin was nearly equipotent against deformylases from both gram-negative and gram-positive bacterial sources lends further support to the idea that a single deformylase inhibitor might have broad-spectrum antibacterial activity. Copyright 1999 Academic Press.

  19. Mechanistic studies of a cell-permeant peptide designed to enhance myosin light chain phosphorylation in polarized intestinal epithelia.

    Science.gov (United States)

    Almansour, Khaled; Taverner, Alistair; Eggleston, Ian M; Mrsny, Randall J

    2018-06-10

    Tight junction (TJ) structures restrict the movement of solutes between adjacent epithelial cells to maintain homeostatic conditions. A peptide, termed PIP 640, with the capacity to regulate the transient opening of intestinal TJ structures through an endogenous mechanism involving the induction of myosin light chain (MLC) phosphorylation at serine 19 (MLC-pS 19 ) has provided a promising new method to enhance the in vivo oral bioavailability of peptide therapeutics. PIP 640 is a decapeptide composed of all D-amino acids (rrdykvevrr-NH 2 ) that contains a central sequence designed to emulates a specific domain of C-kinase potentiated protein phosphatase-1 inhibitor-17 kDa (CPI-17) surrounded by positively-charged amino acids that provide a cell penetrating peptide (CPP)-like character. Here, we examine compositional requirements of PIP 640 with regard to its actions on MLC phosphorylation, its intracellular localization to TJ structures, and its interactions with MLC phosphatase (MLCP) elements that correlate with enhanced solute uptake. These studies showed that a glutamic acid and tyrosine within this peptide are critical for PIP 640 to retain its ability to increase MLC-pS 19 levels and enhance the permeability of macromolecular solutes of the size range of therapeutic peptides without detectable cytotoxicity. On the other hand, exchange of the aspartic acid for alanine and then arginine resulted in an increasingly greater bias toward protein phosphatase-1 (PP1) relative to MLCP inhibition, an outcome that resulted in increased paracellular permeability for solutes in the size range of therapeutic peptides, but with a significant increase in cytotoxicity. Together, these data further our understanding of the composition requirements of PIP 640 with respect to the desired goal of transiently altering the intestinal epithelial cell paracellular barrier properties through an endogenous mechanism, providing a novel approach to enhance the oral bioavailability of

  20. Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans

    KAUST Repository

    Åmand, Helene L.

    2012-11-01

    Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide\\'s ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell. © 2012 Elsevier B.V.

  1. Peptide Integrated Optics.

    Science.gov (United States)

    Handelman, Amir; Lapshina, Nadezda; Apter, Boris; Rosenman, Gil

    2018-02-01

    Bio-nanophotonics is a wide field in which advanced optical materials, biomedicine, fundamental optics, and nanotechnology are combined and result in the development of biomedical optical chips. Silk fibers or synthetic bioabsorbable polymers are the main light-guiding components. In this work, an advanced concept of integrated bio-optics is proposed, which is based on bioinspired peptide optical materials exhibiting wide optical transparency, nonlinear and electrooptical properties, and effective passive and active waveguiding. Developed new technology combining bottom-up controlled deposition of peptide planar wafers of a large area and top-down focus ion beam lithography provides direct fabrication of peptide optical integrated circuits. Finding a deep modification of peptide optical properties by reconformation of biological secondary structure from native phase to β-sheet architecture is followed by the appearance of visible fluorescence and unexpected transition from a native passive optical waveguiding to an active one. Original biocompatibility, switchable regimes of waveguiding, and multifunctional nonlinear optical properties make these new peptide planar optical materials attractive for application in emerging technology of lab-on-biochips, combining biomedical photonic and electronic circuits toward medical diagnosis, light-activated therapy, and health monitoring. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. 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.

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

    Science.gov (United States)

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

    2011-01-01

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

  4. Acylation of Therapeutic Peptides

    DEFF Research Database (Denmark)

    Trier, Sofie; Henriksen, Jonas Rosager; Jensen, Simon Bjerregaard

    ) , which promotes intestinal growth and is used to treat bowel disorders such as inflammatory bowel diseases and short bowel syndrome, and the 32 amino acid salmon calcitonin (sCT), which lowers blood calcium and is employed in the treatment of post-menopausal osteoporosis and hypercalcemia. The two...... peptides are similar in size and structure, but oppositely charged at physiological pH. Both peptides were acylated with linear acyl chains of systematically increasing length, where sCT was furthermore acylated at two different positions on the peptide backbone. For GLP-2, we found that increasing acyl...... remained optimal overall. The results indicate that rational acylation of GLP-2 can increase its in vitro intestinal absorption, alone or in combination with permeation enhancers, and are consistent with the initial project hypothesis. For sCT, an unpredicted effect of acylation largely superseded...

  5. Therapeutic HIV Peptide Vaccine

    DEFF Research Database (Denmark)

    Fomsgaard, Anders

    2015-01-01

    Therapeutic vaccines aim to control chronic HIV infection and eliminate the need for lifelong antiretroviral therapy (ART). Therapeutic HIV vaccine is being pursued as part of a functional cure for HIV/AIDS. We have outlined a basic protocol for inducing new T cell immunity during chronic HIV-1...... infection directed to subdominant conserved HIV-1 epitopes restricted to frequent HLA supertypes. The rationale for selecting HIV peptides and adjuvants are provided. Peptide subunit vaccines are regarded as safe due to the simplicity, quality, purity, and low toxicity. The caveat is reduced immunogenicity...

  6. Descriptors for antimicrobial peptides

    DEFF Research Database (Denmark)

    Jenssen, Håvard

    2011-01-01

    of these are currently being used in quantitative structure--activity relationship (QSAR) studies for AMP optimization. Additionally, some key commercial computational tools are discussed, and both successful and less successful studies are referenced, illustrating some of the challenges facing AMP scientists. Through...... examples of different peptide QSAR studies, this review highlights some of the missing links and illuminates some of the questions that would be interesting to challenge in a more systematic fashion. Expert opinion: Computer-aided peptide QSAR using molecular descriptors may provide the necessary edge...

  7. Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion

    International Nuclear Information System (INIS)

    Paredes, Angel M.; Ferreira, Davis; Horton, Michelle; Saad, Ali; Tsuruta, Hiro; Johnston, Robert; Klimstra, William; Ryman, Kate; Hernandez, Raquel; Chiu Wah; Brown, Dennis T.

    2004-01-01

    Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a 'pore'-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell

  8. Antimicrobial Peptides: An Introduction.

    Science.gov (United States)

    Haney, Evan F; Mansour, Sarah C; Hancock, Robert E W

    2017-01-01

    The "golden era" of antibiotic discovery has long passed, but the need for new antibiotics has never been greater due to the emerging threat of antibiotic resistance. This urgency to develop new antibiotics has motivated researchers to find new methods to combat pathogenic microorganisms resulting in a surge of research focused around antimicrobial peptides (AMPs; also termed host defense peptides) and their potential as therapeutics. During the past few decades, more than 2000 AMPs have been identified from a diverse range of organisms (animals, fungi, plants, and bacteria). While these AMPs share a number of common features and a limited number of structural motifs; their sequences, activities, and targets differ considerably. In addition to their antimicrobial effects, AMPs can also exhibit immunomodulatory, anti-biofilm, and anticancer activities. These diverse functions have spurred tremendous interest in research aimed at understanding the activity of AMPs, and various protocols have been described to assess different aspects of AMP function including screening and evaluating the activities of natural and synthetic AMPs, measuring interactions with membranes, optimizing peptide function, and scaling up peptide production. Here, we provide a general overview of AMPs and introduce some of the methodologies that have been used to advance AMP research.

  9. Taming the Wildness of "Trojan-Horse" Peptides by Charge-Guided Masking and Protease-Triggered Demasking for the Controlled Delivery of Antitumor Agents.

    Science.gov (United States)

    Shi, Nian-Qiu; Qi, Xian-Rong

    2017-03-29

    Cell-penetrating peptide (CPP), also called "Trojan Horse" peptide, has become a successful approach to deliver various payloads into cells for achieving the intracellular access. However, the "Trojan Horse" peptide is too wild, not just to "Troy", but rather widely distributed in the body. Thus, there is an urgent need to tame the wildness of "Trojan Horse" peptide for targeted delivery of antineoplastic agents to the tumor site. To achieve this goal, we exploit a masked CPP-doxorubicin conjugate platform for targeted delivery of chemotherapeutic drugs using charge-guided masking and protease-triggered demasking strategies. In this platform, the cell-penetrating function of the positively CPP (d-form nonaarginine) is abrogated by a negatively shielding peptide (masked CPP), and between them is a cleavable substrate peptide by the protease (MMP-2/9). Protease-triggered demasking would occur when the masked CPP reached the MMP-2/9-riched tumor. The CPP-doxorubicin conjugate (CPP-Dox) and the masked CPP-Dox conjugate (mCPP-Dox) were used as models for the evaluation of masking and demasking processes. It was found that exogenous MMP-2/9 could effectively trigger the reversion of CPP-cargo in this conjugate, and this trigger adhered to the Michaelis-Menten kinetics profile. This conjugate was sensitive to the trigger of endogenous MMP-2/9 and could induce enhanced cytotoxicity toward MMP-2/9-rich tumor cells. In vivo antitumor efficacy revealed that this masked conjugate had considerable antitumor activity and could inhibit the tumor growth at a higher level relative to CPP-cargo. Low toxicity in vivo showed the noticeably decreased wildness of this conjugate toward normal tissues and more controllable entry of antitumor agents into "Troy". On the basis of analyses in vitro and in vivo, this mCPP-cargo conjugate delivery system held an improved selectivity toward MMP-2/9-rich tumors and would be a promising strategy for tumor-targeted treatment.

  10. [Distiller Yeasts Producing Antibacterial Peptides].

    Science.gov (United States)

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

    2015-01-01

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

  11. Ligand-regulated peptide aptamers.

    Science.gov (United States)

    Miller, Russell A

    2009-01-01

    The peptide aptamer approach employs high-throughput selection to identify members of a randomized peptide library displayed from a scaffold protein by virtue of their interaction with a target molecule. Extending this approach, we have developed a peptide aptamer scaffold protein that can impart small-molecule control over the aptamer-target interaction. This ligand-regulated peptide (LiRP) scaffold, consisting of the protein domains FKBP12, FRB, and GST, binds to the cell-permeable small-molecule rapamycin and the binding of this molecule can prevent the interaction of the randomizable linker region connecting FKBP12 with FRB. Here we present a detailed protocol for the creation of a peptide aptamer plasmid library, selection of peptide aptamers using the LiRP scaffold in a yeast two-hybrid system, and the screening of those peptide aptamers for a ligand-regulated interaction.

  12. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

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

  13. Radiolabelled peptides for oncological diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Laverman, Peter; Boerman, Otto C.; Oyen, Wim J.G. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, Nijmegen (Netherlands); Sosabowski, Jane K. [Queen Mary University of London, Centre for Molecular Oncology, Barts Cancer Institute, London (United Kingdom)

    2012-02-15

    Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. The {sup 111}In-labelled somatostatin analogue octreotide (OctreoScan trademark) is the most successful radiopeptide for tumour imaging, and was the first to be approved for diagnostic use. Based on the success of these studies, other receptor-targeting peptides such as cholecystokinin/gastrin analogues, glucagon-like peptide-1, bombesin (BN), chemokine receptor CXCR4 targeting peptides, and RGD peptides are currently under development or undergoing clinical trials. In this review, we discuss some of these peptides and their analogues, with regard to their potential for radionuclide imaging of tumours. (orig.)

  14. Antimicrobial Peptides (AMPs

    Directory of Open Access Journals (Sweden)

    Mehrzad Sadredinamin

    2016-04-01

    Full Text Available Antimicrobial peptides (AMPs are extensive group of molecules that produced by variety tissues of invertebrate, plants, and animal species which play an important role in their immunity response. AMPs have different classifications such as; biosynthetic machines, biological sources, biological functions, molecular properties, covalent bonding patterns, three dimensional structures, and molecular targets.These molecules have multidimensional properties including antimicrobial activity, antiviral activity, antifungal activity, anti-parasite activity, biofilm control, antitumor activity, mitogens activity and linking innate to adaptive immunity that making them promising agents for therapeutic drugs. In spite of this advantage of AMPs, their clinical developments have some limitation for commercial development. But some of AMPs are under clinical trials for the therapeutic purpose such as diabetic foot ulcers, different bacterial infections and tissue damage. In this review, we emphasized on the source, structure, multidimensional properties, limitation and therapeutic applications of various antimicrobial peptides.

  15. Cellular Uptake and Photo-Cytotoxicity of a Gadolinium(III-DOTA-Naphthalimide Complex “Clicked” to a Lipidated Tat Peptide

    Directory of Open Access Journals (Sweden)

    William I. O’Malley

    2016-02-01

    Full Text Available A new bifunctional macrocyclic chelator featuring a conjugatable alkynyl-naphthalimide fluorophore pendant group has been prepared and its Gd(III complex coupled to a cell-penetrating lipidated azido-Tat peptide derivative using Cu(I-catalysed “click” chemistry. The resulting fluorescent conjugate is able to enter CAL-33 tongue squamous carcinoma cells, as revealed by confocal microscopy, producing a very modest anti-proliferative effect (IC50 = 93 µM. Due to the photo-reactivity of the naphthalimide moiety, however, the conjugate’s cytotoxicity is significantly enhanced (IC50 = 16 µM upon brief low-power UV-A irradiation.

  16. Therapeutic peptides for cancer therapy. Part II - cell cycle inhibitory peptides and apoptosis-inducing peptides.

    Science.gov (United States)

    Raucher, Drazen; Moktan, Shama; Massodi, Iqbal; Bidwell, Gene L

    2009-10-01

    Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that arrest the cell cycle by mimicking CDK inhibitors or induce apoptosis directly are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Inhibition of cancer cell proliferation directly using peptides that arrest the cell cycle or induce apoptosis is a promising strategy. Peptides can be designed that interact very specifically with cyclins and/or cyclin-dependent kinases and with members of apoptotic cascades. Use of these peptides is not limited by their design, as a rational approach to peptide design is much less challenging than the design of small molecule inhibitors of specific protein-protein interactions. However, the limitations of peptide therapy lie in the poor pharmacokinetic properties of these large, often charged molecules. Therefore, overcoming the drug delivery hurdles could open the door for effective peptide therapy, thus making an entirely new class of molecules useful as anticancer drugs.

  17. Development and characterization of targeted poly(NIPAm) nanoparticles for delivery of anti-inflammatory peptides in peripheral artery disease and osteoarthritis

    Science.gov (United States)

    McMasters, James F.

    Inflammation is the underlying cause of several severe diseases including cardiovascular disease and osteoarthritis. Peripheral artery disease (PAD) is characterized by atherosclerotic occlusions within the peripheral vasculature. Current treatment for severe PAD involves mechanical widening of the artery via percutaneous transluminal angioplasty. Unfortunately, deployment of the balloon damages the endothelial layer, exposing the underlying collagenous matrix. Circulating platelets can bind to this collagen and become activated, releasing proinflammatory cytokines that promote proliferation of local smooth muscle cells. These proliferating cells eventually reocclude the vessel, resulting in restenosis and necessitating the need for a second procedure to reopen the vessel. Current treatments for moderate osteoarthritis include local injection of anti-inflammatory compounds such as glucocorticoids. Unfortunately, prolonged treatment carries with it significant side effects including osteoporosis, and cardiovascular complications. Our lab has developed an anti-inflammatory cell-penetrating peptide that inhibits mitogen-activated protein kinase activated protein kinase 2 (MK2). MK2 is implicated in the inflammatory cascade of atherosclerosis and osteoarthritis, making it a potentially effective strategy for reducing inflammation in both disease states. Unfortunately, these peptides are untargeted and quickly degraded in the presence of serum proteases, making the development of an effective delivery system of paramount importance. The overall goal of the research presented here is to detail the development of a poly(N-isopropylacrylamide) nanoparticle that is able to effectively load and release anti-inflammatory peptides for the treatment of these inflammatory diseases. In this dissertation, I will discuss the development of a collagen-binding nanoparticle that is able to inhibit platelet binding following angioplasty, thereby halting the initial inflammatory cascade

  18. Solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Jensen, Knud Jørgen

    2013-01-01

    This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective.......This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective....

  19. Improving Peptide Applications Using Nanotechnology.

    Science.gov (United States)

    Narayanaswamy, Radhika; Wang, Tao; Torchilin, Vladimir P

    2016-01-01

    Peptides are being successfully used in various fields including therapy and drug delivery. With advancement in nanotechnology and targeted delivery carrier systems, suitable modification of peptides has enabled achievement of many desirable goals over-riding some of the major disadvantages associated with the delivery of peptides in vivo. Conjugation or physical encapsulation of peptides to various nanocarriers, such as liposomes, micelles and solid-lipid nanoparticles, has improved their in vivo performance multi-fold. The amenability of peptides to modification in chemistry and functionalization with suitable nanocarriers are very relevant aspects in their use and have led to the use of 'smart' nanoparticles with suitable linker chemistries that favor peptide targeting or release at the desired sites, minimizing off-target effects. This review focuses on how nanotechnology has been used to improve the number of peptide applications. The paper also focuses on the chemistry behind peptide conjugation to nanocarriers, the commonly employed linker chemistries and the several improvements that have already been achieved in the areas of peptide use with the help of nanotechnology.

  20. Anticancer peptides from bacteria

    Directory of Open Access Journals (Sweden)

    Tomasz M. Karpiński

    2013-08-01

    Full Text Available Cancer is a leading cause of death in the world. The rapid development of medicine and pharmacology allows to create new and effective anticancer drugs. Among modern anticancer drugs are bacterial proteins. Until now has been shown anticancer activity among others azurin and exotoxin A from Pseudomonas aeruginosa, Pep27anal2 from Streptococcus pneumoniae, diphtheria toxin from Corynebacterium diphtheriae, and recently discovered Entap from Enterococcus sp. The study presents the current data regarding the properties, action and anticancer activity of listed peptides.

  1. A nanomedicine based combination therapy based on QLPVM peptide functionalized liposomal tamoxifen and doxorubicin against Luminal A breast cancer.

    Science.gov (United States)

    Wang, Xiaoyou; Chen, Xianhui; Yang, Xiucong; Gao, Wei; He, Bing; Dai, Wenbing; Zhang, Hua; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Dai, Zhifei; Zhang, Qiang

    2016-02-01

    Though combination chemotherapy or antitumor nanomedicine is extensively investigated, their combining remains in infancy. Additionally, enhanced delivery of estrogen or its analogs to tumor with highly-expressed estrogen-receptor (ER) is seldom considered, despite its necessity for ER-positive breast cancer treatment. Here, nanomedicine based combination therapy using QLPVM conjugated liposomal tamoxifen (TAM) and doxorubicin (DOX) was designed and testified, where the penta-peptide was derived from Ku70 Bax-binding domain. Quantitative, semi-quantitative and qualitative approaches demonstrated the enhanced endocytosis and cytotoxicity of QLPVM conjugated sterically stabilized liposomes (QLPVM-SSLs) in vitro and in vivo. Mechanism studies of QLPVM excluded the possible electrostatic, hydrophobic or receptor-ligand interactions. However, as a weak cell-penetrating peptide, QLPVM significantly induced drug release from QLPVM-SSLs during their interaction with cells, which was favorable for drug internalization. These findings suggested that the nanomedicine based combination therapy using QLPVM-SSL-TAM and QLPVM-SSL-DOX might provide a rational strategy for Luminal A breast cancer. Breast cancer remains a leading cause of mortality in women worldwide. Although combined therapy using hormonal antagonist and chemotherapy is the norm nowadays, the use of these agents together in a single delivery system has not been tested. Here, the authors investigated this approach using QLPVM conjugated liposomes in in-vitro and in-vivo models. The positive findings may provide a novel direction for breast cancer treatment in the near future. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line.

    Directory of Open Access Journals (Sweden)

    Romain Rivalin

    Full Text Available Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease. We have previously identified a neurofilament-derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters by endocytosis in glioblastoma cells, where it induces microtubule destruction and inhibits cell proliferation. Here, we explore the impact of NFL-TBS.40-63 peptide on the mitochondrial network and its functions by using global cell respiration, quantitative PCR analysis of the main actors directing mitochondrial biogenesis, western blot analysis of the oxidative phosphorylation (OXPHOS subunits and confocal microscopy. We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration. This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.

  3. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

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

  4. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    N.D. Zegers (Netty)

    1995-01-01

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

  5. Peptide radiopharmaceuticals in nuclear medicine

    International Nuclear Information System (INIS)

    Blok, D.; Vermeij, P.; Feitsma, R.I.J.; Pauwels, E.J.K.

    1999-01-01

    This article reviews the labelling of peptides that are recognised to be of interest for nuclear medicine or are the subject of ongoing nuclear medicine research. Applications and approaches to the labelling of peptide radiopharmaceuticals are discussed, and drawbacks in their development considered. (orig.)

  6. The Equine PeptideAtlas

    DEFF Research Database (Denmark)

    Bundgaard, Louise; Jacobsen, Stine; Sørensen, Mette Aamand

    2014-01-01

    Progress in MS-based methods for veterinary research and diagnostics is lagging behind compared to the human research, and proteome data of domestic animals is still not well represented in open source data repositories. This is particularly true for the equine species. Here we present a first...... Equine PeptideAtlas encompassing high-resolution tandem MS analyses of 51 samples representing a selection of equine tissues and body fluids from healthy and diseased animals. The raw data were processed through the Trans-Proteomic Pipeline to yield high quality identification of proteins and peptides....... The current release comprises 24 131 distinct peptides representing 2636 canonical proteins observed at false discovery rates of 0.2% at the peptide level and 1.4% at the protein level. Data from the Equine PeptideAtlas are available for experimental planning, validation of new datasets, and as a proteomic...

  7. Vascular targeting with peptide libraries

    Energy Technology Data Exchange (ETDEWEB)

    Pasqualini, R. [La Jolla Cancer Research Center The Burnham Inst., La Jolla CA (United States)

    1999-06-01

    The authors have developed an 'in vivo' selection system in which phage capable of selective homing to different tissues are recovered from a phage display peptide library following intravenous administration. Using this strategy, they have isolate several organ and tumor-homing peptides. They have shown that each of those peptides binds of different receptors that are selectively expressed on the vasculature of the target tissue. The tumor-homing peptides bind to receptors that are up regulated in tumor angiogenic vasculature. Targeted delivery of doxorubicin to angiogenic vasculature using these peptides in animals models decrease toxicity and increased the therapeutic efficacy of the drug. Vascular targeting may facilitate the development of other treatment strategies that rely on inhibition of angio genesis and lead to advances to extend the potential for targeting of drugs, genes and radionuclides in the context of many diseases.

  8. Natriuretic peptides and cerebral hemodynamics

    DEFF Research Database (Denmark)

    Guo, Song; Barringer, Filippa; Zois, Nora Elisabeth

    2014-01-01

    Natriuretic peptides have emerged as important diagnostic and prognostic tools for cardiovascular disease. Plasma measurement of the bioactive peptides as well as precursor-derived fragments is a sensitive tool in assessing heart failure. In heart failure, the peptides are used as treatment...... in decompensated disease. In contrast, their biological effects on the cerebral hemodynamics are poorly understood. In this mini-review, we summarize the hemodynamic effects of the natriuretic peptides with a focus on the cerebral hemodynamics. In addition, we will discuss its potential implications in diseases...... where alteration of the cerebral hemodynamics plays a role such as migraine and acute brain injury including stroke. We conclude that a possible role of the peptides is feasible as evaluated from animal and in vitro studies, but more research is needed in humans to determine the precise response...

  9. Maize Bioactive Peptides against Cancer

    Science.gov (United States)

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

    2017-06-01

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

  10. CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

    Directory of Open Access Journals (Sweden)

    Piekarz Andrew D

    2012-07-01

    Full Text Available Abstract Background The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2 to bind to N-type voltage-activated calcium channels (CaV2.2 [Brittain et al. Nature Medicine 17:822–829 (2011]. Results and discussion Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K that bound with greater affinity to Ca2+ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca2+ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP release compared to vehicle control. Conclusions Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of

  11. Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis

    NARCIS (Netherlands)

    Di Toma, Claudia; Sonke, Theo; Quaedflieg, Peter J.; Janssen, Dick B.

    Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in

  12. Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

    2017-08-24

    Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

  13. Fluorescent peptide biosensor for probing the relative abundance of cyclin-dependent kinases in living cells.

    Directory of Open Access Journals (Sweden)

    Laetitia Kurzawa

    Full Text Available Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics.

  14. Synthesis and evaluation of amphiphilic peptides as nanostructures and drug delivery tools

    Science.gov (United States)

    Sayeh, Naser Ali

    conjugates although one limitation lies in the effort of controlling the rate of drug release. The encapsulated or complexed drugs tend to be released rapidly (before reaching the target site) and in the dendrimer--drug conjugates, it is the chemical linkage that controls the drug release. Thus, future studies in this field are urgently required to create more efficient and stable biomaterials. Peptides are considered as efficient vectors for achieving optimal cellular uptake. The potential use of peptides as drug delivery vectors received much attention by the discovery of several cell-penetrating peptides (CPPs). The first CPPs discovered in 1988, that were sequences from HIV-1 encoded TAT protein, TAT (48--60), and penetrated very efficiently through cell membranes of cultured mammalian cells. CPPs are a class of diverse peptides, typically with 8--25 amino acids, and unlike most peptides, they can cross the cellular membrane with more efficiency. CPPs have also shown to undergo self-assembly and generate nanostructures. The generation of self-assembled peptides and nanostructures occur through various types of interactions between functional groups of amino acid residues, such as electrostatic, hydrophobic, and hydrogen bonding. Appropriate design and functionalization of peptides are critical for generating nanostructures. Chemically CPPs are classified into two major groups: linear and cyclic peptides. It has been previously reported that linear peptides containing hydrophilic and hydrophobic amino acids could act as membrane protein stabilizers. These compounds are short hydrophilic or amphiphilic peptides that have positively charged amino acids, such as arginine, lysine or histidine, which can interact with the negative charge phospholipids layer on the cell membrane and translocate the cargo into the cells. Conjugation to cationic linear CPPs, such as TAT, penetratin, or oligoarginine efficiently improves the cellular uptake of large hydrophilic molecules, but the

  15. Radiopharmaceutical development of radiolabelled peptides

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-15

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

  16. 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...

  17. New vasoactive peptides in cirrhosis

    DEFF Research Database (Denmark)

    Kimer, Nina; Goetze, Jens Peter; Bendtsen, Flemming

    2014-01-01

    BACKGROUND: Patients with cirrhosis have substantial circulatory imbalance between vasoconstrictive and vasodilating forces. The study of circulatory vasoactive peptides may provide important pathophysiological information. This study aimed to assess concentrations, organ extraction and relations...... to haemodynamic changes in the pro-peptides copeptin, proadrenomedullin and pro-atrial natriuretic peptide (proANP) in patients with cirrhosis. MATERIALS AND METHODS: Fifty-four cirrhotic patients and 15 controls were characterized haemodynamically during a liver vein catheterization. Copeptin, proadrenomedullin...... pressure (R=0·32, P0·31, Ppeptide is elevated in cirrhosis. Copeptin, proadrenomedullin and proANP are related to portal pressure and seem associated with systemic haemodynamics. These propeptides may...

  18. Characterization of synthetic peptides by mass spectrometry

    DEFF Research Database (Denmark)

    Prabhala, Bala Krishna; Mirza, Osman Asghar; Højrup, Peter

    2015-01-01

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

  19. Marine Peptides: Bioactivities and Applications

    Directory of Open Access Journals (Sweden)

    Randy Chi Fai Cheung

    2015-06-01

    Full Text Available Peptides are important bioactive natural products which are present in many marine species. These marine peptides have high potential nutraceutical and medicinal values because of their broad spectra of bioactivities. Their antimicrobial, antiviral, antitumor, antioxidative, cardioprotective (antihypertensive, antiatherosclerotic and anticoagulant, immunomodulatory, analgesic, anxiolytic anti-diabetic, appetite suppressing and neuroprotective activities have attracted the attention of the pharmaceutical industry, which attempts to design them for use in the treatment or prevention of various diseases. Some marine peptides or their derivatives have high commercial values and had reached the pharmaceutical and nutraceutical markets. A large number of them are already in different phases of the clinical and preclinical pipeline. This review highlights the recent research in marine peptides and the trends and prospects for the future, with special emphasis on nutraceutical and pharmaceutical development into marketed products.

  20. Cardioprotective peptides from marine sources.

    Science.gov (United States)

    Harnedy, Padraigín A; FitzGerald, Richard J

    2013-05-01

    Elevated blood pressure or hypertension is one of the fastest growing health problems worldwide. Although the etiology of essential hypertension has a genetic component, dietary factors play an important role. With the high costs and adverse side-effects associated with synthetic antihypertensive drugs and the awareness of the link between diet and health there has been increased focus on identification of food components that may contribute to cardiovascular health. In recent years special interest has been paid to the cardioprotective activity of peptides derived from food proteins including marine proteins. These peptides are latent within the sequence of the parent protein and only become active when released by proteolytic digestion during gastrointestinal digestion or through food processing. Current data on antihypertensive activity of marine-derived protein hydrolysates/peptides in animal and human studies is reviewed herein. Furthermore, products containing protein hydrolysates/peptides from marine origin with antihypertensive effects are discussed.

  1. Antimicrobial peptides from Capsicum sp.

    African Journals Online (AJOL)

    ajl yemi

    2011-12-30

    Dec 30, 2011 ... Key words: Antimicrobial peptides, Capsicum sp, Capsicum chinense, chili pepper, agronomical options, ..... of this human activity is resumed by the simple phrase: produce .... It will be interesting to scale the AMPs extraction.

  2. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies...... are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors......, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody...

  3. Development of novel recombinant biomimetic chimeric MPG-based peptide as nanocarriers for gene delivery: Imitation of a real cargo.

    Science.gov (United States)

    Majidi, Asia; Nikkhah, Maryam; Sadeghian, Faranak; Hosseinkhani, Saman

    2016-10-01

    In last decades great efforts have been devoted to the study of development of recombinant peptide based vectors that consist of biological motifs with potential applications in gene therapy. Recombinant Biomimetic Chimeric Vectors (rBCVs) are biopolymeric nanocarriers that are designed to mimic viral features to overcome the cellular obstacles in gene transferring pathway into cell nucleus. In this research, we designed and genetically engineered three novel rBCVs with similar sequences that differed in motifs arrangement and motif abundance: MPG-2H1, 2TMPG-2H1 and 2RMPG-2H1. The MPG as a famous amphipathic cell penetrating peptide is the main segment of these constructs which was studied for the first time in association with truncated histone H1 DNA condensing motif. Through the performance of several physicochemical and biological assays, the rBCVs were remarkably examined regarding transfection efficiency. The main objective of this study is focused on the importance of motif design in transfection efficiency of rBCVs on one hand, and the assessment of correlation between structural features and functionality of motifs on the other hand. The results revealed that all three kinds of rBCVs/pDNA nanoparticles with average sizes of 200nm could overwhelm the cellular obstacles associated with gene transfer, and lead to efficient gene delivery. Furthermore, no significant toxicity was perceived and efficient endosome disruptive activity was obtained. It is noteworthy to say among three mentioned constructs 2RMPG-2H1 showed the highest transfection efficiency. Overall the peptide based vectors hold great promise as a nontoxic and effective gene carrier in vitro and in vivo, besides the rational design possibility as the most vital advantages over the other non-viral gene delivery vectors. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Peptides and proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bachovchin, W.W.; Unkefer, C.J.

    1994-12-01

    Advances in magnetic resonance and vibrational spectroscopy make it possible to derive detailed structural information about biomolecular structures in solution. These techniques are critically dependent on the availability of labeled compounds. For example, NMR techniques used today to derive peptide and protein structures require uniformity {sup 13}C-and {sup 15}N-labeled samples that are derived biosynthetically from (U-6-{sup 13}C) glucose. These experiments are possible now because, during the 1970s, the National Stable Isotope Resource developed algal methods for producing (U-6-{sup 13}C) glucose. If NMR techniques are to be used to study larger proteins, we will need sophisticated labelling patterns in amino acids that employ a combination of {sup 2}H, {sup 13}C, and {sup 15}N labeling. The availability of these specifically labeled amino acids requires a renewed investment in new methods for chemical synthesis of labeled amino acids. The development of new magnetic resonance or vibrational techniques to elucidate biomolecular structure will be seriously impeded if we do not see rapid progress in labeling technology. Investment in labeling chemistry is as important as investment in the development of advanced spectroscopic tools.

  5. Matrix-assisted peptide synthesis on nanoparticles.

    Science.gov (United States)

    Khandadash, Raz; Machtey, Victoria; Weiss, Aryeh; Byk, Gerardo

    2014-09-01

    We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix-assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  6. Material Binding Peptides for Nanotechnology

    Directory of Open Access Journals (Sweden)

    Urartu Ozgur Safak Seker

    2011-02-01

    Full Text Available Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.

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

    Directory of Open Access Journals (Sweden)

    Ranganathan Shoba

    2008-12-01

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

  8. Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing.

    Science.gov (United States)

    Zhang, Fan; Briones, Andrea; Soloviev, Mikhail

    2016-01-01

    This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

  9. Automated solid-phase peptide synthesis to obtain therapeutic peptides

    Directory of Open Access Journals (Sweden)

    Veronika Mäde

    2014-05-01

    Full Text Available The great versatility and the inherent high affinities of peptides for their respective targets have led to tremendous progress for therapeutic applications in the last years. In order to increase the drugability of these frequently unstable and rapidly cleared molecules, chemical modifications are of great interest. Automated solid-phase peptide synthesis (SPPS offers a suitable technology to produce chemically engineered peptides. This review concentrates on the application of SPPS by Fmoc/t-Bu protecting-group strategy, which is most commonly used. Critical issues and suggestions for the synthesis are covered. The development of automated methods from conventional to essentially improved microwave-assisted instruments is discussed. In order to improve pharmacokinetic properties of peptides, lipidation and PEGylation are described as covalent conjugation methods, which can be applied by a combination of automated and manual synthesis approaches. The synthesis and application of SPPS is described for neuropeptide Y receptor analogs as an example for bioactive hormones. The applied strategies represent innovative and potent methods for the development of novel peptide drug candidates that can be manufactured with optimized automated synthesis technologies.

  10. What peptides these deltorphins be.

    Science.gov (United States)

    Lazarus, L H; Bryant, S D; Cooper, P S; Salvadori, S

    1999-02-01

    The deltorphins are a class of highly selective delta-opioid heptapeptides from the skin of the Amazonian frogs Phyllomedusa sauvagei and P. bicolor. The first of these fascinating peptides came to light in 1987 by cloning of the cDNA of from frog skins, while the other members of this family were identified either by cDNA or isolation of the peptides. The distinctive feature of deltorphins is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe, comparable to dermorphin, which is the prototype of a group of mu-selective opioids from the same source. The D-amino acid and the anionic residues, either Glu or Asp, as well as their unique amino acid compositions are responsible for the remarkable biostability, high delta-receptor affinity, bioactivity and peptide conformation. This review summarizes a decade of research from many laboratories that defined which residues and substituents in the deltorphins interact with the delta-receptor and characterized pharmacological and physiological activities in vitro and in vivo. It begins with a historical description of the topic and presents general schema for the synthesis of peptide analogues of deltorphins A, B and C as a means to document the methods employed in producing a myriad of analogues. Structure activity studies of the peptides and their pharmacological activities in vitro are detailed in abundantly tabulated data. A brief compendium of the current level of knowledge of the delta-receptor assists the reader to appreciate the rationale for the design of these analogues. Discussion of the conformation of these peptides addresses how structure leads to further hypotheses regarding ligand receptor interaction. The review ends with a broad discussion of the potential applications of these peptides in clinical and therapeutic settings.

  11. Sequencing Cyclic Peptides by Multistage Mass Spectrometry

    Science.gov (United States)

    Mohimani, Hosein; Yang, Yu-Liang; Liu, Wei-Ting; Hsieh, Pei-Wen; Dorrestein, Pieter C.; Pevzner, Pavel A.

    2012-01-01

    Some of the most effective antibiotics (e.g., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of mass spectrometry based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage mass spectrometry, and show its advantages over single stage mass spectrometry. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria. PMID:21751357

  12. Cyclic peptide therapeutics: past, present and future.

    Science.gov (United States)

    Zorzi, Alessandro; Deyle, Kaycie; Heinis, Christian

    2017-06-01

    Cyclic peptides combine several favorable properties such as good binding affinity, target selectivity and low toxicity that make them an attractive modality for the development of therapeutics. Over 40 cyclic peptide drugs are currently in clinical use and around one new cyclic peptide drug enters the market every year on average. The vast majority of clinically approved cyclic peptides are derived from natural products, such as antimicrobials or human peptide hormones. New powerful techniques based on rational design and in vitro evolution have enabled the de novo development of cyclic peptide ligands to targets for which nature does not offer solutions. A look at the cyclic peptides currently under clinical evaluation shows that several have been developed using such techniques. This new source for cyclic peptide ligands introduces a freshness to the field, and it is likely that de novo developed cyclic peptides will be in clinical use in the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Peptide Vaccine: Progress and Challenges

    Directory of Open Access Journals (Sweden)

    Weidang Li

    2014-07-01

    Full Text Available Conventional vaccine strategies have been highly efficacious for several decades in reducing mortality and morbidity due to infectious diseases. The bane of conventional vaccines, such as those that include whole organisms or large proteins, appear to be the inclusion of unnecessary antigenic load that, not only contributes little to the protective immune response, but complicates the situation by inducing allergenic and/or reactogenic responses. Peptide vaccines are an attractive alternative strategy that relies on usage of short peptide fragments to engineer the induction of highly targeted immune responses, consequently avoiding allergenic and/or reactogenic sequences. Conversely, peptide vaccines used in isolation are often weakly immunogenic and require particulate carriers for delivery and adjuvanting. In this article, we discuss the specific advantages and considerations in targeted induction of immune responses by peptide vaccines and progresses in the development of such vaccines against various diseases. Additionally, we also discuss the development of particulate carrier strategies and the inherent challenges with regard to safety when combining such technologies with peptide vaccines.

  14. Double-Stranded Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2001-01-01

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

  15. CIGB-552: A new penetrating peptide with antitumor action mediated by the increased levels of the COMMD1 protein in cancer cell lines

    International Nuclear Information System (INIS)

    Guerra-Vallespi, M; Fernández-Massó, JR; Oliva-Argüelles, B.; Reyes-Acosta, O.; Garay-Pérez, H.E.; Cabrales-Rico, A.; Tejeda-Gómez, Y.; Mendoza-Fuentes, O.; Soria, Y.; Guillen-Pérez, I.; Palenzuela-Gardon, D.; Vázquez-Blomquist, D.; Musacchio-Lasa, A.; Novoa-Perez, L.I.; Gómez-Rodríguez, Y.; Delgado-Roche, L.; Pimentel, G.; Garza, J.; Basaco, T.; Sánchez, I.; Calderón, C.; Rodríguez, J.C.; Astrada, S.; Bollati-Fogolín, M.; Rivera-Markelova, M.; Fichtner, I.

    2015-01-01

    A second-generation peptide CIGB-552, with cell-penetrating capacity, was developed by the modification of the primary structure of the L-2 peptide. The molecular mechanism underlying its cytotoxic activity remains partially unknown. In this study, it was shown that CIGB-552 binds and increases the levels of COMMD1, a protein involved in copper homeostasis, sodium transport, and the NF-kB signaling pathway. We found that CIGB-552 induces ubiquitination of RelA and inhibits the antiapoptotic activity regulated by NF-κβ, whereas the knockdown of COMMD1 blocks this effect. We also found that CIGB-552 increases the levels of reactive oxygen species (ROS), decreases the cellular antioxidant capacity and induces the peroxidation of proteins and lipids in tumor cells. Altogether, our results bring new insights into the mechanism of action of CIGB-552. Moreover, its anti-tumoral effect was explored by subcutaneous administration in a therapeutic schedule in syngeneic murine tumors and patient-derived xenograft models. Outstandingly, a significant delay of tumor growth was observed after the administration of CIGB-552 in these experimental settings. Our data reinforce the perspectives of CIGB-552 for targeted therapy against cancer. This research granted the 2014 Award of the Cuban National Academy of Sciences. (author)

  16. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery....... To better understand the underlying mechanisms of antibody-antigen interaction here we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class....

  17. Self-assembling peptide semiconductors

    Science.gov (United States)

    Tao, Kai; Makam, Pandeeswar; Aizen, Ruth; Gazit, Ehud

    2017-01-01

    Semiconductors are central to the modern electronics and optics industries. Conventional semiconductive materials bear inherent limitations, especially in emerging fields such as interfacing with biological systems and bottom-up fabrication. A promising candidate for bioinspired and durable nanoscale semiconductors is the family of self-assembled nanostructures comprising short peptides. The highly ordered and directional intermolecular π-π interactions and hydrogen-bonding network allow the formation of quantum confined structures within the peptide self-assemblies, thus decreasing the band gaps of the superstructures into semiconductor regions. As a result of the diverse architectures and ease of modification of peptide self-assemblies, their semiconductivity can be readily tuned, doped, and functionalized. Therefore, this family of electroactive supramolecular materials may bridge the gap between the inorganic semiconductor world and biological systems. PMID:29146781

  18. Antimicrobial Peptide Production and Purification.

    Science.gov (United States)

    Suda, Srinivas; Field, Des; Barron, Niall

    2017-01-01

    Antimicrobial peptides (AMPs) are natural defense compounds which are synthesized as ribosomal gene-encoded pre-peptides and produced by all living organisms. AMPs are small peptides, usually cationic and typically have hydrophobic residues which interact with cell membranes and have either a narrow or broad spectrum of biological activity. AMPs are isolated from the natural host or heterologously expressed in other hosts such as Escherichia coli. The proto-typical lantibiotic Nisin is a widely used AMP that is produced by the food-grade organism Lactococcus lactis. Although AMP production and purification procedures require optimization for individual AMPs, the Nisin production and purification protocol outlined in this chapter can be easily applied with minor modifications for the production and purification of other lantibiotics or AMPs. While Nisin is produced and secreted into the supernatant, steps to recover Nisin from both cell-free supernatant and cell pellet are outlined in detail.

  19. Delivery systems for antimicrobial peptides

    DEFF Research Database (Denmark)

    Nordström, Randi; Malmsten, Martin

    2017-01-01

    Due to rapidly increasing resistance development against conventional antibiotics, finding novel approaches for the treatment of infections has emerged as a key health issue. Antimicrobial peptides (AMPs) have attracted interest in this context, and there is by now a considerable literature...... on the identification such peptides, as well as on their optimization to reach potent antimicrobial and anti-inflammatory effects at simultaneously low toxicity against human cells. In comparison, delivery systems for antimicrobial peptides have attracted considerably less interest. However, such delivery systems...... are likely to play a key role in the development of potent and safe AMP-based therapeutics, e.g., through reducing chemical or biological degradation of AMPs either in the formulation or after administration, by reducing adverse side-effects, by controlling AMP release rate, by promoting biofilm penetration...

  20. Radioactive labelling of peptidic hormones

    International Nuclear Information System (INIS)

    Fromageot, P.; Pradelles, P.; Morgat, J.L.; Levine, H.

    1976-01-01

    The labelling of peptidic hormones requires stability, specificity and sensitivity of the label. Introduction of a radioactive atome is one way to satisfy these criteria. Several processes have been described to prepare radioactive TRF: synthesis of the peptide with labelled aminoacids or introduction of the label into the hormone. In that approach, tritium can be substituted in the imidazole ring, via precursors activating the proper carbon. Monoiodo TRF leads essentially to tritium labelling of the 5 positions whereas monoazo TRF allows the preparation of 3 H TRF labelled in the 2 positions. Di-substituted TRF leads to labelling into the 2 and 5 carbons. Labelled analogs of TRF can be prepared with labelled iodine; further developments of peptide labelling, will be presented. In particular, the homolytic scission of the C-iodine, bond by photochemical activation. The nascent carbon radical can be stabilized by a tritiated scavenger. This approach eliminates the use of heavy metal catalysts

  1. The Pig PeptideAtlas

    DEFF Research Database (Denmark)

    Hesselager, Marianne Overgaard; Codrea, Marius; Sun, Zhi

    2016-01-01

    Biological research of Sus scrofa, the domestic pig, is of immediate relevance for food production sciences, and for developing pig as a model organism for human biomedical research. Publicly available data repositories play a fundamental role for all biological sciences, and protein data...... repositories are in particular essential for the successful development of new proteomic methods. Cumulative proteome data repositories, including the PeptideAtlas, provide the means for targeted proteomics, system-wide observations, and cross-species observational studies, but pigs have so far been...... underrepresented in existing repositories. We here present a significantly improved build of the Pig PeptideAtlas, which includes pig proteome data from 25 tissues and three body fluid types mapped to 7139 canonical proteins. The content of the Pig PeptideAtlas reflects actively ongoing research within...

  2. Novel Formulations for Antimicrobial Peptides

    Directory of Open Access Journals (Sweden)

    Ana Maria Carmona-Ribeiro

    2014-10-01

    Full Text Available Peptides in general hold much promise as a major ingredient in novel supramolecular assemblies. They may become essential in vaccine design, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organs transplants, design of novel materials for dentistry, formulations against diabetes and other important strategical applications. This review discusses how novel formulations may improve the therapeutic index of antimicrobial peptides by protecting their activity and improving their bioavailability. The diversity of novel formulations using lipids, liposomes, nanoparticles, polymers, micelles, etc., within the limits of nanotechnology may also provide novel applications going beyond antimicrobial chemotherapy.

  3. Peptides and the new endocrinology

    Science.gov (United States)

    Schwyzer, Robert

    1982-01-01

    The discovery of regulatory peptides common to the nervous and the endocrine systems (brain, gut, and skin) has brought about a revolution in our concepts of endocrinology and neurology. We are beginning to understand some of the complex interrelationships between soma and psyche that might, someday, be important for an integrated treatment of diseases. Examples of the actions of certain peptides in the periphery and in the central nervous system are given, and their biosynthesis and molecular anatomy as carriers for information are discussed.

  4. Novel Formulations for Antimicrobial Peptides

    Science.gov (United States)

    Carmona-Ribeiro, Ana Maria; Carrasco, Letícia Dias de Melo

    2014-01-01

    Peptides in general hold much promise as a major ingredient in novel supramolecular assemblies. They may become essential in vaccine design, antimicrobial chemotherapy, cancer immunotherapy, food preservation, organs transplants, design of novel materials for dentistry, formulations against diabetes and other important strategical applications. This review discusses how novel formulations may improve the therapeutic index of antimicrobial peptides by protecting their activity and improving their bioavailability. The diversity of novel formulations using lipids, liposomes, nanoparticles, polymers, micelles, etc., within the limits of nanotechnology may also provide novel applications going beyond antimicrobial chemotherapy. PMID:25302615

  5. Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

    International Nuclear Information System (INIS)

    Johns, Douglas G.; Ao, Zhaohui; Heidrich, Bradley J.; Hunsberger, Gerald E.; Graham, Taylor; Payne, Lisa; Elshourbagy, Nabil; Lu, Quinn; Aiyar, Nambi; Douglas, Stephen A.

    2007-01-01

    Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [ 125 I]-ANP from NPR-C with pM-to-nM K i values. DNP displaced [ 125 I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K i > 1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure

  6. Defeating Leishmania resistance to miltefosine (hexadecylphosphocholine) by peptide-mediated drug smuggling: a proof of mechanism for trypanosomatid chemotherapy.

    Science.gov (United States)

    Luque-Ortega, Juan Román; de la Torre, Beatriz G; Hornillos, Valentín; Bart, Jean-Mathieu; Rueda, Cristina; Navarro, Miguel; Amat-Guerri, Francisco; Acuña, A Ulises; Andreu, David; Rivas, Luis

    2012-08-10

    Miltefosine (hexadecylphosphocholine, HePC), the first orally active drug successful against leishmaniasis, is especially active on the visceral form of the disease. Resistance mechanisms are almost exclusively associated to dysfunction in HePC uptake systems. In order to evade the requirements of its cognate receptor/translocator, HePC-resistant Leishmania donovani parasites (R40 strain) were challenged with constructs consisting of an ω-thiol-functionalized HePC analogue conjugated to the cell-penetrating peptide (CPP) Tat(48-60), either through a disulfide or a thioether bond. The conjugates enter and kill both promastigote and intracellular amastigote forms of the R40 strain. Intracellular release of HePC by reduction of the disulfide-based conjugate was confirmed by means of double tagging at both the CPP (Quasar 670) and HePC (BODIPY) moieties. Scission of the conjugate, however, is not mandatory, as the metabolically more stable thioether conjugate retained substantial activity. The disulfide conjugate is highly active on the bloodstream form of Trypanosoma b. brucei, naturally resistant to HePC. Our results provide proof-of-mechanism for the use of CPP conjugates to avert drug resistance by faulty drug accumulation in parasites, as well as the possibility to extend chemotherapy into other parasites intrinsically devoid of membrane translocation systems. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Toxins and antimicrobial peptides: interactions with membranes

    Science.gov (United States)

    Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

    2009-08-01

    The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of resistant pathogens.

  8. 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....

  9. Streptavidin-binding peptides and uses thereof

    Science.gov (United States)

    Szostak, Jack W. (Inventor); Wilson, David S. (Inventor); Keefe, Anthony D. (Inventor)

    2006-01-01

    The invention provides peptides with high affinity for streptavidin. These peptides may be expressed as part of fusion proteins to facilitate the detection, quantitation, and purification of proteins of interest.

  10. Biomedical Applications of Self-Assembling Peptides

    NARCIS (Netherlands)

    Radmalekshahi, Mazda; Lempsink, Ludwijn; Amidi, Maryam; Hennink, Wim E.; Mastrobattista, Enrico

    2016-01-01

    Self-assembling peptides have gained increasing attention as versatile molecules to generate diverse supramolecular structures with tunable functionality. Because of the possibility to integrate a wide range of functional domains into self-assembling peptides including cell attachment sequences,

  11. Computer-Aided Design of Antimicrobial Peptides

    DEFF Research Database (Denmark)

    Fjell, Christopher D.; Hancock, Robert E.W.; Jenssen, Håvard

    2010-01-01

    in antimicrobial activity. Consequently, the majority of peptides put into clinical trials have failed at some point, underlining the importance of a thorough peptide optimization. An important tool in peptide design and optimization is quantitative structure-activity relationship (QSAR) analysis, correlating...... chemical parameters with biological activities of the peptide, using statistical methods. In this review we will discuss two different in silico strategies of computer-aided antibacterial peptide design, a linear correlation model build as an extension of traditional principal component analysis (PCA......) and a non-linear artificial neural network model. Studies on structurally diverse peptides, have concluded that the PCA derived model are able to guide the antibacterial peptide design in a meaningful way, however requiring rather a high homology between the peptides in the test-set and the in silico...

  12. Characterization of cyclic peptides containing disulfide bonds

    OpenAIRE

    Johnson, Mindy; Liu, Mingtao; Struble, Elaine; Hettiarachchi, Kanthi

    2015-01-01

    Unlike linear peptides, analysis of cyclic peptides containing disulfide bonds is not straightforward and demands indirect methods to achieve a rigorous proof of structure. Three peptides that belong to this category, p-Cl-Phe-DPDPE, DPDPE, and CTOP, were analyzed and the results are presented in this paper. The great potential of two dimensional NMR and ESI tandem mass spectrometry was harnessed during the course of peptide characterizations. A new RP-HPLC method for the analysis of trifluor...

  13. Development and use of engineered peptide deformylase in chemoenzymatic peptide synthesis

    NARCIS (Netherlands)

    Di Toma, Claudia

    2012-01-01

    Deze thesis beschrijft het onderzoek naar potentieel van het gebruik van het peptide deformylase (PDF) in chemo enzymatische peptide synthese. PDF is geschikt voor selective N terminale deformylatie van bepaalde N-formyl-peptides zonder gelijktijdige hydrolyse van de peptide binding. Door de

  14. Oxidative Modification of Tryptophan-Containing Peptides

    DEFF Research Database (Denmark)

    Petersen, Jonas; Christensen, Pia Katrine; Nielsen, Mathias T

    2018-01-01

    We herein present a broadly useful method for the chemoselective modification of a wide range of tryptophan-containing peptides. Exposing a tryptophan-containing peptide to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) resulted in a selective cyclodehydration between the peptide backbone...

  15. 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....

  16. Discovery of thrombin activatable fibrinolysis inhibitor (TAFI)

    NARCIS (Netherlands)

    Bertina, R.M.; Tilburg, N.H. van; Haverkate, F.; Bouma, B.N.; Borne, P.A.K. von dem; Meijers, J.C.M.; Campbell, W.; Eaton, D.; Hendriks, D.F.; Willemse, J.L.

    2006-01-01

    CAS: blood clotting factor 11, 9013-55-2; thrombin, 9002-04-4; tissue plasminogen activator, 105913-11-9; protein C, 60202-16-6; Carboxypeptidase U, 3.4.17.20; Protein C; Tissue Plasminogen Activator, 3.4.21.68

  17. Insect Peptides - Perspectives in Human Diseases Treatment.

    Science.gov (United States)

    Chowanski, Szymon; Adamski, Zbigniew; Lubawy, Jan; Marciniak, Pawel; Pacholska-Bogalska, Joanna; Slocinska, Malgorzata; Spochacz, Marta; Szymczak, Monika; Urbanski, Arkadiusz; Walkowiak-Nowicka, Karolina; Rosinski, Grzegorz

    2017-01-01

    Insects are the largest and the most widely distributed group of animals in the world. Their diversity is a source of incredible variety of different mechanisms of life processes regulation. There are many agents that regulate immunology, reproduction, growth and development or metabolism. Hence, it seems that insects may be a source of numerous substances useful in human diseases treatment. Especially important in the regulation of insect physiology are peptides, like neuropeptides, peptide hormones or antimicrobial peptides. There are two main aspects where they can be helpful, 1) Peptides isolated from insects may become potential drugs in therapy of different diseases, 2) A lot of insect peptide hormones show structural or functional homology to mammalian peptide hormones and the comparative studies may give a new look on human disorders. In our review we focused on three group of insect derived peptides: 1) immune-active peptides, 2) peptide hormones and 3) peptides present in venoms. In our review we try to show the considerable potential of insect peptides in searching for new solutions for mammalian diseases treatment. We summarise the knowledge about properties of insect peptides against different virulent agents, anti-inflammatory or anti-nociceptive properties as well as compare insect and mammalian/vertebrate peptide endocrine system to indicate usefulness of knowledge about insect peptide hormones in drug design. The field of possible using of insect delivered peptide to therapy of various human diseases is still not sufficiently explored. Undoubtedly, more attention should be paid to insects due to searching new drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Peptides: Production, bioactivity, functionality, and applications

    DEFF Research Database (Denmark)

    Hajfathalian, Mona; Ghelichi, Sakhi; García Moreno, Pedro Jesús

    2017-01-01

    Production of peptides with various effects from proteins of different sources continues to receive academic attention. Researchers of different disciplines are putting increasing efforts to produce bioactive and functional peptides from different sources such as plants, animals, and food industry...... by-products. The aim of this review is to introduce production methods of hydrolysates and peptides and provide a comprehensive overview of their bioactivity in terms of their effects on immune, cardiovascular, nervous, and gastrointestinal systems. Moreover, functional and antioxidant properties...... of hydrolysates and isolated peptides are reviewed. Finally, industrial and commercial applications of bioactive peptides including their use in nutrition and production of pharmaceuticals and nutraceuticals are discussed....

  19. Natriuretic peptides in cardiometabolic regulation and disease

    DEFF Research Database (Denmark)

    Zois, Nora E; Bartels, Emil D; Hunter, Ingrid

    2014-01-01

    decade. Dysregulation of the natriuretic peptide system has been associated with obesity, glucose intolerance, type 2 diabetes mellitus, and essential hypertension. Moreover, the natriuretic peptides have been implicated in the protection against atherosclerosis, thrombosis, and myocardial ischaemia. All...... these conditions can coexist and potentially lead to heart failure, a syndrome associated with a functional natriuretic peptide deficiency despite high circulating concentrations of immunoreactive peptides. Therefore, dysregulation of the natriuretic peptide system, a 'natriuretic handicap', might be an important...... factor in the initiation and progression of metabolic dysfunction and its accompanying cardiovascular complications. This Review provides a summary of the natriuretic peptide system and its involvement in these cardiometabolic conditions. We propose that these peptides might have an integrating role...

  20. Analysis of peptide uptake and location of root hair-promoting peptide accumulation in plant roots.

    Science.gov (United States)

    Matsumiya, Yoshiki; Taniguchi, Rikiya; Kubo, Motoki

    2012-03-01

    Peptide uptake by plant roots from degraded soybean-meal products was analyzed in Brassica rapa and Solanum lycopersicum. B. rapa absorbed about 40% of the initial water volume, whereas peptide concentration was decreased by 75% after 24 h. Analysis by reversed-phase HPLC showed that number of peptides was absorbed by the roots during soaking in degraded soybean-meal products for 24 h. Carboxyfluorescein-labeled root hair-promoting peptide was synthesized, and its localization, movement, and accumulation in roots were investigated. The peptide appeared to be absorbed by root hairs and then moved to trichoblasts. Furthermore, the peptide was moved from trichoblasts to atrichoblasts after 24 h. The peptide was accumulated in epidermal cells, suggesting that the peptide may have a function in both trichoblasts and atrichoblasts. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

  1. Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions.

    Science.gov (United States)

    Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco; Østergaard, Jesper

    2016-10-10

    Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins. In this work, we show that protein-protein and peptide-peptide interactions can advantageously be investigated by measurement of the diffusion coefficient using Taylor Dispersion Analysis. Through comparison to Dynamic Light Scattering it was shown that Taylor Dispersion Analysis is well suited for the characterization of protein-protein interactions of solutions of α-lactalbumin and human serum albumin. The peptide-peptide interactions of three selected peptides were then investigated in a concentration range spanning from 0.5mg/ml up to 80mg/ml using Taylor Dispersion Analysis. The peptide-peptide interactions determination indicated that multibody interactions significantly affect the PPIs at concentration levels above 25mg/ml for the two charged peptides. Relative viscosity measurements, performed using the capillary based setup applied for Taylor Dispersion Analysis, showed that the viscosity of the peptide solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative, approach to assessment of the colloidal stability of both peptide and protein formulations. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Radio peptide imaging and therapy

    International Nuclear Information System (INIS)

    Buscombe, Jonh

    1997-01-01

    Full text. The concept of the magic bullet retains its attraction to us. If only we could take a drug or radioisotope and inject this intravenously and then will attach to the target cancer. This may allow imaging if labelled with a radio pharmaceutical or possibly even effective therapy. Initially work was started using antibodies of mouse origin. These have shown some utility in targeting tumors but there are problems in that these are essentially non-human proteins, often derived from mice. This leads to the formation of antibodies against that antibody so that repeat administrations lead to reduced efficacy and possibly may carry a risk anaphylaxis for the patient. Two different methods have evolved to deal with this situation. Either make antibodies more human or use smaller fragments, so that they are less likely to cause allergic reactions. The second method is to try and use a synthetic peptide. This will contain a series of amino acids which recognize a certain cell receptor. For example the somatostatin analogue Octreotide is an 8 amino acid peptide which has the same biological actions as natural somatostatin but an increased plasma half life. To this is added a linker a good example being DTPA and then radioisotope for example In-111. There we can have the complex In-111-DTPA-Octreotide which can be used to image somatostatin receptors in vivo. The main advantage over antibodies is that the cost production is less and many different variation of peptides for a particular receptor can be manufactured and assessed to find which is the optimal agent tumour imaging at a fraction of the cost of antibody production. There are two main approaches. Firstly to take a natural peptide hormone such as insulin or VIP and label by a simple method such as iodination with I-123. A group in Vienna have done it and shown good uptake of I-123 Insulin in primary hepatomas and of I-123 VIP in pancreatic cancers. Many natural peptide hormones however have a short plasma half

  3. Peptide-targeted polymer cancerostatics

    Czech Academy of Sciences Publication Activity Database

    Böhmová, Eliška; Pola, Robert

    2016-01-01

    Roč. 65, Suppl. 2 (2016), S153-S164 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) LO1507 Institutional support: RVO:61389013 Keywords : HPMA copolymers * tumor targeting * peptides Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.461, year: 2016 http://www.biomed.cas.cz/physiolres/pdf/65%20Suppl%202/65_S153.pdf

  4. Photosystem Inspired Peptide Hybrid Catalysts

    Science.gov (United States)

    2017-06-07

    materials defined at the molecular level. We propose a novel way to make hybrid catalyst composed of inorganic nanomaterials and peptides. The...Distribution approved for public release. AF Office Of Scientific Research (AFOSR)/ IOA Arlington, Virginia 22203 Air Force Research Laboratory Air...ORGANIZATION NAME(S) AND ADDRESS(ES) SEOUL NATIONAL UNIVERSITY SNUR&DB FOUNDATION RESEARCH PARK CENTER SEOUL, 151742 KR 8. PERFORMING ORGANIZATION REPORT

  5. Peptide stabilized amphotericin B nanodisks

    Science.gov (United States)

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

    2007-01-01

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

  6. Biopharmaceuticals: From peptide to drug

    Science.gov (United States)

    Hannappel, Margarete

    2017-08-01

    Biologics are therapeutic proteins or peptides that are produced by means of biological processes within living organisms and cells. They are highly specific molecules and play a crucial role as therapeutics for the treatment of severe and chronic diseases (e.g. cancer, rheumatoid arthritis, diabetes, autoimmune disorders). The development of new biologics and biologics-based drugs gains more and more importance in the fight against various diseases. A short overview on biotherapeutical drug development is given. Cone snails are a large group of poisonous, predatory sea snails with more than 700 species. They use a very powerful venom which rapidly inactivates and paralyzes their prey. Most bioactive venom components are small peptides (conotoxins, conopeptides) which are precisely directed towards a specific target (e.g. ion channel, receptors). Due to their small size, their precision and speed of action, naturally occurring cone snail venom peptides represent an attractive source for the identification and design of novel biological drug entities. The Jagna cone snail project is an encouraging initiative to map the ecological variety of cone snails around the island of Bohol (Philippines) and to conserve the biological information for potential future application.

  7. Coffee, hunger, and peptide YY.

    Science.gov (United States)

    Greenberg, James A; Geliebter, Allan

    2012-06-01

    There is evidence from several empirical studies suggesting that coffee may help people control body weight. Our objective was to assess the effects of caffeine, caffeinated coffee, and decaffeinated coffee, both alone and in combination with 75 g of glucose, on perceived hunger and satiety and related peptides. We conducted a placebo-controlled single-blinded randomized 4-way crossover trial. Eleven healthy male volunteers (mean age, 23.5 ± 5.7 years; mean BMI, 23.6 ± 4.2 kg/m(2)) ingested 1 of 3 test beverages (caffeine in water, caffeinated coffee, or decaffeinated coffee) or placebo (water), and 60 minutes later they ingested the glucose. Eight times during each laboratory visit, hunger and satiety were assessed by visual analog scales, and blood samples were drawn to measure 3 endogenous peptides associated with hunger and satiety: ghrelin, peptide YY (PYY), and leptin. Compared to placebo, decaffeinated coffee yielded significantly lower hunger during the whole 180-minute study period and higher plasma PYY for the first 90 minutes (p hunger or PYY. Caffeinated coffee showed a pattern between that of decaffeinated coffee and caffeine in water. These findings suggest that one or more noncaffeine ingredients in coffee may have the potential to decrease body weight. Glucose ingestion did not change the effects of the beverages. Our randomized human trial showed that decaffeinated coffee can acutely decrease hunger and increase the satiety hormone PYY.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many...... or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin...

  9. Chemical Methods for Peptide and Protein Production

    Directory of Open Access Journals (Sweden)

    Istvan Toth

    2013-04-01

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

  10. Chemical methods for peptide and protein production.

    Science.gov (United States)

    Chandrudu, Saranya; Simerska, Pavla; Toth, Istvan

    2013-04-12

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

  11. 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

  12. Synthesis of peptide .alpha.-thioesters

    Science.gov (United States)

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

    2008-08-19

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

  13. Peptide YY receptors in the brain

    International Nuclear Information System (INIS)

    Inui, A.; Oya, M.; Okita, M.

    1988-01-01

    Radiolabelled ligand binding studies demonstrated that specific receptors for peptide YY are present in the porcine as well as the canine brains. Peptide YY was bound to brain tissue membranes via high-affinity (dissociation constant, 1.39 X 10(-10)M) and low-affinity (dissociation constant, 3.72 X 10(-8)M) components. The binding sites showed a high specificity for peptide YY and neuropeptide Y, but not for pancreatic polypeptide or structurally unrelated peptides. The specific activity of peptide YY binding was highest in the hippocampus, followed by the pituitary gland, the hypothalamus, and the amygdala of the porcine brain, this pattern being similarly observed in the canine brain. The results suggest that peptide YY and neuropeptide Y may regulate the function of these regions of the brain through interaction with a common receptor site

  14. The human endolymphatic sac expresses natriuretic peptides

    DEFF Research Database (Denmark)

    Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas

    2017-01-01

    : Several natriuretic peptides were found expressed significantly in the ES, including uroguanylin and brain natriuretic peptide, but also peptides regulating vascular tone, including adrenomedullin 2. In addition, both neurophysin and oxytocin (OXT) were found significantly expressed. All peptides were...... verified by immunohistochemistry. CONCLUSION: The present data support the hypothesis that the human ES may have an endocrine/paracrine capacity through expression of several peptides with potent natriuretic activity. Furthermore, the ES may influence the hypothalamo-pituitary-adrenal axis and may regulate...... vasopressin receptors and aquaporin-2 channels in the inner ear via OXT expression. We hypothesize that the ES is likely to regulate inner ear endolymphatic homeostasis, possibly through secretion of several peptides, but it may also influence systemic and/or intracranial blood pressure through direct...

  15. Potent peptidic fusion inhibitors of influenza virus

    Energy Technology Data Exchange (ETDEWEB)

    Kadam, Rameshwar U.; Juraszek, Jarek; Brandenburg, Boerries; Buyck, Christophe; Schepens, Wim B. G.; Kesteleyn, Bart; Stoops, Bart; Vreeken, Rob J.; Vermond, Jan; Goutier, Wouter; Tang, Chan; Vogels, Ronald; Friesen, Robert H. E.; Goudsmit, Jaap; van Dongen, Maria J. P.; Wilson, Ian A.

    2017-09-28

    Influenza therapeutics with new targets and mechanisms of action are urgently needed to combat potential pandemics, emerging viruses, and constantly mutating strains in circulation. We report here on the design and structural characterization of potent peptidic inhibitors of influenza hemagglutinin. The peptide design was based on complementarity-determining region loops of human broadly neutralizing antibodies against the hemagglutinin (FI6v3 and CR9114). The optimized peptides exhibit nanomolar affinity and neutralization against influenza A group 1 viruses, including the 2009 H1N1 pandemic and avian H5N1 strains. The peptide inhibitors bind to the highly conserved stem epitope and block the low pH–induced conformational rearrangements associated with membrane fusion. These peptidic compounds and their advantageous biological properties should accelerate the development of new small molecule– and peptide-based therapeutics against influenza virus.

  16. Designing anticancer peptides by constructive machine learning.

    Science.gov (United States)

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

    2018-04-21

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

  17. Use of galerina marginata genes and proteins for peptide production

    Science.gov (United States)

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2018-04-03

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

  18. Use of Galerina marginata genes and proteins for peptide production

    Energy Technology Data Exchange (ETDEWEB)

    Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.; Luo, Hong

    2017-03-21

    The present invention relates to compositions and methods comprising genes and peptides associated with cyclic peptides and cyclic peptide production in mushrooms. In particular, the present invention relates to using genes and proteins from Galerina species encoding peptides specifically relating to amatoxins in addition to proteins involved with processing cyclic peptide toxins. In a preferred embodiment, the present invention also relates to methods for making small peptides and small cyclic peptides including peptides similar to amanitin. Further, the present inventions relate to providing kits for making small peptides.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  20. Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2

    DEFF Research Database (Denmark)

    Blume, N; Madsen, O D; Kofod, Hans

    1990-01-01

    for antibody binding to the immunizing antigen. Antisera to C-peptide 2, stained islet beta-cells on mouse and rat, but not monkey pancreas sections in immunocytochemical analysis. Preabsorption to the synthetic C-peptide 2, but not the synthetic mouse and rat C-peptide 1 abolished staining. In conclusion we......Mice and rats have two functional non-allelic insulin genes. By using a synthetic peptide representing a common sequence in mouse and rat C-peptide 2 as antigen, we have produced rabbit antisera specific for an epitope which is not present in mouse or rat C-peptide 1. Long-term immunization did...... not seem to increase the end point titre as tested in direct ELISA. The specificity of the antiserum was determined by competitive ELISA and histochemistry on pancreas sections. Only the synthetic C-peptide 2, but not the homologous synthetic C-peptide 1 from mouse and rat competed efficiently in ELISA...

  1. Therapeutic peptides for cancer therapy. Part I - peptide inhibitors of signal transduction cascades.

    Science.gov (United States)

    Bidwell, Gene L; Raucher, Drazen

    2009-10-01

    Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that inhibit signal transduction cascades are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Given our current knowledge of protein sequences, structures and interaction interfaces, therapeutic peptides that inhibit interactions of interest are easily designed. These peptides are advantageous because they are highly specific for the interaction of interest, and they are much more easily developed than small molecule inhibitors of the same interactions. The main hurdle to application of peptides for cancer therapy is their poor pharmacokinetic and biodistribution parameters. Therefore, successful development of peptide delivery vectors could potentially make possible the use of this new and very promising class of anticancer agents.

  2. Antimicrobial Peptides, Infections and the Skin Barrier

    DEFF Research Database (Denmark)

    Clausen, Maja Lisa; Agner, Tove

    2016-01-01

    The skin serves as a strong barrier protecting us from invading pathogens and harmful organisms. An important part of this barrier comes from antimicrobial peptides (AMPs), which are small peptides expressed abundantly in the skin. AMPs are produced in the deeper layers of the epidermis and trans......The skin serves as a strong barrier protecting us from invading pathogens and harmful organisms. An important part of this barrier comes from antimicrobial peptides (AMPs), which are small peptides expressed abundantly in the skin. AMPs are produced in the deeper layers of the epidermis...

  3. Nanoformulated cell-penetrating survivin mutant and its dual actions

    Directory of Open Access Journals (Sweden)

    Sriramoju B

    2014-07-01

    Full Text Available Bhasker Sriramoju, Rupinder K Kanwar, Jagat R Kanwar Nanomedicine Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, School of Medicine, Faculty of Health, Deakin University, Geelong, Australia Abstract: In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells, and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. Keywords: survivin mutant, neurological disorders, protein therapeutics, inhibitor of apoptosis protein family, poly(lactic-co-glycolic acid

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  5. Connecting peptide (c-peptide) and the duration of diabetes mellitus ...

    African Journals Online (AJOL)

    Objective: C-peptide is derived from proinsulin and it is secreted in equimolar concentration with insulin. Plasma C-peptide is more stable than insulin and it provides an indirect measure of insulin secretory reserve and beta cell function. To determine relationship between C-peptide and duration of diabetes mellitus, age, ...

  6. Radiometallating antibodies and autoantigenic peptides

    International Nuclear Information System (INIS)

    Mercer-Smith, J.A.; Lewis, D.; Cole, D.A.; Newmyer, S.L.; Schulte, L.D.; Mixon, P.L.; Schreyer, S.A.; Burns, T.P.; Roberts, J.C.; Figard, S.D.; McCormick, D.J.; Lennon, V.A.; Hayashi, M.; Lavallee, D.K.

    1991-01-01

    We have developed methods to radiolabel large molecules, using porphyrins as bifunctional chelating agents for radiometals. The porphyrins are substituted with an N- benzyl group to activate them for radiometallation under mild reaction conditions. Porphyrins that have one functional group for covalent attachment to other molecules cannot cause crosslinking. We have examined the labeling chemistry for antibodies and have developed methods to label smaller biologically active molecules, such as autoantigenic peptides (fragments of the acetylcholine receptor), which are pertinent to myasthenia gravis research. The methods of covalent attachment of these bifunctional chelating agents to large molecules, the radiometallation chemistry, and biological characterization of the radiolabeled compounds will be discussed

  7. Atrial natriuretic peptides in plasma

    DEFF Research Database (Denmark)

    Goetze, Jens Peter; Hansen, Lasse H; Terzic, Dijana

    2014-01-01

    Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone....... These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid...

  8. Atrial natriuretic peptides in plasma

    DEFF Research Database (Denmark)

    Goetze, Jens P; Holst Hansen, Lasse; Terzic, Dijana

    2015-01-01

    Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone....... These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid...

  9. Synthesis of radioiodinated labeled peptides

    International Nuclear Information System (INIS)

    Matloobi, M.; Rafii, H.; Beigi, D.; Khalaj, A.; Kamali-Dehghan, M.

    2003-01-01

    Optimization of radioiodination of peptides is covered by both a direct method in which a constituent tyrosine residue is labeled and indirect method by using an iodinated derivative (SIB) of N succinimidyl 3-(tri-n-butylstannyl) benzoate (ATE) as the intermediate. Radioiodination of IgG and FMLF were performed by direct method using Chloramine-T as an oxidant but since Formyl-Methyl-Leucyl-Phenylalanine, FMLF, does not lend itself for direct radioiodination we performed labeling of FMLF by indirect method via radioiodined SIB at different pH. (author)

  10. A Tat-conjugated Peptide Nucleic Acid Tat-PNA-DR Inhibits Hepatitis B Virus Replication In Vitro and In Vivo by Targeting LTR Direct Repeats of HBV RNA

    Science.gov (United States)

    Zeng, Zhengyang; Han, Shisong; Hong, Wei; Lang, Yange; Li, Fangfang; Liu, Yongxiang; Li, Zeyong; Wu, Yingliang; Li, Wenxin; Zhang, Xianzheng; Cao, Zhijian

    2016-01-01

    Hepatitis B virus (HBV) infection is a major cause of chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma, all of which are severe threats to human health. However, current clinical therapies for HBV are limited by potential side effects, toxicity, and drug-resistance. In this study, a cell-penetrating peptide-conjugated peptide nucleic acid (PNA), Tat-PNA-DR, was designed to target the direct repeat (DR) sequences of HBV. Tat-PNA-DR effectively inhibited HBV replication in HepG2.2.15 cells. Its anti-HBV effect relied on the binding of Tat-PNA-DR to the DR, whereby it suppressed the translation of hepatitis B e antigen (HBeAg), HBsAg, HBV core, hepatitis B virus x protein, and HBV reverse transcriptase (RT) and the reverse transcription of the HBV genome. Furthermore, Tat-PNA-DR administered by intravenous injection efficiently cleared HBeAg and HBsAg in an acute hepatitis B mouse model. Importantly, it induced an 80% decline in HBV DNA in mouse serum, which was similar to the effect of the widely used clinical drug Lamivudine (3TC). Additionally, a long-term hydrodynamics HBV mouse model also demonstrated Tat-PNA-DR's antiviral effect. Interestingly, Tat-PNA-DR displayed low cytotoxicity, low mouse acute toxicity, low immunogenicity, and high serum stability. These data indicate that Tat-PNA-DR is a unique PNA and a promising drug candidate against HBV. PMID:26978579

  11. A Tat-conjugated Peptide Nucleic Acid Tat-PNA-DR Inhibits Hepatitis B Virus Replication In Vitro and In Vivo by Targeting LTR Direct Repeats of HBV RNA

    Directory of Open Access Journals (Sweden)

    Zhengyang Zeng

    2016-01-01

    Full Text Available Hepatitis B virus (HBV infection is a major cause of chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma, all of which are severe threats to human health. However, current clinical therapies for HBV are limited by potential side effects, toxicity, and drug-resistance. In this study, a cell-penetrating peptide-conjugated peptide nucleic acid (PNA, Tat-PNA-DR, was designed to target the direct repeat (DR sequences of HBV. Tat-PNA-DR effectively inhibited HBV replication in HepG2.2.15 cells. Its anti-HBV effect relied on the binding of Tat-PNA-DR to the DR, whereby it suppressed the translation of hepatitis B e antigen (HBeAg, HBsAg, HBV core, hepatitis B virus x protein, and HBV reverse transcriptase (RT and the reverse transcription of the HBV genome. Furthermore, Tat-PNA-DR administered by intravenous injection efficiently cleared HBeAg and HBsAg in an acute hepatitis B mouse model. Importantly, it induced an 80% decline in HBV DNA in mouse serum, which was similar to the effect of the widely used clinical drug Lamivudine (3TC. Additionally, a long-term hydrodynamics HBV mouse model also demonstrated Tat-PNA-DR's antiviral effect. Interestingly, Tat-PNA-DR displayed low cytotoxicity, low mouse acute toxicity, low immunogenicity, and high serum stability. These data indicate that Tat-PNA-DR is a unique PNA and a promising drug candidate against HBV.

  12. peptide

    Indian Academy of Sciences (India)

    Prakash

    effects can be observed under certain conditions but these are not always .... of proteins with amyloid characteristics in muscle (Jayaraman et al. 2008) ... not enhance the growth of dangerous fibrils generated at pH. 7.4. ..... The lower chart shows Aβ(25-35) aggregation kinetics during the first 4 min of monitoring. Results are ...

  13. Peptide hormones and lung cancer.

    Science.gov (United States)

    Moody, T W

    2006-03-01

    Several peptide hormones have been identified which alter the proliferation of lung cancer. Small cell lung cancer (SCLC), which is a neuroendocrine cancer, produces and secretes gastrin releasing peptide (GRP), neurotensin (NT) and adrenomedullin (AM) as autocrine growth factors. GRP, NT and AM bind to G-protein coupled receptors causing phosphatidylinositol turnover or elevated cAMP in SCLC cells. Addition of GRP, NT or AM to SCLC cells causes altered expression of nuclear oncogenes, such as c-fos, and stimulation of growth. Antagonists have been developed for GRP, NT and AM receptors which function as cytostatic agents and inhibit SCLC growth. Growth factor antagonists, such as the NT1 receptor antagonist SR48692, facilitate the ability of chemotherapeutic drugs to kill lung cancer cells. It remains to be determined if GRP, NT and AM receptors will served as molecular targets, for development of new therapies for the treatment of SCLC patients. Non-small cell lung cancer (NSCLC) cells also have a high density of GRP, NT, AM and epidermal growth factor (EGF) receptors. Several NSCLC patients with EGF receptor mutations respond to gefitinib, a tyrosine kinase inhibitor. Gefitinib relieves NSCLC symptoms, maintaining stable disease in patients who are not eligible for systemic chemotherapy. It is important to develop new therapeutic approaches using translational research techniques for the treatment of lung cancer patients.

  14. Synthetic mimics of antimicrobial peptides.

    Science.gov (United States)

    Som, Abhigyan; Vemparala, Satyavani; Ivanov, Ivaylo; Tew, Gregory N

    2008-01-01

    Infectious diseases and antibiotic resistance are now considered the most imperative global healthcare problem. In the search for new treatments, host defense, or antimicrobial, peptides have attracted considerable attention due to their various unique properties; however, attempts to develop in vivo therapies have been severely limited. Efforts to develop synthetic mimics of antimicrobial peptides (SMAMPs) have increased significantly in the last decade, and this review will focus primarily on the structural evolution of SMAMPs and their membrane activity. This review will attempt to make a bridge between the design of SMAMPs and the fundamentals of SMAMP-membrane interactions. In discussions regarding the membrane interaction of SMAMPs, close attention will be paid to the lipid composition of the bilayer. Despite many years of study, the exact conformational aspects responsible for the high selectivity of these AMPs and SMAMPs toward bacterial cells over mammalian cells are still not fully understood. The ability to design SMAMPs that are potently antimicrobial, yet nontoxic to mammalian cells has been demonstrated with a variety of molecular scaffolds. Initial animal studies show very good tissue distribution along with more than a 4-log reduction in bacterial counts. The results on SMAMPs are not only extremely promising for novel antibiotics, but also provide an optimistic picture for the greater challenge of general proteomimetics.

  15. Driving engineering of novel antimicrobial peptides from simulations of peptide-micelle interactions

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Langham, Allison A; Kaznessis, Yiannis N

    2006-01-01

    Simulations of antimicrobial peptides in membrane mimics can provide the high resolution, atomistic picture that is necessary to decipher which sequence and structure components are responsible for activity and toxicity. With such detailed insight, engineering new sequences that are active but non...... peptides and their interaction with membrane mimics. In this article, we discuss the promise and the challenges of widely used models and detail our recent work on peptide-micelle simulations as an attractive alternative to peptide-bilayer simulations. We detail our results with two large structural...... classes of peptides, helical and beta-sheet and demonstrate how simulations can assist in engineering of novel antimicrobials with therapeutic potential....

  16. Peptide-tagged proteins in aqueous two-phase systems

    OpenAIRE

    Nilsson, Anna

    2002-01-01

    This thesis deals with proteins containing peptide tags for improved partitioning in aqueous two-phase systems. Qualitatively the peptide-tagged protein partitioning could be predicted from peptide data, i.e. partitioning trends found for peptides were also found for the peptide-tagged proteins. However, full effect of the tag as expected from peptide partitioning was not found in the tagged protein. When alkyl-ethylene oxide surfactant was included in a two-polymer system, almost full effect...

  17. Topical Peptide Treatments with Effective Anti-Aging Results

    OpenAIRE

    Silke Karin Schagen

    2017-01-01

    In the last two decades, many new peptides have been developed, and new knowledge on how peptides improve the skin has been uncovered. The spectrum of peptides in the field of cosmetics is continuously growing. This review summarizes some of the effective data on cosmeceutical peptides that work against intrinsic and extrinsic aging. Some peptides have been proven in their efficacy through clinical skin trials. Well-known and documented peptides like copper tripeptide are still under research...

  18. Prediction of twin-arginine signal peptides

    DEFF Research Database (Denmark)

    Bendtsen, Jannick Dyrløv; Nielsen, Henrik; Widdick, D.

    2005-01-01

    expressions, whereas hydrophobicity discrimination of Tat- and Sec- signal peptides is carried out by an artificial neural network. A potential cleavage site of the predicted Tat signal peptide is also reported. The TatP prediction server is available as a public web server at http://www.cbs.dtu.dk/services/TatP/....

  19. Double quick, double click reversible peptide "stapling".

    Science.gov (United States)

    Grison, Claire M; Burslem, George M; Miles, Jennifer A; Pilsl, Ludwig K A; Yeo, David J; Imani, Zeynab; Warriner, Stuart L; Webb, Michael E; Wilson, Andrew J

    2017-07-01

    The development of constrained peptides for inhibition of protein-protein interactions is an emerging strategy in chemical biology and drug discovery. This manuscript introduces a versatile, rapid and reversible approach to constrain peptides in a bioactive helical conformation using BID and RNase S peptides as models. Dibromomaleimide is used to constrain BID and RNase S peptide sequence variants bearing cysteine (Cys) or homocysteine ( h Cys) amino acids spaced at i and i + 4 positions by double substitution. The constraint can be readily removed by displacement of the maleimide using excess thiol. This new constraining methodology results in enhanced α-helical conformation (BID and RNase S peptide) as demonstrated by circular dichroism and molecular dynamics simulations, resistance to proteolysis (BID) as demonstrated by trypsin proteolysis experiments and retained or enhanced potency of inhibition for Bcl-2 family protein-protein interactions (BID), or greater capability to restore the hydrolytic activity of the RNAse S protein (RNase S peptide). Finally, use of a dibromomaleimide functionalized with an alkyne permits further divergent functionalization through alkyne-azide cycloaddition chemistry on the constrained peptide with fluorescein, oligoethylene glycol or biotin groups to facilitate biophysical and cellular analyses. Hence this methodology may extend the scope and accessibility of peptide stapling.

  20. Protein identification by peptide mass fingerprinting

    DEFF Research Database (Denmark)

    Hjernø, Karin

    2007-01-01

      Peptide mass fingerprinting is an effective way of identifying, e.g., gel-separated proteins, by matching experimentally obtained peptide mass data against large databases. However, several factors are known to influence the quality of the resulting matches, such as proteins contaminating the s...

  1. Peptide Mass Fingerprinting of Egg White Proteins

    Science.gov (United States)

    Alty, Lisa T.; LaRiviere, Frederick J.

    2016-01-01

    Use of advanced mass spectrometry techniques in the undergraduate setting has burgeoned in the past decade. However, relatively few undergraduate experiments examine the proteomics tools of protein digestion, peptide accurate mass determination, and database searching, also known as peptide mass fingerprinting. In this experiment, biochemistry…

  2. Practical use of natriuretic peptide measurement

    DEFF Research Database (Denmark)

    Husby, Simon; Lind, Bent; Goetze, Jens P

    2012-01-01

    To elucidate the knowledge regarding B-type natriuretic peptide (BNP)/N-terminal proBNP (NT-proBNP) measurement among doctors using this biomarker.......To elucidate the knowledge regarding B-type natriuretic peptide (BNP)/N-terminal proBNP (NT-proBNP) measurement among doctors using this biomarker....

  3. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

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

  4. Superior Antifouling Performance of a Zwitterionic Peptide Compared to an Amphiphilic, Non-Ionic Peptide.

    Science.gov (United States)

    Ye, Huijun; Wang, Libing; Huang, Renliang; Su, Rongxin; Liu, Boshi; Qi, Wei; He, Zhimin

    2015-10-14

    The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.

  5. Peptide ligands for targeting the extracellular domain of EGFR: Comparison between linear and cyclic peptides.

    Science.gov (United States)

    Williams, Tyrslai M; Sable, Rushikesh; Singh, Sitanshu; Vicente, Maria Graca H; Jois, Seetharama D

    2018-02-01

    Colorectal cancer (CRC) is the third most common solid internal malignancy among cancers. Early detection of cancer is key to increasing the survival rate of colorectal cancer patients. Overexpression of the EGFR protein is associated with CRC. We have designed a series of peptides that are highly specific for the extracellular domain of EGFR, based on our earlier studies on linear peptides. The previously reported linear peptide LARLLT, known to bind to EGFR, was modified with the goals of increasing its stability and its specificity toward EGFR. Peptide modifications, including D-amino acid substitution, cyclization, and chain reversal, were investigated. In addition, to facilitate labeling of the peptide with a fluorescent dye, an additional lysine residue was introduced onto the linear (KLARLLT) and cyclic peptides cyclo(KLARLLT) (Cyclo.L1). The lysine residue was also converted into an azide group in both a linear and reversed cyclic peptide sequences cyclo(K(N3)larllt) (Cyclo.L1.1) to allow for subsequent "click" conjugation. The cyclic peptides showed enhanced binding to EGFR by SPR. NMR and molecular modeling studies suggest that the peptides acquire a β-turn structure in solution. In vitro stability studies in human serum show that the cyclic peptide is more stable than the linear peptide. © 2017 John Wiley & Sons A/S.

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

    DEFF Research Database (Denmark)

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

    2002-01-01

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

  7. Tumor-targeting peptides from combinatorial libraries*

    Science.gov (United States)

    Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S.

    2018-01-01

    Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges infighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. PMID:27210583

  8. Development of novel ligands for peptide GPCRs.

    Science.gov (United States)

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

    2016-12-01

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

  9. Circulating elastin peptides, role in vascular pathology.

    Science.gov (United States)

    Robert, L; Labat-Robert, J

    2014-12-01

    The atherosclerotic process starts with the degradation of elastic fibers. Their presence was demonstrated in the circulation as well as several of their biological properties elucidated. We described years ago a procedure to obtain large elastin peptides by organo-alkaline hydrolysis, κ-elastin. This method enabled also the preparation of specific antibodies used to determine elastin peptides, as well as anti-elastin antibodies in body fluids and tissue extracts. Elastin peptides were determined in a large number of human blood samples. Studies were carried out to explore their pharmacological properties. Similar recent studies by other laboratories confirmed our findings and arose new interest in circulating elastin peptides for their biological activities. This recent trend justified the publication of a review of the biological and pathological activities of elastin peptides demonstrated during our previous studies, subject of this article. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  10. Interpreting peptide mass spectra by VEMS

    DEFF Research Database (Denmark)

    Mathiesen, Rune; Lundsgaard, M.; Welinder, Karen G.

    2003-01-01

    the calculated and the experimental mass spectrum of the called peptide. The program package includes four accessory programs. VEMStrans creates protein databases in FASTA format from EST or cDNA sequence files. VEMSdata creates a virtual peptide database from FASTA files. VEMSdist displays the distribution......Most existing Mass Spectra (MS) analysis programs are automatic and provide limited opportunity for editing during the interpretation. Furthermore, they rely entirely on publicly available databases for interpretation. VEMS (Virtual Expert Mass Spectrometrist) is a program for interactive analysis...... of peptide MS/MS spectra imported in text file format. Peaks are annotated, the monoisotopic peaks retained, and the b-and y-ion series identified in an interactive manner. The called peptide sequence is searched against a local protein database for sequence identity and peptide mass. The report compares...

  11. Intracellular Signalling by C-Peptide

    Directory of Open Access Journals (Sweden)

    Claire E. Hills

    2008-01-01

    Full Text Available C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.

  12. Radiolabeled peptides: experimental and clinical applications

    International Nuclear Information System (INIS)

    Thakur, M.L.; Pallela, V.R.

    1998-01-01

    Radiolabeled receptor specific biomolecules hold unlimited potential in nuclear medicine. During the past few years much attention has been drawn to the development radiolabeled peptides for a variety of diagnostic applications, as well as for therapy of malignant tumors. Although only one peptide, In-111-DTPA-(D)-Phe 1 -octreotide, is available commercially for oncologic imaging, many more have been examined in humans with hematological disorders, and the early results appear to be promising. Impetus generated by these results have prompted investigators to label peptides with such radionuclides as Tc-99m, I-123, F-18, Cu-64, and Y-90. This review is intended to highlight the qualities of peptides, summarize the clinical results, and address some important issues associated with radiolabeling of highly potent peptides. While doing so, various methods of radiolabeling have been described, and their strengths and weaknesses have also been discussed. (author)

  13. Chemical reactions directed Peptide self-assembly.

    Science.gov (United States)

    Rasale, Dnyaneshwar B; Das, Apurba K

    2015-05-13

    Fabrication of self-assembled nanostructures is one of the important aspects in nanoscience and nanotechnology. The study of self-assembled soft materials remains an area of interest due to their potential applications in biomedicine. The versatile properties of soft materials can be tuned using a bottom up approach of small molecules. Peptide based self-assembly has significant impact in biology because of its unique features such as biocompatibility, straight peptide chain and the presence of different side chain functionality. These unique features explore peptides in various self-assembly process. In this review, we briefly introduce chemical reaction-mediated peptide self-assembly. Herein, we have emphasised enzymes, native chemical ligation and photochemical reactions in the exploration of peptide self-assembly.

  14. Harnessing supramolecular peptide nanotechnology in biomedical applications.

    Science.gov (United States)

    Chan, Kiat Hwa; Lee, Wei Hao; Zhuo, Shuangmu; Ni, Ming

    2017-01-01

    The harnessing of peptides in biomedical applications is a recent hot topic. This arises mainly from the general biocompatibility of peptides, as well as from the ease of tunability of peptide structure to engineer desired properties. The ease of progression from laboratory testing to clinical trials is evident from the plethora of examples available. In this review, we compare and contrast how three distinct self-assembled peptide nanostructures possess different functions. We have 1) nanofibrils in biomaterials that can interact with cells, 2) nanoparticles that can traverse the bloodstream to deliver its payload and also be bioimaged, and 3) nanotubes that can serve as cross-membrane conduits and as a template for nanowire formation. Through this review, we aim to illustrate how various peptides, in their various self-assembled nanostructures, possess great promise in a wide range of biomedical applications and what more can be expected.

  15. Antimicrobial peptides interact with peptidoglycan

    Science.gov (United States)

    Neelay, Om P.; Peterson, Christian A.; Snavely, Mary E.; Brown, Taylor C.; TecleMariam, Ariam F.; Campbell, Jennifer A.; Blake, Allison M.; Schneider, Sydney C.; Cremeens, Matthew E.

    2017-10-01

    Traditional therapeutics are losing effectiveness as bacterial resistance increases, and antimicrobial peptides (AMPs) can serve as an alternative source for antimicrobial agents. Their mode of action is commonly hypothesized to involve pore formation in the lipid membrane, thereby leading to cell death. However, bacterial cell walls are much more complex than just the lipid membrane. A large portion of the wall is comprised of peptidoglycan, yet we did not find any report of AMP-peptidoglycan interactions. Consequently, this work evaluated AMP-peptidoglycan and AMP-phospholipid (multilamellar vesicles) interactions through tryptophan fluorescence. Given that peptidoglycan is insoluble and vesicles are large particles, we took advantage of the unique properties of Trp-fluorescence to use one technique for two very different systems. Interestingly, melittin and cecropin A interacted with peptidoglycan to a degree similar to vancomycin, a positive control. Whether these AMP-peptidoglycan interactions relate to a killing mode of action requires further study.

  16. Peptide Antibiotics for ESKAPE Pathogens

    DEFF Research Database (Denmark)

    Thomsen, Thomas Thyge

    is considered poor compared to medicines for lifestyle diseases. According to the WHO we could be moving towards a post-antibiotic era in which previously treatable infections become fatal. Of special importance are multidrug resistant bacteria from the ESKAPE group (Enterococcus faecium, Staphylococcus aureus......Multi-drug resistance to antibiotics represents a global health challenge that results in increased morbidity and mortality rates. The annual death-toll is >700.000 people world-wide, rising to ~10 million by 2050. New antibiotics are lacking, and few are under development as return on investment......, Klebsiella pneumoniae, Acinetobacter, Pseudomonas aeruginosa and Enterobacter). As a consequence of widespread multi-drug resistance, researchers have sought for alternative sources of antimicrobials. Antimicrobial peptides are produced by almost all living organisms as part of their defense or innate immune...

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2006-06-01

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

  19. Designing Antibacterial Peptides with Enhanced Killing Kinetics

    Directory of Open Access Journals (Sweden)

    Faiza H. Waghu

    2018-02-01

    Full Text Available Antimicrobial peptides (AMPs are gaining attention as substitutes for antibiotics in order to combat the risk posed by multi-drug resistant pathogens. Several research groups are engaged in design of potent anti-infective agents using natural AMPs as templates. In this study, a library of peptides with high sequence similarity to Myeloid Antimicrobial Peptide (MAP family were screened using popular online prediction algorithms. These peptide variants were designed in a manner to retain the conserved residues within the MAP family. The prediction algorithms were found to effectively classify peptides based on their antimicrobial nature. In order to improve the activity of the identified peptides, molecular dynamics (MD simulations, using bilayer and micellar systems could be used to design and predict effect of residue substitution on membranes of microbial and mammalian cells. The inference from MD simulation studies well corroborated with the wet-lab observations indicating that MD-guided rational design could lead to discovery of potent AMPs. The effect of the residue substitution on membrane activity was studied in greater detail using killing kinetic analysis. Killing kinetics studies on Gram-positive, negative and human erythrocytes indicated that a single residue change has a drastic effect on the potency of AMPs. An interesting outcome was a switch from monophasic to biphasic death rate constant of Staphylococcus aureus due to a single residue mutation in the peptide.

  20. Peptides in fermented Finnish milk products

    Directory of Open Access Journals (Sweden)

    Minna Kahala

    1993-09-01

    Full Text Available This study was conducted to investigate the rate of proteolysis and peptide profiles of different Finnish fermented milk products. The highest rate of proteolysis was observed in Biokefir, while the greatest change in the rate of proteolysis was observed in Gefilus®. Differences in starters and manufacturing processes reflected on the peptide profiles of the products. Most of the identified peptides originated from either the N- or C-terminal region of β-casein or from the N-terminal region of αs1-casein.

  1. Fourier transform infrared spectroscopy of peptides.

    Science.gov (United States)

    Bakshi, Kunal; Liyanage, Mangala R; Volkin, David B; Middaugh, C Russell

    2014-01-01

    Fourier transform infrared (FTIR) spectroscopy provides data that are widely used for secondary structure characterization of peptides. A wide array of available sampling methods permits structural analysis of peptides in diverse environments such as aqueous solution (including optically turbid media), powders, detergent micelles, and lipid bilayers. In some cases, side chain vibrations can also be resolved and used for tertiary structure and chemical analysis. Data from several low-resolution spectroscopic techniques, including FTIR, can be combined to generate an empirical phase diagram, an overall picture of peptide structure as a function of environmental conditions that can aid in the global interpretation of large amounts of spectroscopic data.

  2. Neoglycolipids for Prolonging the Effects of Peptides

    DEFF Research Database (Denmark)

    van Witteloostuijn, Søren Blok; Mannerstedt, Karin Margareta Sophia; Wismann, Pernille

    2017-01-01

    Novel principles for optimizing the properties of peptide-based drugs are needed in order to leverage their full pharmacological potential. We present the design, synthesis, and evaluation of a library of neoglycolipidated glucagon-like peptide 1 (GLP-1) analogues, which are valuable drug...... was maintained or even improved compared to native GLP-1. This translated into pronounced in vivo efficacy in terms of both decreased acute food intake and improved glucose homeostasis in mice. Thus, we propose neoglycolipidation as a novel, general method for modulating the properties of therapeutic peptides...

  3. How Nature Morphs Peptide Scaffolds into Antibiotics

    Science.gov (United States)

    Nolan, Elizabeth M.; Walsh, Christopher T.

    2010-01-01

    The conventional notion that peptides are poor candidates for orally available drugs because of protease-sensitive peptide bonds, intrinsic hydrophilicity, and ionic charges contrasts with the diversity of antibiotic natural products with peptide-based frameworks that are synthesized and utilized by Nature. Several of these antibiotics, including penicillin and vancomycin, are employed to treat bacterial infections in humans and have been best-selling therapeutics for decades. Others might provide new platforms for the design of novel therapeutics to combat emerging antibiotic-resistant bacterial pathogens. PMID:19058272

  4. Brain natriuretic peptide: Diagnostic potential in dogs

    Directory of Open Access Journals (Sweden)

    Spasojević-Kosić Ljubica

    2009-01-01

    Full Text Available The endocrine role of the heart is evident in the secretion of noradrenaline and natriuretic peptides. The secretion of natriuretic peptides presents a useful mechanism for different conditions of cardiac dysfunction. Brain natriuretic peptide (BNP has been accepted in human cardiology as a biomarker for cardiac insufficiency and coronary arterial disease. The specificity of the BNP structure is specie-specific, so that the testing of diagnostic and prognostic potential in dogs requires the existence of a test that is a homologue for that animal specie. The existence of an adequate method for measuring BNP concentration makes possible its implementation as a screening test in everyday clinical practice. .

  5. Recent updates of marine antimicrobial peptides.

    Science.gov (United States)

    Semreen, Mohammad H; El-Gamal, Mohammed I; Abdin, Shifaa; Alkhazraji, Hajar; Kamal, Leena; Hammad, Saba; El-Awady, Faten; Waleed, Dima; Kourbaj, Layal

    2018-03-01

    Antimicrobial peptides are group of proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens. This class of compounds contributed to solving the microbial resistance dilemma that limited the use of many potent antimicrobial agents. The marine environment is known to be one of the richest sources for antimicrobial peptides, yet this environment is not fully explored. Hence, the scientific research attention should be directed toward the marine ecosystem as enormous amount of useful discoveries could be brought to the forefront. In the current article, the marine antimicrobial peptides reported from mid 2012 to 2017 have been reviewed.

  6. Recent updates of marine antimicrobial peptides

    Directory of Open Access Journals (Sweden)

    Mohammad H. Semreen

    2018-03-01

    Full Text Available Antimicrobial peptides are group of proteins showing broad-spectrum antimicrobial activity that have been known to be powerful agents against a variety of pathogens. This class of compounds contributed to solving the microbial resistance dilemma that limited the use of many potent antimicrobial agents. The marine environment is known to be one of the richest sources for antimicrobial peptides, yet this environment is not fully explored. Hence, the scientific research attention should be directed toward the marine ecosystem as enormous amount of useful discoveries could be brought to the forefront. In the current article, the marine antimicrobial peptides reported from mid 2012 to 2017 have been reviewed.

  7. Cysteine-containing peptides having antioxidant properties

    Science.gov (United States)

    Bielicki, John K [Castro Valley, CA

    2008-10-21

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

  8. Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules.

    Science.gov (United States)

    Binkowski, Brock F; Miller, Russell A; Belshaw, Peter J

    2005-07-01

    We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.

  9. Amide I SFG Spectral Line Width Probes the Lipid-Peptide and Peptide-Peptide Interactions at Cell Membrane In Situ and in Real Time.

    Science.gov (United States)

    Zhang, Baixiong; Tan, Junjun; Li, Chuanzhao; Zhang, Jiahui; Ye, Shuji

    2018-06-13

    The balance of lipid-peptide and peptide-peptide interactions at cell membrane is essential to a large variety of cellular processes. In this study, we have experimentally demonstrated for the first time that sum frequency generation vibrational spectroscopy can be used to probe the peptide-peptide and lipid-peptide interactions in cell membrane in situ and in real time by determination of the line width of amide I band of protein backbone. Using a "benchmark" model of α-helical WALP23, it is found that the dominated lipid-peptide interaction causes a narrow line width of the amide I band, whereas the peptide-peptide interaction can markedly broaden the line width. When WALP23 molecules insert into the lipid bilayer, a quite narrow line width of the amide I band is observed because of the lipid-peptide interaction. In contrast, when the peptide lies down on the bilayer surface, the line width of amide I band becomes very broad owing to the peptide-peptide interaction. In terms of the real-time change in the line width, the transition from peptide-peptide interaction to lipid-peptide interaction is monitored during the insertion of WALP23 into 1,2-dipalmitoyl- sn-glycero-3-phospho-(1'- rac-glycerol) (DPPG) lipid bilayer. The dephasing time of a pure α-helical WALP23 in 1-palmitoyl-2-oleoyl- sn-glycero-3-phospho-(1'- rac-glycerol) and DPPG bilayer is determined to be 2.2 and 0.64 ps, respectively. The peptide-peptide interaction can largely accelerate the dephasing time.

  10. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

  11. Biologically Active and Antimicrobial Peptides from Plants

    Science.gov (United States)

    Salas, Carlos E.; Badillo-Corona, Jesus A.; Ramírez-Sotelo, Guadalupe; Oliver-Salvador, Carmen

    2015-01-01

    Bioactive peptides are part of an innate response elicited by most living forms. In plants, they are produced ubiquitously in roots, seeds, flowers, stems, and leaves, highlighting their physiological importance. While most of the bioactive peptides produced in plants possess microbicide properties, there is evidence that they are also involved in cellular signaling. Structurally, there is an overall similarity when comparing them with those derived from animal or insect sources. The biological action of bioactive peptides initiates with the binding to the target membrane followed in most cases by membrane permeabilization and rupture. Here we present an overview of what is currently known about bioactive peptides from plants, focusing on their antimicrobial activity and their role in the plant signaling network and offering perspectives on their potential application. PMID:25815307

  12. Antimicrobial Peptides for Therapeutic Applications: A Review

    Directory of Open Access Journals (Sweden)

    Tsogbadrakh Mishig-Ochir

    2012-10-01

    Full Text Available Antimicrobial peptides (AMPs have been considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and different mechanisms of action compared to conventional antibiotics. Although AMPs possess considerable benefits as new generation antibiotics, their clinical and commercial development still have some limitations, such as potential toxicity, susceptibility to proteases, and high cost of peptide production. In order to overcome those obstacles, extensive efforts have been carried out. For instance, unusual amino acids or peptido-mimetics are introduced to avoid the proteolytic degradation and the design of short peptides retaining antimicrobial activities is proposed as a solution for the cost issue. In this review, we focus on small peptides, especially those with less than twelve amino acids, and provide an overview of the relationships between their three-dimensional structures and antimicrobial activities. The efforts to develop highly active AMPs with shorter sequences are also described.

  13. Charge Transport Phenomena in Peptide Molecular Junctions

    International Nuclear Information System (INIS)

    Luchini, A.; Petricoin, E.F.; Geho, D.H.; Liotta, L.A.; Long, D.P.; Vaisman, I.I.

    2008-01-01

    Inelastic electron tunneling spectroscopy (IETS) is a valuable in situ spectroscopic analysis technique that provides a direct portrait of the electron transport properties of a molecular species. In the past, IETS has been applied to small molecules. Using self-assembled nano electronic junctions, IETS was performed for the first time on a large polypeptide protein peptide in the phosphorylated and native form, yielding interpretable spectra. A reproducible 10-fold shift of the I/V characteristics of the peptide was observed upon phosphorylation. Phosphorylation can be utilized as a site-specific modification to alter peptide structure and thereby influence electron transport in peptide molecular junctions. It is envisioned that kinases and phosphatases may be used to create tunable systems for molecular electronics applications, such as biosensors and memory devices.

  14. Diagnostic value of C-peptide determination

    International Nuclear Information System (INIS)

    Kober, G.; Rainer, O.H.

    1983-01-01

    C-peptide and insulin serum determinations were performed in 94 glucagon-stimulated diabetics and in 15 healthy persons. A minimal increase of 1.5 ng C-peptide/ml serum after glucagon injection (1 mg i.v.) was found to be a useful parameter for the differentiation of insulin dependent and non-insulin dependent diabetics. The maximal response to glucagon occurred during the first 10-minutes after the injection (blood was drawn at 2-minutes intervals). Serum insulin levels and basal C-peptide concentrations were of no value in predicting insulin-dependency. Basal C-peptide levels were significantly different from control in juvenile insulin dependent diabetics (decrease) only. (Author)

  15. Biologically Active and Antimicrobial Peptides from Plants

    Directory of Open Access Journals (Sweden)

    Carlos E. Salas

    2015-01-01

    Full Text Available Bioactive peptides are part of an innate response elicited by most living forms. In plants, they are produced ubiquitously in roots, seeds, flowers, stems, and leaves, highlighting their physiological importance. While most of the bioactive peptides produced in plants possess microbicide properties, there is evidence that they are also involved in cellular signaling. Structurally, there is an overall similarity when comparing them with those derived from animal or insect sources. The biological action of bioactive peptides initiates with the binding to the target membrane followed in most cases by membrane permeabilization and rupture. Here we present an overview of what is currently known about bioactive peptides from plants, focusing on their antimicrobial activity and their role in the plant signaling network and offering perspectives on their potential application.

  16. Atrial natriuretic peptide (ANP)-granules: ultrastructure ...

    African Journals Online (AJOL)

    AJB SERVER

    2006-12-29

    Dec 29, 2006 ... morphometry and function. Eliane Florencio ... granules is greatest in the right atrium followed by the left atrium and left auricle and right auricle, in this order. ... family: Atrial natriuretic peptide (ANP), Urodilatin, Brain natriuretic ...

  17. Taylor Dispersion Analysis as a promising tool for assessment of peptide-peptide interactions

    DEFF Research Database (Denmark)

    Høgstedt, Ulrich B; Schwach, Grégoire; van de Weert, Marco

    2016-01-01

    solutions increased with concentration. Our results indicate that a viscosity difference between run buffer and sample in Taylor Dispersion Analysis may result in overestimation of the measured diffusion coefficient. Thus, Taylor Dispersion Analysis provides a practical, but as yet primarily qualitative......Protein-protein and peptide-peptide (self-)interactions are of key importance in understanding the physiochemical behavior of proteins and peptides in solution. However, due to the small size of peptide molecules, characterization of these interactions is more challenging than for proteins...

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

    Science.gov (United States)

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

    2006-09-01

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

  19. Aggregation and toxicity of amyloid-beta peptide in relation to peptide sequence variation

    OpenAIRE

    Vandersteen, A.

    2012-01-01

    Generally, aggregation of the amyloid-ß peptide is considered the cause of neuronal death in Alzheimer disease. The heterogenous Aß peptide occurs in various lengths in vivo: Aß40 and Aß42 are the predominant forms while both shorter and longer peptides exist. Aß40 and shorter isoforms are less aggregation-prone and hence considered less dangerous than Aß42 and longer isoforms, which are more aggregation-prone. Up to now research mainly focussed on the predominant Aß peptides and their indivi...

  20. A distributive peptide cyclase processes multiple microviridin core peptides within a single polypeptide substrate.

    Science.gov (United States)

    Zhang, Yi; Li, Kunhua; Yang, Guang; McBride, Joshua L; Bruner, Steven D; Ding, Yousong

    2018-05-03

    Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an important family of natural products. Their biosynthesis follows a common scheme in which the leader peptide of a precursor peptide guides the modifications of a single core peptide. Here we describe biochemical studies of the processing of multiple core peptides within a precursor peptide, rare in RiPP biosynthesis. In a cyanobacterial microviridin pathway, an ATP-grasp ligase, AMdnC, installs up to two macrolactones on each of the three core peptides within AMdnA. The enzyme catalysis occurs in a distributive fashion and follows an unstrict N-to-C overall directionality, but a strict order in macrolactonizing each core peptide. Furthermore, AMdnC is catalytically versatile to process unnatural substrates carrying one to four core peptides, and kinetic studies provide insights into its catalytic properties. Collectively, our results reveal a distinct biosynthetic logic of RiPPs, opening up the possibility of modular production via synthetic biology approaches.

  1. The preparation and characterization of peptide's lung cancer imaging agent

    International Nuclear Information System (INIS)

    Liu Jianfeng; Chu Liping; Wang Yan; Wang Yueying; Liu Jinjian; Wu Hongying

    2010-01-01

    Objective: To screen in vivo lung cancer specific binding seven peptides by T7 phage display peptide library, so as to prepare peptide's lung cancer early diagnostic agent. Methods: Use phage display in vivo technology, the 7-peptide phage that binding the lung cancer specifically was obtained, then the DNA sequence was measured and the seven peptide was synthesized. After labeled by 125 I, the seven peptide was injected into mice via vein and the distribution was observed. Results: One peptide was obtained by four rounds screening, and the peptide can bind lung cancer tissue specifically. Two hours after injection get the best imaging of lung cancer, metabolism of peptide in mice is fast, the distribution in vivo is decrease six hours and almost disappear 20 hours after injection. Conclusion: The peptide can image and diagnose lung cancer better. (authors)

  2. Discovery of peptidic anti-­myotoxins

    DEFF Research Database (Denmark)

    Bjärtun, Johanna; Laustsen, Andreas Hougaard; Munk, Andreas

    More than 2.5 millions envenomations and 125.000 death occur each year due to snakebite. Current antivenoms consist of immunoglobulinesderived from animals, and they are therefore associated with a high risk of adverse reactions in humans. The use of synthetic peptidic antitoxinsmay lead to safer...... and more effective antivenoms. This research reports the discovery of peptidic antitoxins against myotoxin II from B. asper....

  3. Preparation of peptide thioesters through fmoc-based solid-phase peptide synthesis by using amino thioesters

    DEFF Research Database (Denmark)

    Stuhr-Hansen, N.; Wilbek, T.S.; Strømgaard, K.

    2013-01-01

    protected peptide thioester, which was globally deprotected to afford the desired unprotected peptide thioester. The method is compatible with labile groups such as phosphoryl and glycosyl moieties. The synthesis of peptide alkyl thioesters by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis...

  4. Membrane manufacture for peptide separations

    KAUST Repository

    Kim, Dooli; Salazar Moya, Octavio Ruben; Nunes, Suzana Pereira

    2016-01-01

    Nanostructured polymeric membranes are key tools in biomedical applications such as hemodialysis, protein separations, in the food industry, and drinking water supply from seawater. Despite of the success in different separation processes, membrane manufacture itself is at risk, since the most used solvents are about to be banned in many countries due to environmental and health issues. We propose for the first time the preparation of polyethersulfone membranes based on dissolution in the ionic liquid 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DEP). We obtained a series of membranes tailored for separation of solutes with molecular weight of 30, 5, 1.3, and 1.25 kg mol-1 with respective water permeances of 140, 65, 30 and 20 Lm-2h-1bar-1. We demonstrate their superior efficiency in the separation of complex mixtures of peptides with molecular weights in the range of 800 to 3500 gmol-1. Furthermore, the thermodynamics and kinetics of phase separation leading to the pore formation in the membranes were investigated. The rheology of the solutions and the morphology of the prepared membranes were examed and compared to those of polyethersulfone in organic solvents currently used for membrane manufacture.

  5. Membrane manufacture for peptide separations

    KAUST Repository

    Kim, Dooli

    2016-06-07

    Nanostructured polymeric membranes are key tools in biomedical applications such as hemodialysis, protein separations, in the food industry, and drinking water supply from seawater. Despite of the success in different separation processes, membrane manufacture itself is at risk, since the most used solvents are about to be banned in many countries due to environmental and health issues. We propose for the first time the preparation of polyethersulfone membranes based on dissolution in the ionic liquid 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DEP). We obtained a series of membranes tailored for separation of solutes with molecular weight of 30, 5, 1.3, and 1.25 kg mol-1 with respective water permeances of 140, 65, 30 and 20 Lm-2h-1bar-1. We demonstrate their superior efficiency in the separation of complex mixtures of peptides with molecular weights in the range of 800 to 3500 gmol-1. Furthermore, the thermodynamics and kinetics of phase separation leading to the pore formation in the membranes were investigated. The rheology of the solutions and the morphology of the prepared membranes were examed and compared to those of polyethersulfone in organic solvents currently used for membrane manufacture.

  6. Chimeric opioid peptides: tools for identifying opioid receptor types.

    OpenAIRE

    Xie, G X; Miyajima, A; Yokota, T; Arai, K; Goldstein, A

    1990-01-01

    We synthesized several chimeric peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the kappa opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surf...

  7. Constraining cyclic peptides to mimic protein structure motifs

    DEFF Research Database (Denmark)

    Hill, Timothy A.; Shepherd, Nicholas E.; Diness, Frederik

    2014-01-01

    peptides can have protein-like biological activities and potencies, enabling their uses as biological probes and leads to therapeutics, diagnostics and vaccines. This Review highlights examples of cyclic peptides that mimic three-dimensional structures of strand, turn or helical segments of peptides...... and proteins, and identifies some additional restraints incorporated into natural product cyclic peptides and synthetic macrocyclic pepti-domimetics that refine peptide structure and confer biological properties....

  8. Confinement-Dependent Friction in Peptide Bundles

    Science.gov (United States)

    Erbaş, Aykut; Netz, Roland R.

    2013-01-01

    Friction within globular proteins or between adhering macromolecules crucially determines the kinetics of protein folding, the formation, and the relaxation of self-assembled molecular systems. One fundamental question is how these friction effects depend on the local environment and in particular on the presence of water. In this model study, we use fully atomistic MD simulations with explicit water to obtain friction forces as a single polyglycine peptide chain is pulled out of a bundle of k adhering parallel polyglycine peptide chains. The whole system is periodically replicated along the peptide axes, so a stationary state at prescribed mean sliding velocity V is achieved. The aggregation number is varied between k = 2 (two peptide chains adhering to each other with plenty of water present at the adhesion sites) and k = 7 (one peptide chain pulled out from a close-packed cylindrical array of six neighboring peptide chains with no water inside the bundle). The friction coefficient per hydrogen bond, extrapolated to the viscous limit of vanishing pulling velocity V → 0, exhibits an increase by five orders of magnitude when going from k = 2 to k = 7. This dramatic confinement-induced friction enhancement we argue to be due to a combination of water depletion and increased hydrogen-bond cooperativity. PMID:23528088

  9. Biomathematical Description of Synthetic Peptide Libraries

    Science.gov (United States)

    Trepel, Martin

    2015-01-01

    Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org), allowing scientists to plan and analyse their peptide libraries. PMID:26042419

  10. Peptides as Therapeutic Agents for Dengue Virus.

    Science.gov (United States)

    Chew, Miaw-Fang; Poh, Keat-Seong; Poh, Chit-Laa

    2017-01-01

    Dengue is an important global threat caused by dengue virus (DENV) that records an estimated 390 million infections annually. Despite the availability of CYD-TDV as a commercial vaccine, its long-term efficacy against all four dengue virus serotypes remains unsatisfactory. There is therefore an urgent need for the development of antiviral drugs for the treatment of dengue. Peptide was once a neglected choice of medical treatment but it has lately regained interest from the pharmaceutical industry following pioneering advancements in technology. In this review, the design of peptide drugs, antiviral activities and mechanisms of peptides and peptidomimetics (modified peptides) action against dengue virus are discussed. The development of peptides as inhibitors for viral entry, replication and translation is also described, with a focus on the three main targets, namely, the host cell receptors, viral structural proteins and viral non-structural proteins. The antiviral peptides designed based on these approaches may lead to the discovery of novel anti-DENV therapeutics that can treat dengue patients.

  11. Peptide pheromone signaling in Streptococcus and Enterococcus

    Science.gov (United States)

    Cook, Laura C.; Federle, Michael J.

    2014-01-01

    Intercellular chemical signaling in bacteria, commonly referred to as quorum sensing (QS), relies on the production and detection of compounds known as pheromones to elicit coordinated responses among members of a community. Pheromones produced by Gram-positive bacteria are comprised of small peptides. Based on both peptide structure and sensory system architectures, Gram-positive bacterial signaling pathways may be classified into one of four groups with a defining hallmark: cyclical peptides of the Agr type, peptides that contain Gly-Gly processing motifs, sensory systems of the RNPP family, or the recently characterized Rgg-like regulatory family. The recent discovery that Rgg family members respond to peptide pheromones increases substantially the number of species in which QS is likely a key regulatory component. These pathways control a variety of fundamental behaviors including conjugation, natural competence for transformation, biofilm development, and virulence factor regulation. Overlapping QS pathways found in multiple species and pathways that utilize conserved peptide pheromones provide opportunities for interspecies communication. Here we review pheromone signaling identified in the genera Enterococcus and Streptococcus, providing examples of all four types of pathways. PMID:24118108

  12. Human C-peptide. Pt. 1

    International Nuclear Information System (INIS)

    Beischer, W.; Keller, L.; Maas, M.; Schiefer, E.; Pfeiffer, E.F.

    1976-01-01

    Synthetic human C-peptide bearing a tyrosine group at its amino end is labelled with 125 iodine using chloramin T or hydrogen peroxide and lactoperoxidase. The results of the two methods are compared. Antiserum to synthetic human C-peptide (without tyrosine), which was partially coupled to rabbit albumin, is raised in guinea pigs and goats. Goats show to be superior to guinea pips concerning antibody production. The so-called 'hook effect' phenomenon is observed when setting up the standard curves for the radioimmunoassay. Monotonically decreasing standard curves are obtained on dilution of antiserum with a high antibody titer which was produced by repeated immunization in goats. Free C-peptide and C-peptide bound to antiserum are separated using the anion exchange resin amberlite. Using this separation technique we excluded unspecific binding of labelled C-peptide to protein fractions in serum of diabetics. The sensitivity of our radioimmunoassay is approx. 0.3 ng C-peptide/ml serum. Intra- and interassay variability are below 10%. Human proinsulin is the only substance found to crossreact with the antiserum. (orig.) [de

  13. Harnessing supramolecular peptide nanotechnology in biomedical applications

    Directory of Open Access Journals (Sweden)

    Chan KH

    2017-02-01

    Full Text Available Kiat Hwa Chan,1 Wei Hao Lee,2 Shuangmu Zhuo,3 Ming Ni3 1Division of Science, Yale-NUS College, Singapore; 2Department of Chemistry, Krieger School of Arts & Sciences, Johns Hopkins University, Baltimore, MD, USA; 3Fujian Provincial Key Laboratory for Photonics Technology, Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Normal University, Fuzhou, People’s Republic of China Abstract: The harnessing of peptides in biomedical applications is a recent hot topic. This arises mainly from the general biocompatibility of peptides, as well as from the ease of tunability of peptide structure to engineer desired properties. The ease of progression from laboratory testing to clinical trials is evident from the plethora of examples available. In this review, we compare and contrast how three distinct self-assembled peptide nanostructures possess different functions. We have 1 nanofibrils in biomaterials that can interact with cells, 2 nanoparticles that can traverse the bloodstream to deliver its payload and also be bioimaged, and 3 nanotubes that can serve as cross-membrane conduits and as a template for nanowire formation. Through this review, we aim to illustrate how various peptides, in their various self-assembled nanostructures, possess great promise in a wide range of biomedical applications and what more can be expected. Keywords: peptides, self-assembly, nanotechnology

  14. [Peptide phage display in biotechnology and biomedicine].

    Science.gov (United States)

    Kuzmicheva, G A; Belyavskaya, V A

    2016-07-01

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

  15. Peptides with Dual Antimicrobial and Anticancer Activities

    Science.gov (United States)

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

    2017-02-01

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

  16. Comprehensive computational design of ordered peptide macrocycles

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-12-14

    Mixed chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to-date, but there is currently no way to systematically search through the structural space spanned by such compounds for new drug candidates. Natural proteins do not provide a useful guide: peptide macrocycles lack regular secondary structures and hydrophobic cores and have different backbone torsional constraints. Hence the development of new peptide macrocycles has been approached by modifying natural products or using library selection methods; the former is limited by the small number of known structures, and the latter by the limited size and diversity accessible through library-based methods. To overcome these limitations, here we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L and D amino acids. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. We synthesize and characterize by NMR twelve 7-10 residue macrocycles, 9 of which have structures very close to the design models in solution. NMR structures of three 11-14 residue bicyclic designs are also very close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide based macrocycles unparalleled for other molecular systems, and vastly increase the available starting scaffolds for both rational drug design and library selection methods.

  17. Human C-peptide. Pt. 1. Radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Beischer, W; Keller, L; Maas, M; Schiefer, E; Pfeiffer, E F [Ulm Univ. (Germany, F.R.). Abt. Innere Medizin, Endokrinologie und Stoffwechsel

    1976-08-01

    Synthetic human C-peptide bearing a tyrosine group at its amino end is labelled with /sup 125/iodine using chloramin T or hydrogen peroxide and lactoperoxidase. The results of the two methods are compared. Antiserum to synthetic human C-peptide (without tyrosine), which was partially coupled to rabbit albumin, is raised in guinea pigs and goats. Goats show to be superior to guinea pips concerning antibody production. The so-called 'hook effect' phenomenon is observed when setting up the standard curves for the radioimmunoassay. Monotonically decreasing standard curves are obtained on dilution of antiserum with a high antibody titer which was produced by repeated immunization in goats. Free C-peptide and C-peptide bound to antiserum are separated using the anion exchange resin amberlite. Using this separation technique we excluded unspecific binding of labelled C-peptide to protein fractions in serum of diabetics. The sensitivity of our radioimmunoassay is approx. 0.3 ng C-peptide/ml serum. Intra- and interassay variability are below 10%. Human proinsulin is the only substance found to crossreact with the antiserum.

  18. A cocoa peptide protects Caenorhabditis elegans from oxidative stress and β-amyloid peptide toxicity.

    Directory of Open Access Journals (Sweden)

    Patricia Martorell

    Full Text Available BACKGROUND: Cocoa and cocoa-based products contain different compounds with beneficial properties for human health. Polyphenols are the most frequently studied, and display antioxidant properties. Moreover, protein content is a very interesting source of antioxidant bioactive peptides, which can be used therapeutically for the prevention of age-related diseases. METHODOLOGY/PRINCIPAL FINDINGS: A bioactive peptide, 13L (DNYDNSAGKWWVT, was obtained from a hydrolyzed cocoa by-product by chromatography. The in vitro inhibition of prolyl endopeptidase (PEP was used as screening method to select the suitable fraction for peptide identification. Functional analysis of 13L peptide was achieved using the transgenic Caenorhabditis elegans strain CL4176 expressing the human Aβ₁₋₄₂ peptide as a pre-clinical in vivo model for Alzheimer's disease. Among the peptides isolated, peptide 13L (1 µg/mL showed the highest antioxidant activity (P≤0.001 in the wild-type strain (N2. Furthermore, 13L produced a significant delay in body paralysis in strain CL4176, especially in the 24-47 h period after Aβ₁₋₄₂ peptide induction (P≤0.0001. This observation is in accordance with the reduction of Aβ deposits in CL4176 by western blot. Finally, transcriptomic analysis in wild-type nematodes treated with 13L revealed modulation of the proteosomal and synaptic functions as the main metabolic targets of the peptide. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the cocoa 13L peptide has antioxidant activity and may reduce Aβ deposition in a C. elegans model of Alzheimer's disease; and therefore has a putative therapeutic potential for prevention of age-related diseases. Further studies in murine models and humans will be essential to analyze the effectiveness of the 13L peptide in higher animals.

  19. Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    Science.gov (United States)

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

  20. Topical ocular delivery to laser-induced choroidal neovascularization by dual internalizing RGD and TAT peptide-modified nanoparticles

    Directory of Open Access Journals (Sweden)

    Chu YC

    2017-02-01

     > primary NPs. iRGD and TAT dual-modified NPs thus displayed significant targeting and penetration ability both in vitro and in vivo, indicating that it is a promising drug delivery system for managing CNV via topical ocular administration. Keywords: nanoparticles, ocular drug delivery, choroidal neovascularization, RGD, cell-penetrating peptides

  1. Protein interaction networks by proteome peptide scanning.

    Directory of Open Access Journals (Sweden)

    Christiane Landgraf

    2004-01-01

    Full Text Available A substantial proportion of protein interactions relies on small domains binding to short peptides in the partner proteins. Many of these interactions are relatively low affinity and transient, and they impact on signal transduction. However, neither the number of potential interactions mediated by each domain nor the degree of promiscuity at a whole proteome level has been investigated. We have used a combination of phage display and SPOT synthesis to discover all the peptides in the yeast proteome that have the potential to bind to eight SH3 domains. We first identified the peptides that match a relaxed consensus, as deduced from peptides selected by phage display experiments. Next, we synthesized all the matching peptides at high density on a cellulose membrane, and we probed them directly with the SH3 domains. The domains that we have studied were grouped by this approach into five classes with partially overlapping specificity. Within the classes, however, the domains display a high promiscuity and bind to a large number of common targets with comparable affinity. We estimate that the yeast proteome contains as few as six peptides that bind to the Abp1 SH3 domain with a dissociation constant lower than 100 microM, while it contains as many as 50-80 peptides with corresponding affinity for the SH3 domain of Yfr024c. All the targets of the Abp1 SH3 domain, identified by this approach, bind to the native protein in vivo, as shown by coimmunoprecipitation experiments. Finally, we demonstrate that this strategy can be extended to the analysis of the entire human proteome. We have developed an approach, named WISE (whole interactome scanning experiment, that permits rapid and reliable identification of the partners of any peptide recognition module by peptide scanning of a proteome. Since the SPOT synthesis approach is semiquantitative and provides an approximation of the dissociation constants of the several thousands of interactions that are

  2. Interaction of antimicrobial peptides with lipid membranes

    Energy Technology Data Exchange (ETDEWEB)

    Hanulova, Maria

    2008-12-15

    This study aims to investigate the difference in the interaction of antimicrobial peptides with two classes of zwitterionic peptides, phosphatidylethanolamines (PE) and phosphatidylcholines (PC). Further experiments were performed on model membranes prepared from specific bacterial lipids, lipopolysaccharides (LPS) isolated from Salmonella minnesota. The structure of the lipid-peptide aqueous dispersions was studied by small-and wide-angle X-ray diffraction during heating and cooling from 5 to 85 C. The lipids and peptides were mixed at lipid-to-peptide ratios 10-10000 (POPE and POPC) or 2-50 (LPS). All experiments were performed at synchrotron soft condensed matter beamline A2 in Hasylab at Desy in Hamburg, Germany. The phases were identified and the lattice parameters were calculated. Alamethicin and melittin interact in similar ways with the lipids. Pure POPC forms only lamellar phases. POPE forms lamellar phases at low temperatures that upon heating transform into a highly curved inverse hexagonal phase. Insertion of the peptide induced inverse bicontinuous cubic phases which are an ideal compromise between the curvature stress and the packing frustration. Melittin usually induced a mixture of two cubic phases, Im3m and Pn3m, with a ratio of lattice parameters close to 1.279, related to the underlying minimal surfaces. They formed during the lamellar to hexagonal phase transition and persisted during cooling till the onset of the gel phase. The phases formed at different lipid-to-peptide ratios had very similar lattice parameters. Epitaxial relationships existed between coexisting cubic phases and hexagonal or lamellar phases due to confinement of all phases to an onion vesicle, a vesicle with several layers consisting of different lipid phases. Alamethicin induced the same cubic phases, although their formation and lattice parameters were dependent on the peptide concentration. The cubic phases formed during heating from the lamellar phase and their onset

  3. Interaction of antimicrobial peptides with lipid membranes

    International Nuclear Information System (INIS)

    Hanulova, Maria

    2008-12-01

    This study aims to investigate the difference in the interaction of antimicrobial peptides with two classes of zwitterionic peptides, phosphatidylethanolamines (PE) and phosphatidylcholines (PC). Further experiments were performed on model membranes prepared from specific bacterial lipids, lipopolysaccharides (LPS) isolated from Salmonella minnesota. The structure of the lipid-peptide aqueous dispersions was studied by small-and wide-angle X-ray diffraction during heating and cooling from 5 to 85 C. The lipids and peptides were mixed at lipid-to-peptide ratios 10-10000 (POPE and POPC) or 2-50 (LPS). All experiments were performed at synchrotron soft condensed matter beamline A2 in Hasylab at Desy in Hamburg, Germany. The phases were identified and the lattice parameters were calculated. Alamethicin and melittin interact in similar ways with the lipids. Pure POPC forms only lamellar phases. POPE forms lamellar phases at low temperatures that upon heating transform into a highly curved inverse hexagonal phase. Insertion of the peptide induced inverse bicontinuous cubic phases which are an ideal compromise between the curvature stress and the packing frustration. Melittin usually induced a mixture of two cubic phases, Im3m and Pn3m, with a ratio of lattice parameters close to 1.279, related to the underlying minimal surfaces. They formed during the lamellar to hexagonal phase transition and persisted during cooling till the onset of the gel phase. The phases formed at different lipid-to-peptide ratios had very similar lattice parameters. Epitaxial relationships existed between coexisting cubic phases and hexagonal or lamellar phases due to confinement of all phases to an onion vesicle, a vesicle with several layers consisting of different lipid phases. Alamethicin induced the same cubic phases, although their formation and lattice parameters were dependent on the peptide concentration. The cubic phases formed during heating from the lamellar phase and their onset

  4. Growth hormone-releasing peptides.

    Science.gov (United States)

    Ghigo, E; Arvat, E; Muccioli, G; Camanni, F

    1997-05-01

    Growth hormone-releasing peptides (GHRPs) are synthetic, non-natural peptides endowed with potent stimulatory effects on somatotrope secretion in animals and humans. They have no structural homology with GHRH and act via specific receptors present either at the pituitary or the hypothalamic level both in animals and in humans. The GHRP receptor has recently been cloned and, interestingly, it does not show sequence homology with other G-protein-coupled receptors known so far. This evidence strongly suggests the existence of a natural GHRP-like ligand which, however, has not yet been found. The mechanisms underlying the GHRP effect are still unclear. At present, several data favor the hypothesis that GHRPs could act by counteracting somatostatinergic activity both at the pituitary and the hypothalamic level and/or, at least partially, via a GHRH-mediated mechanism. However, the possibility that GHRPs act via an unknown hypothalamic factor (U factor) is still open. GHRP-6 was the first hexapeptide to be extensively studied in humans. More recently, a heptapeptide, GHRP-1, and two other hexapeptides, GHRP-2 and Hexarelin, have been synthesized and are now available for human studies. Moreover, non-peptidyl GHRP mimetics have been developed which act via GHRP receptors and their effects have been clearly demonstrated in animals and in humans in vivo. Among non-peptidyl GHRPs, MK-0677 seems the most interesting molecule. The GH-releasing activity of GHRPs is marked and dose-related after intravenous, subcutaneous, intranasal and even oral administration. The effect of GHRPs is reproducible and undergoes partial desensitization, more during continuous infusion, less during intermittent administration: in fact, prolonged administration of GHRPs increases IGF-1 levels both in animals and in humans. The GH-releasing effect of GHRPs does not depend on sex but undergoes age-related variations. It increases from birth to puberty, persists at a similar level in adulthood and

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

    KAUST Repository

    Rydberg, Hanna A

    2014-04-18

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

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

    KAUST Repository

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

    2014-01-01

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

  7. Urodilatin. A renal natriuretic peptide

    International Nuclear Information System (INIS)

    Carstens, Jan

    1998-01-01

    Development and validation of a radioimmunoassay for endogenous URO in urine and synthetic URO in plasma is described. The first obstacle to overcome was to produce an antibody specific for URO. A polyclonal URO antibody with a cross-reactivity with the structural highly homologous atrial natriuretic peptide (ANP) was developed by immunization of rabbits with the whole URO(95-126). Purification of the polyclonal URO antiserum with CNBr-activated Sepharose affinity chromatography was a simple way of producing a URO-specific antibody without cross-reactivity with ANP analogues. A reliable 125 I-labelled URO tracer was made with the Iodo-Gen method. Prior to the assay, the urine samples were prepared by ethanol with a recovery of unlabelled URO between 80 - 100% and the plasma samples were Sep-Pak C 18 extracted with a recovery of about 50%. The radioimmunoassay is performed in 3 days, using polyethylene glycol for separation. The sensitivity of the assay was improved by sample preparation and concentration, reducing the amount of tracer and late addition, reducing the amount of antibody and increasing the incubation time and lowering the temperature of incubation. The infusion rate of 20 ng URO kg -1 min -1 was most potential and well tolerated in healthy subjects. The short-term natriuretic and diuretic effects were closely associated with a significant diminished sodium reabsorption in the distal nephron. Further studies are needed to exploit the therapeutical potential of URO, for example in patients with sodium-water retaining disorders. The therapeutical dose range will probably be narrow due to the blood pressure lowering effect of URO with infusion rates higher than 20-30 ng kg -1 min -1 . (EHS)

  8. Human antimicrobial peptides and cancer.

    Science.gov (United States)

    Jin, Ge; Weinberg, Aaron

    2018-05-30

    Antimicrobial peptides (AMPs) have long been a topic of interest for entomologists, biologists, immunologists and clinicians because of these agents' intriguing origins in insects, their ubiquitous expression in many life forms, their capacity to kill a wide range of bacteria, fungi and viruses, their role in innate immunity as microbicidal and immunoregulatory agents that orchestrate cross-talk with the adaptive immune system, and, most recently, their association with cancer. We and others have theorized that surveillance through epithelial cell-derived AMPs functions to keep the natural flora of microorganisms in a steady state in different niches such as the skin, the intestines, and the mouth. More recently, findings related to specific activation pathways of some of these AMPs have led investigators to associate them with pro-tumoral activity; i.e., contributing to a tumorigenic microenvironment. This area is still in its infancy as there are intriguing yet contradictory findings demonstrating that while some AMPs have anti-tumoral activity and are under-expressed in solid tumors, others are overexpressed and pro-tumorigenic. This review will introduce a new paradigm in cancer biology as it relates to AMP activity in neoplasia to address the following questions: Is there evidence that AMPs contribute to tumor promoting microenvironments? Can an anti-AMP strategy be of use in cancer therapy? Do AMPs, expressed in and released from tumors, contribute to compositional shifting of bacteria in cancerous lesions? Can specific AMP expression characteristics be used one day as early warning signs for solid tumors? Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. THE USE OF DEDICATED PEPTIDE LIBRARIES PERMITS THE DISCOVERY OF HIGH-AFFINITY BINDING PEPTIDES

    NARCIS (Netherlands)

    DEKOSTER, HS; AMONS, R; BENCKHUIJSEN, WE; FEIJLBRIEF, M; SCHELLEKENS, GA; DRIJFHOUT, JW

    1995-01-01

    The motif for peptide binding to monoclonal antibody mAb A16, which is known to be directed against glycoprotein D of Herpes simplex virus type 1, was determined using two dedicated peptide libraries. As a starting point for this study we used an A-16 binding lead sequence, which had previously been

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

    DEFF Research Database (Denmark)

    Singhal, Abhishek; Bagnacani, Valentina; Corradini, Roberto

    2014-01-01

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

  11. Peptide-MHC class I stability is a stronger predictor of CTL immunogenicity than peptide affinity

    DEFF Research Database (Denmark)

    Harndahl, Mikkel Nors; Rasmussen, Michael; Nielsen, Morten

    2012-01-01

    Peptide-MHC class I stability is a stronger predictor of CTL immunogenicity than peptide affinity Mikkel Harndahla, Michael Rasmussena, Morten Nielsenb, Soren Buusa,∗ a Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Denmark b Center for Biological Seq...... al., 2007. J. Immunol. 178, 7890–7901. doi:10.1016/j.molimm.2012.02.025...

  12. New dendrimer - peptide host - guest complexes : towards dendrimers as peptide carriers

    NARCIS (Netherlands)

    Boas, U.; Sontjens, S.H.M.; Jensen, K.J.; Christensen, J.B.; Meijer, E.W.

    2002-01-01

    Adamantyl urea and adamantyl thiourea modified poly(propylene imine) dendrimers act as hosts for N-terminal tert-butoxycarbonyl (Boc)-protected peptides and form chloroform-soluble complexes. investigations with NMR spectroscopy show that the peptide is bound to the dendrimer by ionic interactions

  13. The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles.

    Directory of Open Access Journals (Sweden)

    Dimitris Missirlis

    Full Text Available BACKGROUND: Peptide amphiphiles (PAs are a class of amphiphilic molecules able to self-assemble into nanomaterials that have shown efficient in vivo targeted delivery. Understanding the interactions of PAs with cells and the mechanisms of their internalization and intracellular trafficking is critical in their further development for therapeutic delivery applications. METHODOLOGY/PRINCIPAL FINDINGS: PAs of a novel, cell- and tissue-penetrating peptide were synthesized possessing two different lipophilic tail architectures and their interactions with prostate cancer cells were studied in vitro. Cell uptake of peptides was greatly enhanced post-modification. Internalization occurred via lipid-raft mediated endocytosis and was common for the two analogs studied. On the contrary, we identified the non-peptidic part as the determining factor of differences between intracellular trafficking and retention of PAs. PAs composed of di-stearyl lipid tails linked through poly(ethylene glycol to the peptide exhibited higher exocytosis rates and employed different recycling pathways compared to ones consisting of di-palmitic-coupled peptides. As a result, cell association of the former PAs decreased with time. CONCLUSIONS/SIGNIFICANCE: Control over peptide intracellular localization and retention is possible by appropriate modification with synthetic hydrophobic tails. We propose this as a strategy to design improved peptide-based delivery systems.

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  15. A microbially derived tyrosine-sulfated peptide mimics a plant peptide hormone.

    Science.gov (United States)

    Pruitt, Rory N; Joe, Anna; Zhang, Weiguo; Feng, Wei; Stewart, Valley; Schwessinger, Benjamin; Dinneny, José R; Ronald, Pamela C

    2017-07-01

    The biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces a sulfated peptide named RaxX, which shares similarity to peptides in the PSY (plant peptide containing sulfated tyrosine) family. We hypothesize that RaxX mimics the growth-stimulating activity of PSY peptides. Root length was measured in Arabidopsis and rice treated with synthetic RaxX peptides. We also used comparative genomic analyses and reactive oxygen species burst assays to evaluate the activity of RaxX and PSY peptides. Here we found that a synthetic sulfated RaxX derivative comprising 13 residues (RaxX13-sY), highly conserved between RaxX and PSY, induces root growth in Arabidopsis and rice in a manner similar to that triggered by PSY. We identified residues that are required for activation of immunity mediated by the rice XA21 receptor but that are not essential for root growth induced by PSY. Finally, we showed that a Xanthomonas strain lacking raxX is impaired in virulence. These findings suggest that RaxX serves as a molecular mimic of PSY peptides to facilitate Xoo infection and that XA21 has evolved the ability to recognize and respond specifically to the microbial form of the peptide. © 2017 UT-Battelle LLC. New Phytologist © 2017 New Phytologist Trust.

  16. Peptide and Peptide-Dependent Motions in MHC Proteins: Immunological Implications and Biophysical Underpinnings

    Directory of Open Access Journals (Sweden)

    Cory M. Ayres

    2017-08-01

    Full Text Available Structural biology of peptides presented by class I and class II MHC proteins has transformed immunology, impacting our understanding of fundamental immune mechanisms and allowing researchers to rationalize immunogenicity and design novel vaccines. However, proteins are not static structures as often inferred from crystallographic structures. Their components move and breathe individually and collectively over a range of timescales. Peptides bound within MHC peptide-binding grooves are no exception and their motions have been shown to impact recognition by T cell and other receptors in ways that influence function. Furthermore, peptides tune the motions of MHC proteins themselves, which impacts recognition of peptide/MHC complexes by other proteins. Here, we review the motional properties of peptides in MHC binding grooves and discuss how peptide properties can influence MHC motions. We briefly review theoretical concepts about protein motion and highlight key data that illustrate immunological consequences. We focus primarily on class I systems due to greater availability of data, but segue into class II systems as the concepts and consequences overlap. We suggest that characterization of the dynamic “energy landscapes” of peptide/MHC complexes and the resulting functional consequences is one of the next frontiers in structural immunology.

  17. Primary structure and conformational analysis of peptide methionine-tyrosine, a peptide related to neuropeptide Y and peptide YY isolated from lamprey intestine

    DEFF Research Database (Denmark)

    Conlon, J M; Bjørnholm, B; Jørgensen, Flemming Steen

    1991-01-01

    A peptide belonging to the pancreatic-polypeptide-fold family of regulatory peptides has been isolated from the intestine of an Agnathan, the sea lamprey (Petromyzon marinus). The primary structure of the peptide (termed peptide methionine-tyrosine) was established as Met-Pro-Pro-Lys-Pro-Asp-Asn-...... in a preferred structure in which the conformation of the beta-turn between the two helical domains (residues 9-14) is appreciably different....

  18. Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.

    Science.gov (United States)

    Bell, Thomas J; Eberwine, James

    2015-01-01

    RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data.

  19. Antimicrobial peptides design by evolutionary multiobjective optimization.

    Directory of Open Access Journals (Sweden)

    Giuseppe Maccari

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

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides...... (n=40), systemic lupus erythematosus (n=20), Sjögren's syndrome (n=40)) were screened for antibody reactivity. Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide......, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative...

  1. Acetone-Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling.

    Science.gov (United States)

    Assem, Naila; Ferreira, David J; Wolan, Dennis W; Dawson, Philip E

    2015-07-20

    Macrocyclization is a broadly applied approach for overcoming the intrinsically disordered nature of linear peptides. Herein, it is shown that dichloroacetone (DCA) enhances helical secondary structures when introduced between peptide nucleophiles, such as thiols, to yield an acetone-linked bridge (ACE). Aside from stabilizing helical structures, the ketone moiety embedded in the linker can be modified with diverse molecular tags by oxime ligation. Insights into the structure of the tether were obtained through co-crystallization of a constrained S-peptide in complex with RNAse S. The scope of the acetone-linked peptides was further explored through the generation of N-terminus to side chain macrocycles and a new approach for generating fused macrocycles (bicycles). Together, these studies suggest that acetone linking is generally applicable to peptide macrocycles with a specific utility in the synthesis of stabilized helices that incorporate functional tags. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Chimeric opioid peptides: Tools for identifying opioid receptor types

    International Nuclear Information System (INIS)

    Xie, G.; Miyajima, A.; Yokota, T.; Arai, K.; Goldstein, A.

    1990-01-01

    The authors synthesized several chimeric [125J-labelled] peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the κ opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating μ, δ, and κ opioid receptors and for identifying opioid receptors on transfected cells in expression cloning procedures. The general approach using chimeric peptides should be applicable to other peptide receptors

  3. Urinary Peptide Levels in Patients with Chronic Renal Failure

    Directory of Open Access Journals (Sweden)

    Mungli Prakash

    2010-10-01

    Full Text Available Introduction: Peptide levels in urine are found to be decreased in renal failure. In the current study urinary peptide levels were determined in chronic renal failure (CRF patients. Method: 86 CRF patients and 80 healthy controls were selected for the study. Urinary proteins and peptide levels were determined by spectrophotometer based Lowry and Bradford methods. Urinary creatinine levels were determined by clinical chemistry analyzer. Results: There was significant decrease in urinary peptide levels in CRF patients and Urinary % peptides were significantly decreased in CRF patients as compared to healthy controls. Urinary % peptides correlated negatively with proteinuria. Conclusion: we have found decrease in urinary peptides and % urinary peptides in CRF patients and possibly measurement of % urinary peptides may possibly serve as better indicator in early detection of impairment in renal function.

  4. Evaluation of MAP-specific peptides following vaccination of goats

    DEFF Research Database (Denmark)

    Lybeck, Kari; Sjurseth, Siri K.; Melvang, Heidi Mikkelsen

    species or 2) selected based on “experience”. Peptides predicted to bind bovine MHC II by in silico analysis were included in further studies, resulting in two panels 1) genome-based and 2) selected. Initially, two groups of 15 healthy goats were vaccinated with one of the two panels (50 µg/peptide in CAF......01 adjuvant/CAF04 for boosting). Four MAP-infected goats were also vaccinated. In a second vaccination trail, groups of 8 healthy goat kids were vaccinated with genome-based peptides, selected peptides or selected peptides linked together in a recombinant protein (20 µg/peptide or 50 µg protein...... peptides. IFN-γ responses in healthy goats after the first vaccination were low, but testing of T cell lines from MAP-infected goats identified peptides inducing strong proliferative responses. Peptides for a second vaccination were selected by combining results from this study with a parallel cattle study...

  5. Peptide pool immunization and CD8+ T cell reactivity

    DEFF Research Database (Denmark)

    Rasmussen, Susanne B; Harndahl, Mikkel N; Buus, Anette Stryhn

    2013-01-01

    Mice were immunized twice with a pool of five peptides selected among twenty 8-9-mer peptides for their ability to form stable complexes at 37°C with recombinant H-2K(b) (half-lives 10-15h). Vaccine-induced immunity of splenic CD8(+) T cells was studied in a 24h IFNγ Elispot assay. Surprisingly...... peptides induced normal peptide immunity i.e. the specific T cell reactivity in the Elispot culture was strictly dependent on exposure to the immunizing peptide ex vivo. However, immunization with two of the peptides, a VSV- and a Mycobacterium-derived peptide, resulted in IFNγ spot formation without...... peptide in the Elispot culture. Immunization with a mixture of the VSV-peptide and a "normal" peptide also resulted in IFNγ spot formation without addition of peptide to the assay culture. Peptide-tetramer staining of CD8(+) T cells from mice immunized with a mixture of VSV-peptide and "normal" peptide...

  6. Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils.

    Directory of Open Access Journals (Sweden)

    Heike L Rittner

    2009-04-01

    Full Text Available In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR and/or toll like receptor (TLR agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively. Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim

  7. Peptide inhibition of human cytomegalovirus infection

    Directory of Open Access Journals (Sweden)

    Morris Cindy A

    2011-02-01

    Full Text Available Abstract Background Human cytomegalovirus (HCMV is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB, a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection. Results Using the Wimley-White Interfacial Hydrophobicity Scale (WWIHS, several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF were infected with the Towne-GFP strain of HCMV (0.5 MOI, preincubated with peptides at a range of concentrations (78 nm to 100 μM, and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 μM, and 51% and 62% inhibition at concentrations of 5 μM and 2.5 μM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100

  8. Comprehensive computational design of ordered peptide macrocycles

    Science.gov (United States)

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

    2018-01-01

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

  9. Folding very short peptides using molecular dynamics.

    Directory of Open Access Journals (Sweden)

    Bosco K Ho

    2006-04-01

    Full Text Available Peptides often have conformational preferences. We simulated 133 peptide 8-mer fragments from six different proteins, sampled by replica-exchange molecular dynamics using Amber7 with a GB/SA (generalized-Born/solvent-accessible electrostatic approximation to water implicit solvent. We found that 85 of the peptides have no preferred structure, while 48 of them converge to a preferred structure. In 85% of the converged cases (41 peptides, the structures found by the simulations bear some resemblance to their native structures, based on a coarse-grained backbone description. In particular, all seven of the beta hairpins in the native structures contain a fragment in the turn that is highly structured. In the eight cases where the bioinformatics-based I-sites library picks out native-like structures, the present simulations are largely in agreement. Such physics-based modeling may be useful for identifying early nuclei in folding kinetics and for assisting in protein-structure prediction methods that utilize the assembly of peptide fragments.

  10. New peptides players in metabolic disorders

    Directory of Open Access Journals (Sweden)

    Agata Mierzwicka

    2016-08-01

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

  11. Encoded libraries of chemically modified peptides.

    Science.gov (United States)

    Heinis, Christian; Winter, Greg

    2015-06-01

    The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries. Copyright © 2015. Published by Elsevier Ltd.

  12. Dinosaur peptides suggest mechanisms of protein survival.

    Science.gov (United States)

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

    2011-01-01

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

  13. Guanylin peptides: cyclic GMP signaling mechanisms

    Directory of Open Access Journals (Sweden)

    Forte L.R.

    1999-01-01

    Full Text Available Guanylate cyclases (GC serve in two different signaling pathways involving cytosolic and membrane enzymes. Membrane GCs are receptors for guanylin and atriopeptin peptides, two families of cGMP-regulating peptides. Three subclasses of guanylin peptides contain one intramolecular disulfide (lymphoguanylin, two disulfides (guanylin and uroguanylin and three disulfides (E. coli stable toxin, ST. The peptides activate membrane receptor-GCs and regulate intestinal Cl- and HCO3- secretion via cGMP in target enterocytes. Uroguanylin and ST also elicit diuretic and natriuretic responses in the kidney. GC-C is an intestinal receptor-GC for guanylin and uroguanylin, but GC-C may not be involved in renal cGMP pathways. A novel receptor-GC expressed in the opossum kidney (OK-GC has been identified by molecular cloning. OK-GC cDNAs encode receptor-GCs in renal tubules that are activated by guanylins. Lymphoguanylin is highly expressed in the kidney and heart where it may influence cGMP pathways. Guanylin and uroguanylin are highly expressed in intestinal mucosa to regulate intestinal salt and water transport via paracrine actions on GC-C. Uroguanylin and guanylin are also secreted from intestinal mucosa into plasma where uroguanylin serves as an intestinal natriuretic hormone to influence body Na+ homeostasis by endocrine mechanisms. Thus, guanylin peptides control salt and water transport in the kidney and intestine mediated by cGMP via membrane receptors with intrinsic guanylate cyclase activity.

  14. Dinosaur Peptides Suggest Mechanisms of Protein Survival

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-16

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

  15. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    F counterselection was developed to directly select for compounds able to disrupt selected interactions. We have subsequently constructed a cyclic peptide library for intracellular synthesis of cyclic peptides using known technology. Several cyclic peptides were able to interfere with oligomerization of Dna......N (), DnaB and DnaX (). Three peptides identified as inhibitors of DnaN have been purified. Two of these peptides inhibited growth as well as DNA replication in S. aureus. The minimal inhibitory concentration (MIC) of the peptides was approximately 50 g/ml. Overexpression of DnaN reduced the inhibitory...

  16. Insulin and C-peptide in human brain neurons (insulin/C-peptide/brain peptides/immunohistochemistry/radioimmunoassay)

    International Nuclear Information System (INIS)

    Dorn, A.; Bernstein, H.G.; Rinne, A.; Hahn, H.J.; Ziegler, M.

    1983-01-01

    The regional distribution and cellular localization of insulin and C-peptide immunoreactivities were studied in human cadaver brains using the indirect immunofluorescence method, the peroxidase-antiperoxidase technique, and radioimmunoassay. Products of the immune reactions to both polypeptides were observed in most nerve cells in all areas of the brain examined. Immunostaining was mainly restricted to the cell soma and proximal dendrites. Radioimmunoassay revealed that human brain contains insulin and C-peptide in concentrations much higher than the blood, the highest being in the hypothalamus. These findings support the hypothesis that the 'brain insulin' is - at least in part - produced in the CNS. (author)

  17. Antimicrobial Peptides: Multifunctional Drugs for Different Applications

    Directory of Open Access Journals (Sweden)

    Lea-Jessica Albrecht

    2012-02-01

    Full Text Available Antimicrobial peptides (APs are an important part of the innate immune system in epithelial and non-epithelial surfaces. So far, many different antimicrobial peptides from various families have been discovered in non-vertebrates and vertebrates. They are characterized by antibiotic, antifungal and antiviral activities against a variety of microorganisms. In addition to their role as endogenous antimicrobials, APs participate in multiple aspects of immunity. They are involved in septic and non-septic inflammation, wound repair, angiogenesis, regulation of the adaptive immune system and in maintaining homeostasis. Due to those characteristics AP could play an important role in many practical applications. Limited therapeutic efficiency of current antimicrobial agents and the emerging resistance of pathogens require alternate antimicrobial drugs. The purpose of this review is to highlight recent literature on functions and mechanisms of APs. It also shows their current practical applications as peptide therapeutics and bioactive polymers and discusses the possibilities of future clinical developments.

  18. Metal Ion Controlled Polymorphism of a Peptide

    DEFF Research Database (Denmark)

    Hemmingsen, Lars Bo Stegeager; Jancso, Attila; Szunyogh, Daniel

    2011-01-01

    ions on fully or partially unstructured proteins, or the effect of metal ions on protein aggregation. Metal ions may be employed to fold (or misfold) individual peptides in a controlled manner depending on the potential metal ion coordinating amino acid side chains (Cys, His, Asp, Glu......In this work a metal ion binding model dodecapeptide was investigated in terms of its capacity to adopt different structures depending on the metal ion to peptide stoichiometry. The dodecapeptide is much simpler than real proteins, yet displays sufficient complexity to model the effect of metal......, …) in the peptide, and the ligand and structural preferences of the metal ion (in our studies Zn2+, Cd2+, Hg2+, Cu+/2+). Simultaneously, new species such as metal ion bridged ternary complexes or even oligomers may be formed. In recent previous studies we have observed similar polymorphism of zinc finger model...

  19. Radiolabelled RGD peptides for imaging and therapy

    Energy Technology Data Exchange (ETDEWEB)

    Gaertner, F.C.; Schwaiger, M.; Beer, A.J. [Technische Universitaet Muenchen, Department of Nuclear Medicine, Klinikum rechts der Isar, Munich (Germany); Kessler, H. [Technische Universitaet Muenchen, Institute for Advanced Study and Center of Integrated Protein Science, Department of Chemistry, Garching (Germany); King Abdulaziz University, Chemistry Department, Faculty of Science, Jeddah (Saudi Arabia); Wester, H.-J. [Institute for Pharmaceutical Radiochemistry, Garching (Germany)

    2012-02-15

    Imaging of angiogenesis has become increasingly important with the rising use of targeted antiangiogenic therapies like bevacizumab (Avastin). Non-invasive assessment of angiogenic activity is in this respect interesting, e.g. for response assessment of such targeted antiangiogenic therapies. One promising approach of angiogenesis imaging is imaging of specific molecular markers of the angiogenic cascade like the integrin {alpha}{sub v}{beta}{sub 3}. For molecular imaging of integrin expression, the use of radiolabelled peptides is still the only approach that has been successfully translated into the clinic. In this review we will summarize the current data on imaging of {alpha}{sub v}{beta}{sub 3} expression using radiolabelled RGD peptides with a focus on tracers already in clinical use. A perspective will be presented on the future clinical use of radiolabelled RGD peptides including an outlook on potential applications for radionuclide therapy. (orig.)

  20. Peptide Based Radiopharmaceuticals: Specific Construct Approach

    Energy Technology Data Exchange (ETDEWEB)

    Som, P; Rhodes, B A; Sharma, S S

    1997-10-21

    The objective of this project was to develop receptor based peptides for diagnostic imaging and therapy. A series of peptides related to cell adhesion molecules (CAM) and immune regulation were designed for radiolabeling with 99mTc and evaluated in animal models as potential diagnostic imaging agents for various disease conditions such as thrombus (clot), acute kidney failure, and inflection/inflammation imaging. The peptides for this project were designed by the industrial partner, Palatin Technologies, (formerly Rhomed, Inc.) using various peptide design approaches including a newly developed rational computer assisted drug design (CADD) approach termed MIDAS (Metal ion Induced Distinctive Array of Structures). In this approach, the biological function domain and the 99mTc complexing domain are fused together so that structurally these domains are indistinguishable. This approach allows construction of conformationally rigid metallo-peptide molecules (similar to cyclic peptides) that are metabolically stable in-vivo. All the newly designed peptides were screened in various in vitro receptor binding and functional assays to identify a lead compound. The lead compounds were formulated in a one-step 99mTc labeling kit form which were studied by BNL for detailed in-vivo imaging using various animals models of human disease. Two main peptides usingMIDAS approach evolved and were investigated: RGD peptide for acute renal failure and an immunomodulatory peptide derived from tuftsin (RMT-1) for infection/inflammation imaging. Various RGD based metallopeptides were designed, synthesized and assayed for their efficacy in inhibiting ADP-induced human platelet aggregation. Most of these peptides displayed biological activity in the 1-100 µM range. Based on previous work by others, RGD-I and RGD-II were evaluated in animal models of acute renal failure. These earlier studies showed that after acute ischemic injury the renal cortex displays

  1. Sacubitril/valsartan: beyond natriuretic peptides.

    Science.gov (United States)

    Singh, Jagdeep S S; Burrell, Louise M; Cherif, Myriam; Squire, Iain B; Clark, Andrew L; Lang, Chim C

    2017-10-01

    Natriuretic peptides, especially B-type natriuretic peptide (BNP), have primarily been regarded as biomarkers in heart failure (HF). However, they are also possible therapeutic agents due to their potentially beneficial physiological effects. The angiotensin receptor-neprilysin inhibitor, sacubitril/valsartan, simultaneously augments the natriuretic peptide system (NPS) by inhibiting the enzyme neprilysin (NEP) and inhibits the renin-angiotensin-aldosterone system (RAAS) by blocking the angiotensin II receptor. It has been shown to improve mortality and hospitalisation outcomes in patients with HF due to left ventricular systolic dysfunction. The key advantage of sacubitril/valsartan has been perceived to be its ability to augment BNP, while its other effects have largely been overlooked. This review highlights the important effects of sacubitril/valsartan, beyond just the augmentation of BNP. First we discuss how NPS physiology differs between healthy individuals and those with HF by looking at mechanisms like the overwhelming effects of RAAS on the NPS, natriuretic peptide receptor desensitisation and absolute natriuretic deficiency. Second, this review explores other hormones that are augmented by sacubitril/valsartan such as bradykinin, substance P and adrenomedullin that may contribute to the efficacy of sacubitril/valsartan in HF. We also discuss concerns that sacubitril/valsartan may interfere with amyloid-β homeostasis with potential implications on Alzheimer's disease and macular degeneration. Finally, we explore the concept of 'autoinhibition' which is a recently described observation that humans have innate NEP inhibitory capability when natriuretic peptide levels rise above a threshold. There is speculation that autoinhibition may provide a surge of natriuretic and other vasoactive peptides to rapidly reverse decompensation. We contend that by pre-emptively inhibiting NEP, sacubitril/valsartan is inducing this surge earlier during decompensation

  2. Development of peptide and protein based radiopharmaceuticals.

    Science.gov (United States)

    Wynendaele, Evelien; Bracke, Nathalie; Stalmans, Sofie; De Spiegeleer, Bart

    2014-01-01

    Radiolabelled peptides and proteins have recently gained great interest as theranostics, due to their numerous and considerable advantages over small (organic) molecules. Developmental procedures of these radiolabelled biomolecules start with the radiolabelling process, greatly defined by the amino acid composition of the molecule and the radionuclide used. Depending on the radionuclide selection, radiolabelling starting materials are whether or not essential for efficient radiolabelling, resulting in direct or indirect radioiodination, radiometal-chelate coupling, indirect radiofluorination or (3)H/(14)C-labelling. Before preclinical investigations are performed, quality control analyses of the synthesized radiopharmaceutical are recommended to eliminate false positive or negative functionality results, e.g. changed receptor binding properties due to (radiolabelled) impurities. Therefore, radionuclidic, radiochemical and chemical purity are investigated, next to the general peptide attributes as described in the European and the United States Pharmacopeia. Moreover, in vitro and in vivo stability characteristics of the peptides and proteins also need to be explored, seen their strong sensitivity to proteinases and peptidases, together with radiolysis and trans-chelation phenomena of the radiopharmaceuticals. In vitro biomedical characterization of the radiolabelled peptides and proteins is performed by saturation, kinetic and competition binding assays, analyzing KD, Bmax, kon, koff and internalization properties, taking into account the chemical and metabolic stability and adsorption events inherent to peptides and proteins. In vivo biodistribution can be adapted by linker, chelate or radionuclide modifications, minimizing normal tissue (e.g. kidney and liver) radiation, and resulting in favorable dosimetry analyses. Finally, clinical trials are initiated, eventually leading to the marketing of radiolabelled peptides and proteins for PET/SPECT-imaging and therapy

  3. Peptoid-Peptide hybrid backbone architectures

    DEFF Research Database (Denmark)

    Olsen, Christian Adam

    2010-01-01

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

  4. Nuclear oncology with monoclonal antibodies and peptides

    International Nuclear Information System (INIS)

    Hosono, Makoto

    1998-01-01

    Imaging and therapy using radiolabeled monoclonal antibodies have proved useful in many clinical studies. However, immunogenicity of mouse antibodies to human and insufficient tumor-to-normal tissue ratios remained to be solved. Chimerization and humanization by genetic engineering, and multistep targeting techniques have enabled lower immunogenicity and higher tumor-to-normal tissue contrast. Peptides like somatostatin-analogs have been reportedly useful in imaging tumors, which are either somatostatin receptor positive or negative. Elevated normal tissue accumulation of radiolabeled peptides is a drawback in aiming internal radiation therapy. (author). 51 refs

  5. Peptide receptor radionuclide therapy of neuroendocrine tumours

    International Nuclear Information System (INIS)

    Bodei, L.; Giammarile, F.

    2009-01-01

    Neuroendocrine tumours are considered relatively rare tumours that have the characteristic property of secreting bioactive substances, such as amines and hormones. They constitute a heterogeneous group, characterized by good prognosis, but important disparities of the evolutionary potential. In the aggressive forms, the therapeutic strategies are limited. The metabolic or internal radiotherapy, using radiolabelled peptides, which can act at the same time on the primary tumour and its metastases, constitutes a tempting therapeutic alternative, currently in evolution. The prospects are related to the development of new radiopharmaceuticals, with the use of other peptide analogues whose applications will overflow the framework of the neuro-endocrine tumours. (authors)

  6. Antimicrobial peptides in innate immune responses

    DEFF Research Database (Denmark)

    Sorensen, O.E.; Borregaard, N.; Cole, A.M.

    2008-01-01

    Antimicrobial peptides (AMPs) are ancient effector molecules in the innate immune response of eukaryotes. These peptides are important for the antimicrobial efficacy of phagocytes and for the innate immune response mounted by epithelia of humans and other mammals. AMPs are generated either by de...... novo synthesis or by proteolytic cleavage from antimicrobially inactive proproteins. Studies of human diseases and animal studies have given important clues to the in vivo role of AMPs. It is now evident that dysregulation of the generation of AMPs in innate immune responses plays a role in certain...

  7. Radiometallating antibodies and biologically active peptides

    International Nuclear Information System (INIS)

    Mercer-Smith, J.A.; Roberts, J.C.; Lewis, D.; Newmyer, S.L.; Schulte, L.D.; Burns, T.P.; Mixon, P.L.; Jeffery, A.L.; Schreyer, S.A.; Cole, D.A.; Figard, S.D.; Lennon, V.A.; Hayashi, M.; Lavallee, D.K.

    1990-01-01

    We have developed methods to radiolabel large molecules, using porphyrins as bifunctional chelating agents for radiometals. The porphyrins are substituted with an N-benzyl group to activate them for radiometallation under mild reaction conditions. Porphyrins that have on functional group for covalent attachment to other molecules cannot cause crosslinking. We have examined the labeling chemistry for antibodies, and we have also developed methods to label smaller biologically active molecules, such as autoantigenic peptides. The autoantigenic peptides, fragments of the acetylcholine receptor, are under investigation for myasthenia gravis research. The methods of covalent attachment of these bifunctional chelating agents to large molecules and the radiometallation chemistry will be discussed

  8. Novel Zn2+-chelating peptides selected from a fimbria-displayed random peptide library

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Schembri, Mark; Klemm, Per

    2001-01-01

    The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces by virtue of the Fim......H adhesin. FimH is a component of the fimbrial organelle that can accommodate and display a diverse range of peptide sequences on the E. coli cell surface. In this study we have constructed a random peptide library in FimH. The library, consisting of similar to 40 million individual clones, was screened...

  9. Facilitation of peptide fibre formation by arginine-phosphate ...

    Indian Academy of Sciences (India)

    WINTEC

    Peptide; self-assembly; arginine; microscopy. ... The latter property, in particular, observed in .... this process repeated till the gummy compound be- ..... micrograph of Congo red-stained image of individual peptide fibre from aged solution of 4.

  10. Post-staining electroblotting for efficient and reliable peptide blotting.

    Science.gov (United States)

    Lee, Der-Yen; Chang, Geen-Dong

    2015-01-01

    Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

  11. Effect of the renal natriuretic peptide, ularitide, alone or combined ...

    African Journals Online (AJOL)

    Effect of the renal natriuretic peptide, ularitide, alone or combined with ... inhibitor, Omapatrilat, on experimental volume overloadinduced congestive heart failure in ... N-terminal pro–brain natriuretic peptide (NT-proBNP) and high-sensitivity ...

  12. Microwave heating in peptide side chain modification via cysteine alkylation.

    Science.gov (United States)

    Calce, Enrica; De Luca, Stefania

    2016-09-01

    Microwave irradiation has been successfully applied to a selective synthetic procedure for introducing molecular substituents on peptides, providing a noticeable reduction of the reaction time and also an increased crude peptide purity for some compounds.

  13. Constructing bioactive peptides with pH-dependent activities.

    Science.gov (United States)

    Tu, Zhigang; Volk, Melanie; Shah, Khushali; Clerkin, Kevin; Liang, Jun F

    2009-08-01

    Many bioactive peptides are featured by their arginine and lysine rich contents. In this study, lysine and arginine residues in lytic peptides were selectively replaced by histidines. Although resulting histidine-containing lytic peptides had decreased activity, they did show pH-dependent cytotoxicity. The activity of the constructed histidine-containing lytic peptides increased 2-8 times as the solution pH changed from 7.4 to 5.5. More importantly, these histidine-containing peptides maintain the same cell killing mechanism as their parent peptides by causing cell lysis. Both the activity and pH-sensitivity of histidine-containing peptides are tunable by adjusting histidine substitution numbers and positions. This study has presented a general strategy to create bioactive peptides with desired pH-sensitivity to meet the needs of various applications such as cancer treatments.

  14. B-type natriuretic peptide secretion following scuba diving

    DEFF Research Database (Denmark)

    Passino, Claudio; Franzino, Enrico; Giannoni, Alberto

    2011-01-01

    To examine the neurohormonal effects of a scuba dive, focusing on the acute changes in the plasma concentrations of the different peptide fragments from the B-type natriuretic peptide (BNP) precursor....

  15. Screening And Optimizing Antimicrobial Peptides By Using SPOT-Synthesis

    Science.gov (United States)

    López-Pérez, Paula M.; Grimsey, Elizabeth; Bourne, Luc; Mikut, Ralf; Hilpert, Kai

    2017-04-01

    Peptide arrays on cellulose are a powerful tool to investigate peptide interactions with a number of different molecules, for examples antibodies, receptors or enzymes. Such peptide arrays can also be used to study interactions with whole cells. In this review, we focus on the interaction of small antimicrobial peptides with bacteria. Antimicrobial peptides (AMPs) can kill multidrug-resistant (MDR) human pathogenic bacteria and therefore could be next generation antibiotics targeting MDR bacteria. We describe the screen and the result of different optimization strategies of peptides cleaved from the membrane. In addition, screening of antibacterial activity of peptides that are tethered to the surface is discussed. Surface-active peptides can be used to protect surfaces from bacterial infections, for example implants.

  16. Kidney protection during peptide receptor radionuclide therapy with somatostatin analogues.

    NARCIS (Netherlands)

    Rolleman, E.J.; Melis, M.; Valkema, R.; Boerman, O.C.; Krenning, E.P.; Jong, M. de

    2010-01-01

    This review focuses on the present status of kidney protection during peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues. This treatment modality for somatostatin receptor-positive tumours is limited by renal reabsorption and retention of radiolabelled peptides

  17. Topical Peptide Treatments with Effective Anti-Aging Results

    Directory of Open Access Journals (Sweden)

    Silke Karin Schagen

    2017-05-01

    Full Text Available In the last two decades, many new peptides have been developed, and new knowledge on how peptides improve the skin has been uncovered. The spectrum of peptides in the field of cosmetics is continuously growing. This review summarizes some of the effective data on cosmeceutical peptides that work against intrinsic and extrinsic aging. Some peptides have been proven in their efficacy through clinical skin trials. Well-known and documented peptides like copper tripeptide are still under research to obtain more details on their effectiveness, and for the development of new treatments. Palmitoyl pentapeptide-4 and Carnosine are other well-researched cosmeceuticals. Additionally, there are many more peptides that are used in cosmetics. However, study results for some are sparse, or have not been published in scientific journals. This article summarizes topical peptides with proven efficacy in controlled in vivo studies.

  18. Co-factor activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  19. Activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  20. TfR Binding Peptide Screened by Phage Display Technology ...

    African Journals Online (AJOL)

    Purpose: To screen an hTfR affinity peptide and investigate its activity in vitro. Methods: hTfR ... Keywords: Peptide, hTfR, Transferrin receptor, Phage display technology, Enhanced green ..... mediated uptake of peptides that bind the human.

  1. Recent progress in fluorine-18 labelled peptide radiopharmaceuticals

    Energy Technology Data Exchange (ETDEWEB)

    Okarvi, S.M. [Cyclotron and Radiopharmaceuticals Department, King Faisal Specialist Hospital and Research Centre, Riyadh (Saudi Arabia)

    2001-07-01

    The application of biologically active peptides labelled with positron-emitting nuclides has emerged as a useful and interesting field in nuclear medicine. Small synthetic receptor-binding peptides are currently the preferred agents over proteins and antibodies for diagnostic imaging of various tumours. Due to the smaller size of peptides, both higher target-to-background ratios and rapid blood clearance can often be achieved with radiolabelled peptides. Hence, short-lived positron emission tomography (PET) isotopes are potential candidates for labelling peptides. Among a number of positron-emitting nuclides, fluorine-18 appears to be the best candidate for labelling bioactive peptides by virtue of its favourable physical and nuclear characteristics. The major disadvantage of labelling peptides with {sup 18}F is the laborious and time-consuming preparation of the {sup 18}F labelling agents. In recent years, various techniques have been developed which allow efficient labelling of peptides with {sup 18}F without affecting their receptor-binding properties. Moreover, the development of a variety of prosthetic groups has facilitated the efficient and site-specific labelling of peptides with {sup 18}F. The {sup 18}F-labelled peptides hold enormous clinical potential owing to their ability to quantitatively detect and characterise a wide variety of human diseases when using PET. Recently, a number of {sup 18}F-labelled bioactive peptides have shown great promise as diagnostic imaging agents. This review presents the recent developments in {sup 18}F-labelled biologically active peptides used in PET. (orig.)

  2. Facilitating protein solubility by use of peptide extensions

    Science.gov (United States)

    Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

    2013-09-17

    Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

  3. Identification of binding peptides of the ADAM15 disintegrin domain ...

    Indian Academy of Sciences (India)

    Madhsudhan

    ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of ...

  4. the natriuretic peptides: an expanding role in clinical medicine

    African Journals Online (AJOL)

    Enrique

    body's defence against hypertension and plasma volume expansion.2 ... brain natriuretic peptide (B-type), secreted by the ventricle, and C-type peptide, ... Natriuretic peptides, on the other hand, are also stimulated in left ventricular dys- .... tions and in healthy controls as a com- .... stretching of the right ventricle causes.

  5. Bioactive Peptides in Milk Products. | Tirelli | Journal of Food ...

    African Journals Online (AJOL)

    Some peptides produced in vitro or in vivo by enzymatic hydrolysis of caseins and whey protein can affect some biological functions of the body and therefore they are called bioactive peptides. In this paper the physiological significance of bioactive peptides is reviewed and the analytical methods for their purification and ...

  6. Chamber-dependent circadian expression of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Gøtze, Jens Peter; Georg, Birgitte; Jørgensen, Henrik L

    2010-01-01

    Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) have important local functions within the myocardium, where they protect against accelerated fibrosis. As circadian expression of cardiac natriuretic peptides could be of importance in local cardiac protection against disease, we...

  7. Cleaving Double-Stranded DNA with Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1997-01-01

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

  8. Recent progress in fluorine-18 labelled peptide radiopharmaceuticals

    International Nuclear Information System (INIS)

    Okarvi, S.M.

    2001-01-01

    The application of biologically active peptides labelled with positron-emitting nuclides has emerged as a useful and interesting field in nuclear medicine. Small synthetic receptor-binding peptides are currently the preferred agents over proteins and antibodies for diagnostic imaging of various tumours. Due to the smaller size of peptides, both higher target-to-background ratios and rapid blood clearance can often be achieved with radiolabelled peptides. Hence, short-lived positron emission tomography (PET) isotopes are potential candidates for labelling peptides. Among a number of positron-emitting nuclides, fluorine-18 appears to be the best candidate for labelling bioactive peptides by virtue of its favourable physical and nuclear characteristics. The major disadvantage of labelling peptides with 18 F is the laborious and time-consuming preparation of the 18 F labelling agents. In recent years, various techniques have been developed which allow efficient labelling of peptides with 18 F without affecting their receptor-binding properties. Moreover, the development of a variety of prosthetic groups has facilitated the efficient and site-specific labelling of peptides with 18 F. The 18 F-labelled peptides hold enormous clinical potential owing to their ability to quantitatively detect and characterise a wide variety of human diseases when using PET. Recently, a number of 18 F-labelled bioactive peptides have shown great promise as diagnostic imaging agents. This review presents the recent developments in 18 F-labelled biologically active peptides used in PET. (orig.)

  9. Aggregation properties of a short peptide that mediates amyloid fibril ...

    Indian Academy of Sciences (India)

    Short peptides have been identified from amyloidogenic proteins that form amyloid fibrils in isolation. The ... proteins. These peptide fibrils have the conformational features of β-structure that .... water and immediately deposited on freshly cleaved surface of mica .... with the peptide via electrostatic interactions. NaCl would.

  10. RECENT ADVANCES TOWARDS THE RATIONAL DESIGN OF PEPTIDE DRUGS

    OpenAIRE

    YEŞİLADA, Akgül; ÖZKANLI, Fügen

    2004-01-01

    In this review, after a short introduction to definition and physiological roles of regulatory peptides, problems faced during the development of peptide drugs, studies directed to solve these problems and rational design of peptide drugs with special emphesis on peptidomimetics are mentioned

  11. Peptide modification in T cell immunology - from molecule to animal

    NARCIS (Netherlands)

    Haan, Ellen Christine de

    2003-01-01

    Chemical knowledge can be applied in the field of immunology. It provides a better understanding of how a peptide interacts with proteins and cells of the immune system. However, it is not possible to predict the outcome of peptide administration in an animal. Peptides are used in experimental

  12. Peptides and proteins in dendritic assemblies

    NARCIS (Netherlands)

    Baal, van I.

    2007-01-01

    Multiple, simultaneous interactions are often used in biology to enhance the affinity and specificity of binding, an effect referred to as multivalency. This multivalency can be mimicked by anchoring multiple peptides and proteins onto synthetic dendritic scaffolds. The aim of this research was to

  13. Peptide oligomers for holographic data storage

    DEFF Research Database (Denmark)

    Berg, Rolf Henrik; Hvilsted, Søren; Ramanujam, P.S.

    1996-01-01

    SEVERAL classes of organic materials (such as photoanisotropic liquid-crystalline polymers(1-4) and photorefractive polymers(5-7)) are being investigated for the development of media for optical data storage. Here we describe a new family of organic materials-peptide oligomers containing azobenzene...

  14. Hormone action. Part I. Peptide hormones

    International Nuclear Information System (INIS)

    Birnbaumer, L.; O'Malley, B.W.

    1985-01-01

    The major sections of this book on the hormonal action of peptide hormones cover receptor assays, identification of receptor proteins, methods for identification of internalized hormones and hormone receptors, preparation of hormonally responsive cells and cell hybrids, purification of membrane receptors and related techniques, assays of hormonal effects and related functions, and antibodies in hormone action

  15. Fingerprinting desmosine-containing elastin peptides

    DEFF Research Database (Denmark)

    Schräder, Christoph U; Heinz, Andrea; Majovsky, Petra

    2015-01-01

    , and DES-/IDES-containing peptides to determine characteristic product ions. It was found that all investigated compounds yielded the same product ion clusters at elevated collision energies. Elemental composition determination using the exact masses of these ions revealed molecular formulas of the type Cx...

  16. Isolated Gramicidin Peptides Probed by IR Spectroscopy

    NARCIS (Netherlands)

    Rijs, A. M.; Kabelac, M.; Abo-Riziq, A.; Hobza, P.; de Vries, M. S.

    2011-01-01

    We report double-resonant IR/UV ion-dip spectroscopy of neutral gramicidin peptides in the gas phase. The IR spectra of gramicidin A and C, recorded in both the 1000 cm(-1) to 1800 cm(-1) and the 2700 to 3750 cm(-1) region, allow structural analysis. By studying this broad IR range, various local

  17. Peptide Hormones in the Gastrointestinal Tract

    DEFF Research Database (Denmark)

    Rehfeld, Jens F.

    2015-01-01

    Gastrointestinal hormones are peptides released from endocrine cells and neurons in the digestive tract. More than 30 hormone genes are currently known to be expressed in the gastrointestinal tract, which makes the gut the largest hormone-producing organ in the body. Modern biology makes it feasi...

  18. Imidazolidinone adducts of peptides and hemoglobin

    International Nuclear Information System (INIS)

    San George, R.C.; Hoberman, H.D.

    1986-01-01

    Acetaldehyde reacts selectively with the terminal amino groups of the α and β chains of hemoglobin to form stable adducts, the structures of which, based on 13 C NMR studies, are proposed to be diastereomeric 2-methyl imidazolidin-4-ones. In this scheme, acetaldelhyde forms a reversible Schiff base with the α-amino groups of the polypeptide chains which cyclize with the amide nitrogen of the first peptide bond to form the stable imidazolidinone adducts. In support of this mechanism, the authors found that in following the reaction of the peptide val-gly-gly with [1,2- 13 C] acetaldehyde, 13 C NMR resonances attributed to a Schiff base (δ = 170 ppm) were observed which slowly disappeared prior to appearance of resonances from a pair of stable adducts (δ = 70 and 71 ppm) believed to be the diastereomeric imidazolidinones. Schiff base formation appeared to limit the overall rate. Tetraglycine reacted in a similar manner but with a resonance from a single stable adduct observed representing the enantiomeric imidazolidinone adducts of this peptide. Peptides with proline in position 2 should be incapable of forming imidazolidinones, and the authors found that ala-pro-gly did in fact fail to form a stable adduct with acetaldehyde. The 2-methyl imidazolidin-4-one adducts of hemoglobin may be useful in determining the contribution of the amino terminal groups to the structure and functional properties of hemoglobins

  19. It's the peptide-MHC affinity, stupid.

    Science.gov (United States)

    Kammertoens, Thomas; Blankenstein, Thomas

    2013-04-15

    Adoptively transferred T cells can reject large established tumors, but recurrence due to escape variants frequently occurs. In this issue of Cancer Cell, Engels et al. demonstrate that the affinity of the target peptide to the MHC molecule determines whether large tumors will relapse following adoptive T cell therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Peptide-stabilized, fluorescent silver nanoclusters

    DEFF Research Database (Denmark)

    Gregersen, Simon; Vosch, Tom André Jos; Jensen, Knud Jørgen

    2016-01-01

    Few-atom silver nanoclusters (AgNCs) can exhibit strong fluorescence; however, they require ligands to prevent aggregation into larger nanoparticles. Fluorescent AgNCs in biopolymer scaffolds have so far mainly been synthesized in solution, and peptides have only found limited use compared to DNA...