WorldWideScience

Sample records for synthetic domain peptides

  1. Synthetic antifreeze peptide

    OpenAIRE

    1991-01-01

    A synthetic antifreeze peptide and a synthetic gene coding for the antifreeze peptide have been produced. The antifreeze peptide has a greater number of repeating amino acid sequences than is present in the native antifreeze peptides from winter flounder upon which the synthetic antifreeze peptide was modeled. Each repeating amino acid sequence has two polar amino acid residues which are spaced a controlled distance apart so that the antifreeze peptide may inhibit ice formation. The synthetic...

  2. [SYNTHETIC PEPTIDE VACCINES].

    Science.gov (United States)

    Sergeyev, O V; Barinsky, I F

    2016-01-01

    An update on the development and trials of synthetic peptide vaccines is reviewed. The review considers the successful examples of specific protection as a result of immunization with synthetic peptides using various protocols. The importance of conformation for the immunogenicity of the peptide is pointed out. An alternative strategy of the protection of the organism against the infection using synthetic peptides is suggested.

  3. Functional analysis of synthetic DELLA domain peptides and bioactive gibberellin assay using surface plasmon resonance technology.

    Science.gov (United States)

    Zhao, Zhuoya; Xing, Zenan; Zhou, Min; Chen, Yi; Li, Chenzhong; Wang, Ruozhong; Xu, Wenzhong; Ma, Mi

    2015-11-01

    DELLA proteins and phytohormone gibberellin act together to control convergence point of plant development. A gibberellin-bound nuclear receptor that interacts with the N-terminal domain of DELLA proteins is required for gibberellin induced degradation of DELLA proteins. N-terminal DELLA domain includes two conserved motifs: DELLA and VHYNP. However, their respective functions remain unclear. Meanwhile, the identification and detection of several bioactive gibberellins from the more than 100 gibberellin metabolites are overwhelmingly difficult for their similar structures. Using in vitro biochemical approach, our work demonstrates for the first time that the synthetic GAI N-terminal DELLA domain peptides have similar bioactive function as the expressed protein to interact with AtGID1a receptor. Furthermore, our results reveal that DELLA motif is vitally important region and DELLA segment is essentially required region to recognize AtGID1a receptor. Finally, based on bioactive GA-dependent of the interaction between AtGID1a and DELLA protein, we generated a new method that could identify and detect bioactive GAs accurately and rapidly with surface plasmon resonance assays. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. A synthetic peptide derived from the D1 domain of flagellin induced the expression of proinflammatory cytokines in fish macrophages.

    Science.gov (United States)

    González-Stegmaier, Roxana; Guzmán, Fanny; Albericio, Fernando; Villarroel-Espíndola, Franz; Romero, Alex; Mulero, Victoriano; Mercado, Luis

    2015-11-01

    Flagellin is the main protein component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in fish and mammals. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines, and other immunological functions. We have previously reported that recombinant flagellin from Vibrio anguillarum and its ND1 domain are able to upregulate the expression of genes encoding major the proinflammatory mediators in gilthead seabream and rainbow trout macrophages. Considering the key role of D1 domain of flagellin for binding to TLR5M and its immunostimulatory activity, we designed and chemically synthesized a peptide derived of this region. The effects of the synthetic peptide were evaluated in vitro using head kidney macrophages from gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species the expression of genes encoding the proinflammatory cytokines interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), and the chemokine IL-8, was induced upon stimulation of macrophages with the D1 domain synthetic peptide. IL-1β and IL-8 were the most upregulated genes and to a lesser extent TNF-α. Interestingly, however, the induction activity of the synthetic peptide was higher in gilthead seabream than in rainbow trout macrophages. The results were confirmed at the protein levels for IL-8. Collectively, these results suggest that synthetic peptide derived from flagelling could be a promising approach for the immunostimulation and vaccination of farmed fish. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. A synthetic peptide derived from domain III envelope glycoprotein of Dengue virus induces neutralizing antibody.

    Science.gov (United States)

    Mary, J Asnet; Jittmittraphap, Akanitt; Chattanadee, Siriporn; Leaungwutiwong, Pornsawan; Shenbagarathai, R

    2018-02-01

    Dengue virus (DENV) is an arthropod-borne human pathogen that represents a severe public health threat in both endemic and non-endemic regions. So far, there is no licensed vaccine or specific drugs available for dengue fever. A fifteen-amino-acid-long peptide that includes the NGR motif was chemically synthesized and conjugated with keyhole limpet hemocyanin. A standard immunization protocol was followed for the production of polyclonal antibodies by immunizing rabbits against the synthetic peptide. The immune response elicited high-titer polyclonal antibodies with the reactivity of the anti-peptide antibody against both synthetic peptide and four serotypes of DENV confirmed by DOT-ELISA. Neutralizing activity of anti-peptide antibody was found to be cross-reactive and effective resulting in 60% reduction of infectivity at 1:200 dilution in all four serotypes of DENV. Our findings have the potential to further improve our understanding of virus-host interactions and provide new insights into neutralizing antibodies and could also be used as a drug target.

  6. Evaluation of tetravalent and conserved synthetic peptides vaccines derived from Dengue virus Envelope domain I and II.

    Science.gov (United States)

    Rocha, Raissa Prado; Livonesi, Márcia Cristina; Fumagalli, Marcilio Jorge; Rodrigues, Naiara Ferreira; da Costa, Lauro César Felipe; Dos Santos, Michelle Cristina Silva Gomes; de Oliveira Rocha, Eliseu Soares; Kroon, Erna Geessien; Malaquias, Luiz Cosme Cotta; Coelho, Luiz Felipe Leomil

    2014-08-08

    Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus (DENV) serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing a secondary infection with a different serotype progress to the severe form of the disease, called dengue hemorrhagic fever. In this study, the vaccine potential of three tetravalent and conserved synthetic peptides derived from DENV envelope domain I (named Pep01) and II (named Pep02 and Pep03) was evaluated. Human dengue IgM/IgG positive serum (n=16) showed reactivity against Pep01, Pep02 and Pep03 in different degrees. Mice immunization experiments showed that these peptides were able to induce a humoral response characterized by antibodies with low neutralizing activity. The spleen cells derived from mice immunized with the peptides showed a significant cytotoxic activity (only for Pep02 and Pep03), a high expression of IL-10 (Ppeptides, and specially the Pep03, can induce a humoral response characterized by antibodies with low neutralizing activities and probably a T cell response that could be beneficial to induce an effective immune response against all DENV serotypes and do not contributed to the immunopathogenesis. However, further studies in peptide sequence will be required to induce the production of neutralizing antibodies against all four DENV serotypes and also to improve immunogenicity of these peptides. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Synthetic antibiofilm peptides.

    Science.gov (United States)

    de la Fuente-Núñez, César; Cardoso, Marlon Henrique; de Souza Cândido, Elizabete; Franco, Octavio Luiz; Hancock, Robert E W

    2016-05-01

    Bacteria predominantly exist as multicellular aggregates known as biofilms that are associated with at least two thirds of all infections and exhibit increased adaptive resistance to conventional antibiotic therapies. Therefore, biofilms are major contributors to the global health problem of antibiotic resistance, and novel approaches to counter them are urgently needed. Small molecules of the innate immune system called host defense peptides (HDPs) have emerged as promising templates for the design of potent, broad-spectrum antibiofilm agents. Here, we review recent developments in the new field of synthetic antibiofilm peptides, including mechanistic insights, synergistic interactions with available antibiotics, and their potential as novel antimicrobials against persistent infections caused by biofilms. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    NARCIS (Netherlands)

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

    2002-01-01

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

  9. 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

  10. 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

  11. Administration of a Synthetic Peptide Derived from the E-domain Region of Mechano-Growth Factor Delays Decompensation Following Myocardial Infarction.

    Science.gov (United States)

    Shioura, Km; Pena, Jr; Goldspink, Ph

    2014-06-24

    Insulin like growth factor-I (IGF-1) isoforms differ structurally in their E-domain regions and their temporal expression profile in response to injury. We and others have reported that Mechano-growth factor (MGF), which is equivalent to human IGF-1c and rodent IGF-1Eb isoforms, is expressed acutely following myocardial infarction (MI) in the mouse heart. To examine the function of the E-domain region, we have used a stabilized synthetic peptide analog corresponding to the unique 24 amino acid region E-domain of MGF. Here we deliver the human MGF E-domain peptide to mice during the acute phase (within 12 hours) and the chronic phase (8 weeks) post-MI. We assessed the impact of peptide delivery on cardiac function and cardiovascular hemodynamics by pressure-volume (P-V) loop analysis and gene expression by quantitative RT-PCR. A significant decline in both systolic and diastolic hemodynamics accompanied by pathologic hypertrophy occurred by 10 weeks post-MI in the untreated group. Delivery of the E-domain peptide during the acute phase post-MI ameliorated the decline in hemodynamics, delayed decompensation but did not prevent pathologic hypertrophy. Delivery during the chronic phase post-MI significantly improved systolic function, predominantly due to the effects on vascular resistance and prevented decompensation. While pathologic hypertrophy persisted there was a significant decline in atrial natriuretic factor (ANF) expression in the E-domain peptide treated hearts. Taken together our data suggest that administration of the MGF E-domain peptide derived from the propeptide form of IGF-1Ec may be used to facilitate the actions of IGF-I produced by the tissue during the progression of heart failure to improve cardiovascular function.

  12. Synthetic antifreeze peptide and synthetic gene coding for its production

    OpenAIRE

    1991-01-01

    A synthetic antifreeze peptide and a synthetic gene coding for the antifreeze peptide have been produced. The antifreeze peptide has a greater number of repeating amino acid sequences than is present in the native antifreeze peptides from winter flounder upon which the synthetic antifreeze peptide was modeled. Each repeating amino acid sequence has two polar amino acid residues which are spaced a controlled distance apart so that the antifreeze peptide may inhibit ice formation. The synthetic...

  13. Epitope mapping of the monoclonal antibody MM12.10 to external MDR1 P-glycoprotein domain by synthetic peptide scanning and phage display technologies.

    Science.gov (United States)

    Romagnoli, G; Poloni, F; Flego, M; Moretti, F; Di Modugno, F; Chersi, A; Falasca, G; Signoretti, C; Castagna, M; Cianfriglia, M

    1999-05-01

    Epitope mapping of MDR1-P-glycoprotein using specific monoclonal antibodies (mAbs) may help in delineating P-glycoprotein topology and hence in elucidating the relationship between its structural organization and drug-efflux pump function. In this work, by using synthetic peptide scanning and phage display technologies, the binding sites of the mAb MM12.10, a novel antibody to intact human multidrug resistant (MDR) cells, were studied. The results we obtained confirm that two regions localized on the predicted fourth and sixth loops are indeed external and that MDR1 peptides covering the inner domain of the current 12 transmembrane segment (TMs) model of P-glycoprotein could form part of the MM12.10 epitope.

  14. Characterization of Synthetic Peptides by Mass Spectrometry.

    Science.gov (United States)

    Prabhala, Bala K; Mirza, Osman; Højrup, Peter; Hansen, Paul R

    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.

  15. [3H]Azidodantrolene photoaffinity labeling, synthetic domain peptides and monoclonal antibody reactivity identify the dantrolene binding sequence on RyR1

    Energy Technology Data Exchange (ETDEWEB)

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

    2002-06-14

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

  16. Synthetic peptides for diagnostic use

    NARCIS (Netherlands)

    Meloen, R.H.; Langedijk, J.P.M.; Langeveld, J.P.M.

    1997-01-01

    Synthetic peptides representing relevant B-cell epitopes are, potentially, ideal antigens to be used in diagnostic assays because of their superior properties with respect to quality control as compared to those of biologically derived molecules and the much higher specificity that sometimes can be

  17. Characterization of Synthetic Peptides by Mass Spectrometry

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  18. The synthetic peptide P111-136 derived from the C-terminal domain of heparin affin regulatory peptide inhibits tumour growth of prostate cancer PC-3 cells

    Science.gov (United States)

    2011-01-01

    Background Heparin affin regulatory peptide (HARP), also called pleiotrophin, is a heparin-binding, secreted factor that is overexpressed in several tumours and associated to tumour growth, angiogenesis and metastasis. The C-terminus part of HARP composed of amino acids 111 to 136 is particularly involved in its biological activities and we previously established that a synthetic peptide composed of the same amino acids (P111-136) was capable of inhibiting the biological activities of HARP. Here we evaluate the ability of P111-136 to inhibit in vitro and in vivo the growth of a human tumour cell line PC-3 which possess an HARP autocrine loop. Methods A total lysate of PC-3 cells was incubated with biotinylated P111-136 and pulled down for the presence of the HARP receptors in Western blot. In vitro, the P111-136 effect on HARP autocrine loop in PC-3 cells was determined by colony formation in soft agar. In vivo, PC-3 cells were inoculated in the flank of athymic nude mice. Animals were treated with P111-136 (5 mg/kg/day) for 25 days. Tumour volume was evaluated during the treatment. After the animal sacrifice, the tumour apoptosis and associated angiogenesis were evaluated by immunohistochemistry. In vivo anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay. Results Using pull down experiments, we identified the HARP receptors RPTPβ/ζ, ALK and nucleolin as P111-136 binding proteins. In vitro, P111-136 inhibits dose-dependently PC-3 cell colony formation. Treatment with P111-136 inhibits significantly the PC-3 tumour growth in the xenograft model as well as tumour angiogenesis. The angiostatic effect of P111-136 on HARP was also confirmed using an in vivo Matrigel™ plug assay in mice Conclusions Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on in vitro and in vivo growth of PC-3 cells. This inhibition could be linked to a direct or indirect binding of this peptide to the HARP receptors (ALK, RPTP

  19. A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; McCarthy, J B; Furcht, L T

    1993-01-01

    of focal adhesion and stress fiber formation requires additional interactions. Heparin-binding fragments of fibronectin can provide this signal. The COOH-terminal heparin-binding domain of fibronectin contains five separate heparin-binding amino acid sequences. We show here that all five sequences...

  20. 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.

  1. Natural and synthetic peptides with antifungal activity.

    Science.gov (United States)

    Ciociola, Tecla; Giovati, Laura; Conti, Stefania; Magliani, Walter; Santinoli, Claudia; Polonelli, Luciano

    2016-08-01

    In recent years, the increase of invasive fungal infections and the emergence of antifungal resistance stressed the need for new antifungal drugs. Peptides have shown to be good candidates for the development of alternative antimicrobial agents through high-throughput screening, and subsequent optimization according to a rational approach. This review presents a brief overview on antifungal natural peptides of different sources (animals, plants, micro-organisms), peptide fragments derived by proteolytic cleavage of precursor physiological proteins (cryptides), synthetic unnatural peptides and peptide derivatives. Antifungal peptides are schematically reported based on their structure, antifungal spectrum and reported effects. Natural or synthetic peptides and their modified derivatives may represent the basis for new compounds active against fungal infections.

  2. 14C-labeling of synthetic peptides

    International Nuclear Information System (INIS)

    Chersi, A.; Trinca, M.L.; Camera, M.

    1988-01-01

    Two methods are described for the labelling of synthetic peptides using iodo[ 14 C]acetic acid. The first procedure may be employed when the synthetic fragment contains a cysteine with a free sulfhydryl group. Alternatively, a commercial amino-protected cysteine may be carboxymethylated using radioactive iodoacetic acid. This derivative can be added to the growing peptide chain in the manual or automatic solid-phase synthesis of the fragment. 9 refs.; 1 figure; 1 table

  3. 14C-labeling of synthetic peptides.

    Science.gov (United States)

    Chersi, A; Trinca, M L; Camera, M

    1988-06-13

    Two methods are described for the labeling of synthetic peptides using iodo[14C]acetic acid. The first procedure may be employed when the synthetic fragment contains a cysteine with a free sulfhydryl group. Alternatively, a commercial amino-protected cysteine may be carboxymethylated using radioactive iodoacetic acid. This derivative can be added to the growing peptide chain in the manual or automatic solid-phase synthesis of the fragment.

  4. Antibody Production with Synthetic Peptides.

    Science.gov (United States)

    Lee, Bao-Shiang; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Jenny; Gupta, Shalini

    2016-01-01

    Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column.

  5. Synthetic peptides containing B- and T-cell epitope of dengue virus-2 E domain III provoked B- and T-cell responses.

    Science.gov (United States)

    Li, Shanfeng; Peng, Liang; Zhao, Wei; Zhong, Hua; Zhang, Fuchun; Yan, Ziqiang; Cao, Hong

    2011-05-09

    Our previous work applied a combination of bioinformatics approaches and in vitro assays to identify the dengue-2 virus (DENV-2)-specific B- and T-cell epitopes. In this report, we first evaluated the antigenicity of both B- and T-cell epitopes reacting with different sera against DENV-2 by ELISA as well as the ability of T-cell epitope to activate CD4(+) T-cell producing IFN-γ using ELISPOT, which showed a specific reactivity between either B- or T-cell epitope and DENV-2 antisera, and a significant increase of IFN-γ producing cells in DENV-2 infected mice. Then, a multi-epitope peptide containing the above B-, T-cell epitopes of envelope domain III (EDIII) of DENV-2 and pan-DR epitope (PADRE) was bioinformatically designed and synthesized. The verification of its immunogenicity and protective effect was performed in in vitro and in vivo experiments. The results showed that a high level of antibody in mice elicited by the multi-epitope peptide was detected by ELISA and the anti-peptide sera binding to the vero cells infected with DEN-2 was observed with immunofluorescence test. More importantly, the peptide could induce lymphoproliferation in vitro and a predominant Th1 type of immune response was examined by flow cytometry. We also found that the virus replication in the mice vaccinated with the multi-epitope peptide was obviously less than that of the control groups. These results may provide some important information for the development of dengue vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Preparation and utilization of fluorescent synthetic peptides.

    Science.gov (United States)

    Chersi, A; Sezzi, M L; Romano, T F; Evangelista, M; Nista, A

    1990-06-20

    In this study, several methods for controlled labelling of synthetic peptides by the use of fluorescent compounds (fluorescein isothiocyanate and dimethylaminonaphthalene sulfonyl chloride) were investigated. The first reagent yielded monofluoresceinated, active compounds only when the peptides lacked lysine residues. Monolabelling of peptides in solution with dimethylaminonaphthalenesulphonyl chloride was hindered by the broad reactivity of the reagent, but was achieved by reacting the fluorochrome on protected resin-bound peptides in solid-phase synthesis. The remarkable stability of the linkage allowed the cleavage of the peptide from the resin and deprotection of side-chain functions without hydrolysis of the labelled group. The binding of antipeptide antibodies to the labelled fragments was then estimated using different techniques.

  7. Defined carriers for synthetic antigens: Hinge Peptides

    Czech Academy of Sciences Publication Activity Database

    Hlaváček, Jan; Niederhafner, Petr; Gut, Vladimír; Hulačová, Hana; Maloň, Petr

    2005-01-01

    Roč. 29, č. 1 (2005), s. 68 ISSN 0939-4451. [International Congress on Amino Acids and Proteins /9./. 08.08.2005-12.08.2005, Gert Lubec] R&D Projects: GA ČR(CZ) GA203/03/1362 Institutional research plan: CEZ:AV0Z40550506 Keywords : synthetic carrier * antigen * hinge peptide Subject RIV: CC - Organic Chemistry

  8. Advances in synthetic peptides reagent discovery

    Science.gov (United States)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Stratis-Cullum, Dimitra N.

    2013-05-01

    Bacterial display technology offers a number of advantages over competing display technologies (e.g, phage) for the rapid discovery and development of peptides with interaction targeted to materials ranging from biological hazards through inorganic metals. We have previously shown that discovery of synthetic peptide reagents utilizing bacterial display technology is relatively simple and rapid to make laboratory automation possible. This included extensive study of the protective antigen system of Bacillus anthracis, including development of discovery, characterization, and computational biology capabilities for in-silico optimization. Although the benefits towards CBD goals are evident, the impact is far-reaching due to our ability to understand and harness peptide interactions that are ultimately extendable to the hybrid biomaterials of the future. In this paper, we describe advances in peptide discovery including, new target systems (e.g. non-biological materials), advanced library development and clone analysis including integrated reporting.

  9. Structural and Functional Studies of Experimental HIV Synthetic Peptide Immunogens

    Science.gov (United States)

    1997-10-01

    Work performed in this grant continues to address 2 major problems in HIV synthetic peptide vaccine development: (1) the ability of synthetic...In technical aim #1, intranasal immunization with HIV synthetic peptide immunogens was found to be effective for the induction of serum anti-peptide

  10. Synthetic peptide antagonists of glucagon

    International Nuclear Information System (INIS)

    Unson, C.G.; Andreu, D.; Gurzenda, E.M.; Merrifield, R.B.

    1987-01-01

    Several glucagon analogs were synthesized in an effort to find derivatives that would bind with high affinity to the glucagon receptor of rat liver membranes but would not activate membrane-bound adenylate cyclase and, therefore, would serve as antagonists of the hormone. Measurements on a series of glucagon/secretin hybrids indicated that replacement of Asp 9 in glucagon by Glu 9 , found in secretin, was the important sequence difference in the N terminus of the two hormones. Further deletion of His 1 and introduction of a C-terminal amide resulted in des-His 1 -[Glu 9 ]glucagon amide, which had a 40% binding affinity relative to that of native glucagon but caused no detectable adenylate cyclase activation in the rat liver membrane. This antagonist completely inhibited the effect of a concentration of glucagon that alone gave a full agonist response. It had an inhibition index of 12. The pA 2 was 7.2. An attempt was made to relate conformation with receptor binding. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C 18 -silica columns

  11. Activity of synthetic peptides against Chlamydia.

    Science.gov (United States)

    Donati, Manuela; Cenacchi, Giovanna; Biondi, Roberta; Papa, Valentina; Borel, Nicole; Vecchio Nepita, Edoardo; Magnino, Simone; Pasquinelli, Gianandrea; Levi, Aurora; Franco, Octavio L

    2017-11-01

    The in vitro activity of six synthetic peptides against 36 strains of Chlamydia from different origins was investigated. Clavanin MO (CMO) proved to be the most active peptide, reducing the inclusion number of all Chlamydia strains from eight different species tested by ≥50% at 10 µg mL -1 . Mastoparan L showed an equal activity against C. trachomatis, C. pneumoniae, C. suis, and C. muridarum, but did not exert any inhibitory effect against C. psittaci, C. pecorum, C. abortus, and C. avium even at 80 µg mL -1 . These data suggest that CMO could be a promising compound in the prevention and treatment of chlamydial infections. © 2017 Wiley Periodicals, Inc.

  12. Novel domain wall dynamics in synthetic antiferromagnets

    Science.gov (United States)

    Yang, See-Hun; Parkin, Stuart

    2017-08-01

    In this article, we review fascinating new mechanisms on recently observed remarkable current driven domain wall motion in nanowires formed from perpendicularly magnetized synthetic antiferromagnets interfaced with heavy metallic layers, sources of spin-orbit torques. All the associated torques such as volumetric adiabatic and non-adiabatic spin-transfer-torque, spin-orbit torques, shape anisotropy field torques, Dzyaloshinkii-Moriya interaction torques and most importantly a new powerful torque, exchange coupling torque, will be discussed based on an analytical model that provides an intuitive description of domain wall dynamics in synthetic ferromagnets as well as synthetic antiferromagnets. In addition, the current driven DW motion in the presence of in-plane fields will be investigated, thus deepening our knowledge about the role of the exchange coupling torque, which will be of potential use for application to various novel spintronic devices.

  13. Structural and Functional Studies of Experimental HIV Synthetic Peptides Immunogens.

    Science.gov (United States)

    1998-09-01

    Work performed in this grant continues to address major hurdles in the development of an effective synthetic peptide HIV vaccine: 1) the ability of...isolates and 2) the design of synthetic peptide immunogens capable of being recognized by MHC Class I and II molecules in outbred populations. In

  14. Antimicrobial activity of synthetic salivary peptides against voice prosthetic microorganisms

    NARCIS (Netherlands)

    Elving, GJ; van der Mei, HC; Busscher, HJ; Amerongen, AV; Veerman, ECI; Van Weissenbruch, R; Albers, FWJ

    Objectives: To investigate whether synthetic salivary antimicrobial peptides have an inhibitory effect on the growth of bacteria and yeasts isolated from used silicone rubber voice prostheses. Methods: The antimicrobial activities of six synthetic salivary peptides (histatin 5, dhvar1, dhvar4,

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

    NARCIS (Netherlands)

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

    1993-01-01

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

  16. The effects of newly formed synthetic peptide on bone regeneration in rat calvarial defects.

    Science.gov (United States)

    Choi, Jung-Yoo; Jung, Ui-Won; Kim, Chang-Sung; Eom, Tae-Kwan; Kang, Eun-Jung; Cho, Kyoo-Sung; Kim, Chong-Kwan; Choi, Seong-Ho

    2010-02-01

    Significant interest has emerged in the design of cell scaffolds that incorporate peptide sequences that correspond to known signaling domains in extracellular matrix and bone morphogenetic protein. The purpose of this study was to evaluate the bone regenerative effects of the synthetic peptide in a critical-size rat calvarial defect model. Eight millimeter diameter standardized, circular, transosseus defects created on the cranium of forty rats were implanted with synthetic peptide, collagen, or both synthetic peptide and collagen. No material was was implanted the control group. The healing of each group was evaluated histologically and histomorphometrically after 2- and 8-week healing intervals. Surgical implantation of the synthetic peptide and collagen resulted in enhanced local bone formation at both 2 and 8 weeks compared to the control group. When the experimental groups were compared to each other, they showed a similar pattern of bone formation. The defect closure and new bone area were significantly different in synthetic peptide and collagen group at 8 weeks. Concerning the advantages of biomaterials, synthetic peptide can be an effective biomaterial for damaged periodontal regeneration.

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

    Science.gov (United States)

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

    2017-02-06

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

  18. STM studies of synthetic peptide monolayers

    Science.gov (United States)

    Bergeron, David J.; Clauss, Wilfried; Pilloud, Denis L.; Leslie Dutton, P.; Johnson, Alan T.

    1998-08-01

    We have used scanning probe microscopy to investigate self-assembled monolayers of chemically synthesized peptides. We find that the peptides form a dense uniform monolayer, above which is found a sparse additional layer. Using scanning tunneling microscopy, submolecular resolution can be obtained, revealing the alpha helices which constitute the peptide. The nature of the images is not significantly affected by the incorporation of redox cofactors (hemes) in the peptides.

  19. Immunoreactivity of synthetic peptides derived from proteins of Cryptococcus gattii.

    Science.gov (United States)

    de Serpa Brandão, Rafael Melo Santos; Soares Martins, Liline Maria; de Andrade, Hélida Monteiro; Faria, Angélica Rosa; Soares Leal, Maria José; da Silva, Adalberto Socorro; Wanke, Bodo; dos Santos Lazéra, Márcia; Vainstein, Marilene Henning; Mendes, Rinaldo Poncio; Moris, Daniela Vanessa; de Souza Cavalcante, Ricardo; do Monte, Semiramis Jamil Hadad

    2014-01-01

    To determine the immunoreactivity of synthetic Cryptococcus-derived peptides. A total of 63 B-cell epitopes from previously identified Cryptococcus gattii immunoreactive proteins were synthesized and evaluated as antigens in ELISAs. The peptides were first evaluated for their ability to react against sera from immunocompetent subjects carrying cryptococcal meningitis. Peptides that yielded high sensitivity and specificity in the first test were then retested with sera from individuals with other fungal pathologies for cross-reactivity determination. Six of 63 synthetic peptides were recognized by antibodies in immunoassays, with a specificity of 100%, sensitivity of 78% and low cross-reactivity. We successfully determined the immunoreactivity of selected synthetic peptides of C. gattii derived proteins.

  20. 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....

  1. Advances in synthetic peptide immuno-regulatory epitopes.

    Science.gov (United States)

    Creticos, Peter Socrates

    2014-01-01

    Synthetic peptide immuno-regulatory epitopes (SPIRE) represent a new class of therapeutics for allergen immunotherapy that offer the potential to suppress the IgE-mediated allergic disease process through induction of T-cell tolerance. These synthetic T-cell-tolerizing peptides have been designed to induce immunologic tolerance via binding to MHC class II molecules on antigen presenting cells, with subsequent upregulation of regulatory T-cells.

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

    NARCIS (Netherlands)

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

    1997-01-01

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

  3. Kinase activity and specificity assay using synthetic peptides.

    Science.gov (United States)

    Wu, Xu Na; Schulze, Waltraud X

    2015-01-01

    Phosphorylation of substrate proteins by protein kinases can lead to activation or inactivation of signaling pathways or metabolic processes. Precise understanding of activity and specificity of protein kinases are important questions in characterization of kinase functions. Here, we describe a procedure to study kinase activity and specificity using kinase-GFP complexes purified from plant material and synthetic peptides as substrates. Magnetic GFP beads allow purifying receptor-like kinase-GFP complexes from microsomal fractions. Kinase-GFP complexes are then incubated with ATP and the synthetic peptides for kinase reaction. Phosphorylation of substrate peptides is then identified and quantified by mass spectrometry.

  4. Synthetic multivalent antifungal peptides effective against fungi.

    Directory of Open Access Journals (Sweden)

    Rajamani Lakshminarayanan

    Full Text Available Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010 which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is non-haemolytic and non-toxic to mice by intraperitoneal (200 mg/kg or intravenous (100 mg/kg routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2-4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides.

  5. How Peptide Molecular Structure and Charge Influence the Nanostructure of Lipid Bicontinuous Cubic Mesophases: Model Synthetic WALP Peptides Provide Insights.

    Science.gov (United States)

    van 't Hag, Leonie; Li, Xu; Meikle, Thomas G; Hoffmann, Søren V; Jones, Nykola C; Pedersen, Jan Skov; Hawley, Adrian M; Gras, Sally L; Conn, Charlotte E; Drummond, Calum J

    2016-07-12

    Nanostructured bicontinuous lipidic cubic phases are used for the encapsulation of proteins in a range of applications such as in meso crystallization of transmembrane proteins and as drug delivery vehicles. The retention of the nanoscale order of the cubic phases subsequent to protein incorporation, as well as retention of the protein structure and function, is essential for all of these applications. Herein synthetic peptides (WALP21, WALPS53, and WALPS73) with a common α-helical hydrophobic domain, but varying hydrophilic loop size, were designed to systematically examine the effect of peptide structure and charge on bicontinuous cubic phases. The effect of the cubic phases on the secondary structure of the peptides was also investigated. The incorporation of the WALP peptides in cubic phases formed by a range of lipids showed that hydrophobic mismatch of the peptides with the lipid bilayers, the hydrophilic domain size, and peptide charge were all significant factors determining the response of the lipid nanomaterial to protein insertion. As charge repulsion had the most significant effect on the phase transitions observed, we suggest that buffer pH and salt concentration must be carefully considered to ensure cubic mesophase retention. Importantly, the WALP peptides were found to have a different conformation depending on the local lipid environment. Such structural changes could potentially affect membrane protein function, which is crucial for both current and prospective applications.

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

    Science.gov (United States)

    Albada, Bauke; Metzler-Nolte, Nils

    2016-10-12

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

  7. The latest developments in synthetic peptides with immunoregulatory activities.

    Science.gov (United States)

    Zhou, Chun-lei; Lu, Rong; Lin, Gang; Yao, Zhi

    2011-02-01

    In the past few years, many researches have provided us with much data demonstrating the abilities of synthetic peptides to impact immune response in vitro and in vivo. These peptides were designed according to the structure of some important protein molecules which play a key role in immune response, so they act with specific targets. The class I and II MHC-derived peptides inhibit the TCR recognition of antigen peptide-MHC complex. Rationally designed CD80 and CD154-binding peptides block the interaction between cell surface costimulatory molecules on antigen-presenting cells (APCs) and T cells. Some peptides were designed to inhibit the activities of cell signal proteins, including JNK, NF-κB and NFAT. Some peptide antagonists competitively bind to important cytokines and inhibit their activities, such as TNF-α, TGF-β and IL-1β inhibitory peptides. Adhesion molecule ICAM-1 derived peptides block the T cell adhesion and activation. These immunoregulatory peptides showed therapeutic effect in several animal models, including collagen-induced arthritis (CIA), autoimmune cystitis model, murine skin transplant model and cardiac allograft model. These results give us important implications for the development of a novel therapy for immune mediated diseases. Copyright © 2010 Elsevier Inc. All rights reserved.

  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...... attempts have been made to couple peptide immunogens to different carrier proteins [e.g. keyhole limper haemocyanin (KLH) or ovalbumin]. This leads to very complex structures, however. We used a controlled conjugation of a peptide to a single long-chain fatty acid like palmitic acid by a thioester...... 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. Bioprinting synthetic self-assembling peptide hydrogels for biomedical applications

    International Nuclear Information System (INIS)

    Loo, Yihua; Hauser, Charlotte A E

    2016-01-01

    Three-dimensional (3D) bioprinting is a disruptive technology for creating organotypic constructs for high-throughput screening and regenerative medicine. One major challenge is the lack of suitable bioinks. Short synthetic self-assembling peptides are ideal candidates. Several classes of peptides self-assemble into nanofibrous hydrogels resembling the native extracellular matrix. This is a conducive microenvironment for maintaining cell survival and physiological function. Many peptides also demonstrate stimuli-responsive gelation and tuneable mechanical properties, which facilitates extrusion before dispensing and maintains the shape fidelity of the printed construct in aqueous media. The inherent biocompatibility and biodegradability bodes well for in vivo applications as implantable tissues and drug delivery matrices, while their short length and ease of functionalization facilitates synthesis and customization. By applying self-assembling peptide inks to bioprinting, the dynamic complexity of biological tissue can be recreated, thereby advancing current biomedical applications of peptide hydrogel scaffolds. (paper)

  10. Advances in Synthetic Peptides Reagent Discovery

    Science.gov (United States)

    2013-07-01

    spectrophotometry technologies. The commercially engineered red fluorescent protein , dsRed, is used in the current advanced library development...sensor technologies utilize antibodies, which have high affinity and specificity to a given target, but are very costly, difficult to mass -produce...include various types of biological molecules, such as nucleic acids, peptides, and proteins , and have been extensively reviewed[2, 3]. Our work has

  11. Synthetic Advances in Insulin-like Peptides Enable Novel Bioactivity.

    Science.gov (United States)

    Liu, Fa; Li, Pengyun; Gelfanov, Vasily; Mayer, John; DiMarchi, Richard

    2017-08-15

    Insulin is a miraculous hormone that has served a seminal role in the treatment of insulin-dependent diabetes for nearly a century. Insulin resides within in a superfamily of structurally related peptides that are distinguished by three invariant disulfide bonds that anchor the three-dimensional conformation of the hormone. The additional family members include the insulin-like growth factors (IGF) and the relaxin-related set of peptides that includes the so-called insulin-like peptides. Advances in peptide chemistry and rDNA-based synthesis have enabled the preparation of multiple insulin analogues. The translation of these methods from insulin to related peptides has presented unique challenges that pertain to differing biophysical properties and unique amino acid compositions. This Account presents a historical context for the advances in the chemical synthesis of insulin and the related peptides, with division into two general categories where disulfide bond formation is facilitated by native conformational folding or alternatively orthogonal chemical reactivity. The inherent differences in biophysical properties of insulin-like peptides, and in particular within synthetic intermediates, have constituted a central limitation to achieving high yield synthesis of properly folded peptides. Various synthetic approaches have been advanced in the past decade to successfully address this challenge. The use of chemical ligation and metastable amide bond surrogates are two of the more important synthetic advances in the preparation of high quality synthetic precursors to high potency peptides. The discovery and application of biomimetic connecting peptides simplifies proper disulfide formation and the subsequent traceless removal by chemical methods dramatically simplifies the total synthesis of virtually any two-chain insulin-like peptide. We report the application of these higher synthetic yield methodologies to the preparation of insulin-like peptides in support of

  12. Liposome-based synthetic long peptide vaccines for cancer immunotherapy

    NARCIS (Netherlands)

    Varypataki, E.M.

    2016-01-01

    Synthetic long peptides (SLP) derived from cancer-associated antigens hold great promise as well-defined antigens for cancer immunotherapy. Clinical studies showed that SLP vaccines have functional potency when applied to pre-malignant stage patients, but need to be improved for use as a therapeutic

  13. Evaluation of dermal wound healing activity of synthetic peptide SVVYGLR.

    Science.gov (United States)

    Uchinaka, Ayako; Kawaguchi, Naomasa; Ban, Tsuyoshi; Hamada, Yoshinosuke; Mori, Seiji; Maeno, Yoshitaka; Sawa, Yoshiki; Nagata, Kohzo; Yamamoto, Hirofumi

    2017-09-23

    SVVYGLR peptide (SV peptide) is a 7-amino-acid sequence with angiogenic properties that is derived from osteopontin in the extracellular matrix and promotes differentiation of fibroblasts to myofibroblast-like cells and the production of collagen type Ⅲ by cardiac fibroblasts. However, the effects of SV peptide on dermal cells and tissue are unknown. In this study, we evaluated the effects of this peptide in a rat model of dermal wound healing. The synthetic SV peptide was added to dermal fibroblasts or keratinocytes, and their cellular motility was evaluated. In an in vivo wound healing exeriment, male rats aged 8 weeks were randomly assigned to the SV peptide treatment, non-treated control, or phosphate-buffered saline (PBS) groups. Wound healing was assessed by its repair rate and histological features. Scratch assay and cell migration assays using the Chemotaxicell method showed that SV peptide significantly promoted the cell migration in both fibroblasts and keratinocytes. In contrast the proliferation potency of these cells was not affected by SV peptide. In the rat model, wound healing progressed faster in the SV peptide-treated group than in the control and PBS groups. The histopathological analyses showed that the SV peptide treatment stimulated the migration of fibroblasts to the wound area and increased the number of myofibroblasts. Immunohistochemical staining showed a marked increase of von Willebland factor-positive neomicrovessels in the SV peptide-treated group. In conclusion, SV peptide has a beneficial function to promote wound healing by stimulating granulation via stimulating angiogenesis, cell migration, and the myofibroblastic differentiation of fibroblasts. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. 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 ...

  15. Mapping receptor-ligand interactions with synthetic peptide arrays: exploring the structure and function of membrane receptors.

    Science.gov (United States)

    Volkmer, Rudolf; Kretzschmar, Ines; Tapia, Victor

    2012-04-01

    Development of synthetic peptide array technology started in the early 1990s. The technique originally developed by Ronald Frank has become a powerful tool for high throughput approaches in biology and chemistry mapping protein interaction sites. In this review we focus on peptide arrays applied to investigate receptor-ligand interactions, such as peroxisomal membrane receptor proteins, the maltose importer machinery and receptor proteins recognizing short linear motifs of their partners. We present several systematic sets of peptide arrays useful for mapping protein-protein- or receptor-ligand binding sites. Besides a more technical description of the peptide array preparation we discuss in detail the reliability and improvement of mapping protein-protein interactions by synthetic peptide arrays. At least proteomic approaches for mapping protein-protein interactions by peptide arrays are shown especially for the case of protein interaction domains. Copyright © 2011 Elsevier GmbH. All rights reserved.

  16. Generation of Synthetic Turbulence in Arbitrary Domains

    DEFF Research Database (Denmark)

    Gilling, Lasse; Nielsen, Søren R.K.; Sørensen, Niels

    2009-01-01

    A new method for generating synthetic turbulence is presented. The method is intended for generating a turbulent velocity field with a fine spatial resolution but only covering a small moving part of the rotor area of a wind turbine. For this application the Mann and Sandia methods cannot be used......-spectra a realization of a velocity field is determined by factorization and Fourier transform as in the Sandia method....

  17. Chemoselective silicification of synthetic peptides and polyamines

    Directory of Open Access Journals (Sweden)

    Maryna Abacilar

    2015-01-01

    Full Text Available Biosilicification sets the standard for the localized in vitro precipitation of silica at low orthosilicate concentrations in aqueous environment under ambient conditions. Numerous parameters must be controlled for the development of new technologies in designing inventive nanosilica structures, which are able to challenge the biological templates. A long neglected requirement that came into focus in the recent years are the cellular techniques of preventing unintentional lithification of cellular structures since numerous cellular components such as membranes, DNA, and proteins are known to precipitate nanosilica. The diatom metabolism makes use of techniques that restrict silicification to an armor of silica around the cell wall while avoiding the petrifying gaze of Medusa, which turns the whole cell into stone. Step by step, biochemistry unveils the hierarchical interplay of an arsenal of low-molecular weight molecules, proteins, and the cytoskeletal architecture and it becomes clearer why the organisms invest much metabolic effort for an obviously simple chemical reaction like the precipitation of amorphous silica. The discrimination between different soluble components in the silicification process (chemoselective silicification is not only vitally important for the diatom but poses an interesting challenge for in vitro experiments. Until now, silica precipitation studies were mainly focused on the amount, the morphology, and composition of the precipitate while disregarding a quantitative analysis of the remaining soluble components. Here, we turn the tables and quantify the soluble components by 1H NMR in the progress of precipitation and present experiments which quantify the additivity, and potential cooperativity of long chain polyamines (LCPAs and cationic peptides in the silicification process.

  18. Chemoselective silicification of synthetic peptides and polyamines.

    Science.gov (United States)

    Abacilar, Maryna; Daus, Fabian; Geyer, Armin

    2015-01-01

    Biosilicification sets the standard for the localized in vitro precipitation of silica at low orthosilicate concentrations in aqueous environment under ambient conditions. Numerous parameters must be controlled for the development of new technologies in designing inventive nanosilica structures, which are able to challenge the biological templates. A long neglected requirement that came into focus in the recent years are the cellular techniques of preventing unintentional lithification of cellular structures since numerous cellular components such as membranes, DNA, and proteins are known to precipitate nanosilica. The diatom metabolism makes use of techniques that restrict silicification to an armor of silica around the cell wall while avoiding the petrifying gaze of Medusa, which turns the whole cell into stone. Step by step, biochemistry unveils the hierarchical interplay of an arsenal of low-molecular weight molecules, proteins, and the cytoskeletal architecture and it becomes clearer why the organisms invest much metabolic effort for an obviously simple chemical reaction like the precipitation of amorphous silica. The discrimination between different soluble components in the silicification process (chemoselective silicification) is not only vitally important for the diatom but poses an interesting challenge for in vitro experiments. Until now, silica precipitation studies were mainly focused on the amount, the morphology, and composition of the precipitate while disregarding a quantitative analysis of the remaining soluble components. Here, we turn the tables and quantify the soluble components by (1)H NMR in the progress of precipitation and present experiments which quantify the additivity, and potential cooperativity of long chain polyamines (LCPAs) and cationic peptides in the silicification process.

  19. Synthetic peptides derived from the sequence of a lasso peptide microcin J25 show antibacterial activity.

    Science.gov (United States)

    Soudy, Rania; Wang, Liru; Kaur, Kamaljit

    2012-03-01

    Microcin J25 (MccJ25) is a plasmid-encoded, ribosomally synthesized antibacterial peptide with a unique lasso structure. The lasso structure, produced with the aid of two processing enzymes, provides exceptional stability to MccJ25. We report the synthesis of six peptides (1-6), derived from the MccJ25 sequence, that are designed to form folded conformation by disulfide bond formation and electrostatic or hydrophobic interactions. Two peptides (1 and 6) display good activity against Salmonella newport, and are the first synthetic derivatives of MccJ25 that are bactericidal. Peptide 1 displays potent activity against several Salmonella strains including two MccJ25 resistant strains. The solution conformation and the stability studies of the active peptides suggest that they do not fold into a lasso conformation and peptide 1 displays antimicrobial activity by inhibition of target cell respiration. Like MccJ25, the synthetic MccJ25 derivatives display minimal toxicity to mammalian cells suggesting that these peptides act specifically on bacterial cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Synthetic cationic amphiphilic α-helical peptides as antimicrobial agents.

    Science.gov (United States)

    Wiradharma, Nikken; Khoe, Ulung; Hauser, Charlotte A E; Seow, See Voon; Zhang, Shuguang; Yang, Yi-Yan

    2011-03-01

    Antimicrobial peptides (AMPs) secreted by the innate immune system are prevalent as the effective first-line of defense to overcome recurring microbial invasions. They have been widely accepted as the blueprints for the development of new antimicrobial agents for the treatment of drug resistant infections. However, there is also a growing concern that AMPs with a sequence that is too close to the host organism's AMP may inevitably compromise its own natural defense. In this study, we design a series of synthetic (non-natural) short α-helical AMPs to expand the arsenal of the AMP families and to gain further insights on their antimicrobial activities. These cationic and amphiphilic peptides have a general sequence of (XXYY)(n) (X: hydrophobic residue, Y: cationic residue, and n: the number of repeat units), and are designed to mimic the folding behavior of the naturally-occurring α-helical AMPs. The synthetic α-helical AMPs with 3 repeat units, (FFRR)(3), (LLRR)(3), and (LLKK)(3), are found to be more selective towards microbial cells than rat red blood cells, with minimum inhibitory concentration (MIC) values that are more than 10 times lower than their 50% hemolytic concentrations (HC(50)). They are effective against Gram-positive B. subtilis and yeast C. albicans; and the studies using scanning electron microscopy (SEM) have elucidated that these peptides possess membrane-lytic activities against microbial cells. Furthermore, non-specific immune stimulation assays of a typical peptide shows negligible IFN-α, IFN-γ, and TNF-α inductions in human peripheral blood mononuclear cells, which implies additional safety aspects of the peptide for both systemic and topical use. Therefore, the peptides designed in this study can be promising antimicrobial agents against the frequently-encountered Gram-positive bacteria- or yeast-induced infections. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Synthetic Molecular Evolution of Membrane-Active Peptides

    Science.gov (United States)

    Wimley, William

    The physical chemistry of membrane partitioning largely determines the function of membrane active peptides. Membrane-active peptides have potential utility in many areas, including in the cellular delivery of polar compounds, cancer therapy, biosensor design, and in antibacterial, antiviral and antifungal therapies. Yet, despite decades of research on thousands of known examples, useful sequence-structure-function relationships are essentially unknown. Because peptide-membrane interactions within the highly fluid bilayer are dynamic and heterogeneous, accounts of mechanism are necessarily vague and descriptive, and have little predictive power. This creates a significant roadblock to advances in the field. We are bypassing that roadblock with synthetic molecular evolution: iterative peptide library design and orthogonal high-throughput screening. We start with template sequences that have at least some useful activity, and create small, focused libraries using structural and biophysical principles to design the sequence space around the template. Orthogonal high-throughput screening is used to identify gain-of-function peptides by simultaneously selecting for several different properties (e.g. solubility, activity and toxicity). Multiple generations of iterative library design and screening have enabled the identification of membrane-active sequences with heretofore unknown properties, including clinically relevant, broad-spectrum activity against drug-resistant bacteria and enveloped viruses as well as pH-triggered macromolecular poration.

  2. Identification of binding peptides of the ADAM15 disintegrin domain ...

    Indian Academy of Sciences (India)

    Madhsudhan

    Karkkainen et al. 2006). In this study, the recombinant ADAM15 disintegrin domain (RADD) was expressed in E. coli, and 4 specific binding peptides of RADD were obtained by positive panning and subtractive selection using a phage display 12-mer.

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

    Science.gov (United States)

    Pakalns, Teika

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

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential...

  5. Advantages of a synthetic peptide immunogen over a protein immunogen in the development of an anti-pilus vaccine for Pseudomonas aeruginosa.

    Science.gov (United States)

    Kao, Daniel J; Hodges, Robert S

    2009-07-01

    The type IV pilus is an important adhesin in the establishment of infection by Pseudomonas aeruginosa. We have previously reported on a synthetic peptide vaccine targeting the receptor-binding domain of the main structural subunit of the pilus, PilA. The receptor-binding domain is a 14-residue disulfide loop at the C-terminal end of the pilin protein. The objective of this study was to compare the immunogenicity of a peptide-conjugate to a protein subunit immunogen to determine which was superior for use in an anti-pilus vaccine. BALB/c mice were immunized with the native PAK strain pilin protein and a synthetic peptide of the receptor-binding domain conjugated to keyhole limpet haemocyanin. A novel pilin protein with a scrambled receptor-binding domain was used to characterize receptor-binding domain-specific antibodies. The titres against the native pilin of the animals immunized with the synthetic peptide-conjugate were higher than the titres of animals immunized with the pilin protein. In addition, the affinities of anti-peptide sera for the intact pilin receptor-binding domain were significantly higher than affinities of anti-pilin protein sera. These results have significant implications for vaccine design and show that there are significant advantages in using a synthetic peptide-conjugate over a subunit pilin protein for an anti-pilus vaccine.

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    Monoclonal antibodies against a synthetic peptide (aa 138-152) from HIV-1 Nef protein were produced and characterized. Three hybridoma lines producing monoclonal antibodies (MAbs) against the synthetic peptide were generated by fusion between P3-X63 Ag8.653 myeloma cells and BALB/c splenocytes from...... mice immunized with the synthetic peptide coupled to keyhole limpet hemocyanin (KLH). The hybridomas were screened and selected by ELISA with the peptide coupled to bovine serum albumin (BSA) immobilized to the polystyrene surface and specificity for the peptide was confirmed by competitive ELISA...

  7. An implementation of synthetic aperture focusing technique in frequency domain.

    Science.gov (United States)

    Stepinski, Tadeusz

    2007-07-01

    A new implementation of a synthetic aperture focusing technique (SAFT) based on concepts used in synthetic aperture radar and sonar is presented in the paper. The algorithm, based on the convolution model of the imaging system developed in frequency domain, accounts for the beam pattern of the finite-sized transducer used in the synthetic aperture. The 2D fast Fourier transform (FFT) is used for the calculation of a 2D spectrum of the ultrasonic data. The spectrum is then interpolated to convert the polar coordinate system used for the acquisition of ultrasonic signals to the rectangular coordinates used for the presentation of imaging results. After compensating the transducer lobe amplitude profile using a Wiener filter, the transformed spectrum is subjected to the 2D inverse Fourier transform to get the time-domain image again. The algorithm is computationally attractive due to the use of 2D FFT. The performance of the proposed frequency-domain algorithm and the classical time-domain SAFT are compared in the paper using simulated and real ultrasonic data.

  8. A study on the C-peptide radioimmunoassay with synthetized connecting peptide

    International Nuclear Information System (INIS)

    Nakagawa, Shoichi; Sasaki, Takashi; Nakayama, Hidetaka; Watanabe, Takuji; Aoki, Shin

    1976-01-01

    A method of C-peptide radioimmunoassay with the synthetized connecting peptide by Yanaihara was tested for the determination of serum C-peptide immunoreactivity (CPR) in normal people and in diabetics with or without insulin treatment. The CPR value obtained by this method was not interfered with by the presence of serum proteins or by the insulin of people with or without insulin treatment judged by the dilution test and the recovery test. The normal fasting CPR was 2.80 +- 0.78 ng/ml with the synthetized C-peptide as a standard. The CPR value increased and reached a maximum 90 minutes after the ingestion of 50 g of glucose. The increase after the glucose loading reduced corresponding to the severity of diabetes, and some juvenile-onset diabetes showed no response. Adult-type diabetics under insulin treatment, however, showed weak but significant CPR response. The increment of CPR and immunoreactive insulin after glucose loading in normal people and non-treated diabetics was well correlated (γ=0.8262). Judged from the above mentioned results, CPR determination in insulin-treated diabetics was thought to be a useful method for the assessment of the insulin-secreting ability of beta-cells of the pancreas. (J.P.N.)

  9. Synthetic Domain Theory and Models of Linear Abadi & Plotkin Logic

    DEFF Research Database (Denmark)

    Møgelberg, Rasmus Ejlers; Birkedal, Lars; Rosolini, Guiseppe

    2008-01-01

    Plotkin suggested using a polymorphic dual intuitionistic/linear type theory (PILLY) as a metalanguage for parametric polymorphism and recursion. In recent work the first two authors and R.L. Petersen have defined a notion of parametric LAPL-structure, which are models of PILLY, in which one can...... reason using parametricity and, for example, solve a large class of domain equations, as suggested by Plotkin.In this paper, we show how an interpretation of a strict version of Bierman, Pitts and Russo's language Lily into synthetic domain theory presented by Simpson and Rosolini gives rise...

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    Monoclonal antibodies against a synthetic peptide (aa 138-152) from HIV-1 Nef protein were produced and characterized. Three hybridoma lines producing monoclonal antibodies (MAbs) against the synthetic peptide were generated by fusion between P3-X63 Ag8.653 myeloma cells and BALB/c splenocytes from...... mice immunized with the synthetic peptide coupled to keyhole limpet hemocyanin (KLH). The hybridomas were screened and selected by ELISA with the peptide coupled to bovine serum albumin (BSA) immobilized to the polystyrene surface and specificity for the peptide was confirmed by competitive ELISA...... with the peptide free in solution. The reactions of the MAbs with a 5-aa motif (WCYKL) included in the sequence were examined with synthetic peptides and two of the MAbs reacted with the motif. The recognitions of recombinant full-length Nef protein were also tested. One MAb reacted with the protein in both ELISA...

  11. Frequency-domain synthetic aperture focusing for helical ultrasonic imaging

    Science.gov (United States)

    Jin, H.; Chen, J.; Wu, E.; Yang, K.

    2017-04-01

    The synthetic aperture focusing technique (SAFT) is widely used to provide significant improvement in the lateral resolution of ultrasonic images. Frequency-domain SAFT has shown higher accuracy and greater efficiency than time-domain SAFT. However, frequency-domain SAFT should be helix-based for ultrasonic scanning of cylindrical structures such as pipes and axletrees. In this study, a frequency-domain SAFT is proposed for 3D helical ultrasonic imaging applications. This technique adjusts the phase spectra of the images to complete the synthetic aperture focusing process. The focused image is precise because the proposed algorithm is established on the basis of the wave equation in a helical coordinate system. In addition, the algorithm can efficiently separate out point scatterers and present volume scatterers. The experimental results show that the proposed algorithm yields lower side lobes and enhances the angular resolution of the ultrasonic image to approximately 1°- 1.5°, which is much better than the performance of time-domain SAFT. The maximum deviations are only 0.6 mm, 0.5°, and 0.4 mm along the r-axes, θ-axes, and z-axes, respectively, which are appropriate for normal ultrasonic nondestructive testing.

  12. [Effects and mechanism of action of synthetic peptide octarphin].

    Science.gov (United States)

    Nekrasova, Iu N; Sadovnikov, V B; Zolotarev, Iu A; Navolotskaia, E V

    2010-01-01

    We have synthesized the peptide TPLVTLFK corresponding to the β-endorphin fragment 12-19 (the name given by the authors - octarphin), and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol) and its binding to the murine peritoneal macrophages has been studied. [(3)H]Octarphin was found to bind to macrophages with high affinity (K(d) = 2.3 ± 0.2 nM) and specificity. The specific binding of [(3)H]octarphin is inhibited by unlabeled β-endorphin and selective agonist of non-opioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K(i) = 2.7 ± 0.2 and 2.4 ± 0.2 nM respectively) and not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin and [Met(5)]enkephalin (K(i) > 10 μM). Inhibiting activity of unlabeled analogs of octarphin is more then 100 times lower the unlabeled octarphin. Octarphin stimulates activity of murine immunocompetent cells in vitro and in vivo: at the concentration of 1-10 nM enhances the adhesion and spreading of peritoneal macrophages as well as their capacity to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro. Intraperitoneal administration of peptide at dose 20 μg/animal on day 7,3 and 1 prior to the isolation of cells increases activity of peritoneal macrophages as well as T- and B-spleen lymphocytes.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Takahiro Ochiya

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

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

    Science.gov (United States)

    Ochiya, Takahiro; Takenaga, Keizo; Asagiri, Masataka; Nakano, Kazumi; Satoh, Hitoshi; Watanabe, Toshiki; Imajoh-Ohmi, Shinobu; Endo, Hideya

    2015-01-01

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

  16. Synthetic peptides derived from human antimicrobial peptide ubiquicidin accumulate at sites of infections and eradicate (multi-drug resistant) Staphylococcus aureus in mice.

    Science.gov (United States)

    Brouwer, Carlo P J M; Bogaards, Sylvia J P; Wulferink, Marty; Velders, Markwin P; Welling, Mick M

    2006-11-01

    The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely recognized as an evolutionary preserved part of the innate immune system. Based on evidence in animal models and humans, AMPs are now positioned as novel anti-infective agents. The current study aimed to evaluate the potential antimicrobial activity of ubiquicidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the alpha-helix or beta-sheet domains of the human cationic peptide ubiquicidin (UBI 1-59), allowed selection of AMPs for possible treatment of MRSA infections. The strongest antibacterial activity was observed for the entire peptide UBI 1-59 and for synthetic fragments comprising amino acids 31-38. The availability, chemical synthesis opportunities, and size of these small peptides, combined with their strong antimicrobial activity towards MRSA make these compounds promising candidates for antimicrobial therapy and detection of infections in man.

  17. Synergistic Antipseudomonal Effects of Synthetic Peptide AMP38 and Carbapenems.

    Science.gov (United States)

    Rudilla, Héctor; Fusté, Ester; Cajal, Yolanda; Rabanal, Francesc; Vinuesa, Teresa; Viñas, Miguel

    2016-09-12

    The aim was to explore the antimicrobial activity of a synthetic peptide (AMP38) and its synergy with imipenem against imipenem-resistant Pseudomonas aeruginosa. The main mechanism of imipenem resistance is the loss or alteration of protein OprD. Time-kill and minimal biofilm eradication concentration (MBEC) determinations were carried out by using clinical imipenem-resistant strains. AMP38 was markedly synergistic with imipenem when determined in imipenem-resistant P. aeruginosa. MBEC obtained for the combination of AMP38 and imipenem was of 62.5 μg/mL, whereas the MBEC of each antimicrobial separately was 500 μg/mL. AMP38 should be regarded as a promising antimicrobial to fight MDR P. aeruginosa infections. Moreover, killing effect and antibiofilm activity of AMP38 plus imipenem was much higher than that of colistin plus imipenem.

  18. Performance of synthetic antiferromagnetic racetrack memory: domain wall versus skyrmion

    KAUST Repository

    Tomasello, R

    2017-06-20

    A storage scheme based on racetrack memory, where the information can be coded in a domain or a skyrmion, seems to be an alternative to conventional hard disk drive for high density storage. Here, we perform a full micromagnetic study of the performance of synthetic antiferromagnetic (SAF) racetrack memory in terms of velocity and sensitivity to defects by using experimental parameters. We find that, to stabilize a SAF skyrmion, the Dzyaloshinskii–Moriya interaction in the top and the bottom ferromagnet should have an opposite sign. The velocity of SAF skyrmions and SAF Néel domain walls are of the same order and can reach values larger than 1200 m s−1 if a spin–orbit torque from the spin-Hall effect with opposite sign is applied to both ferromagnets. The presence of disordered anisotropy in the form of randomly distributed grains introduces a threshold current for both SAF skyrmions and SAF domain walls motions.

  19. Epitope and functional specificity of monoclonal antibodies to mouse gamma interferon: the synthetic peptide approach

    International Nuclear Information System (INIS)

    Russell, J.K.; Hayes, M.P.; Carter, J.M.; Torres, B.A.; Dunn, B.M.; Johnson, H.M.

    1986-01-01

    Four anti-recombinant mouse gamma interferon (α-IFNγ) monoclonal antibodies were generated using hamster spleen cells. Binding of 125 I-IFNγ by these protein A-bound antibodies was specifically blocked by cold IFNγ. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFNγ, while a corresponding C-terminal (95-133) peptide had no effect on binding. One of the N-terminal specific monoclonal antibodies inhibited both the antiviral and macrophage priming (for tumor cell killing) activities of IFNγ, while the other two had no effect on either biological function. Blocking experiments with cold IFNγ and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFNγ. Polyclonal antibodies to either the N-terminal or C-terminal peptides also inhibited both the antiviral and macrophage priming activities of IFNγ. All of the antibodies that inhibited IFNγ function also blocked binding of IFNγ to membrane receptor on cells, while antibodies that did not inhibit function also did not block binding. The data suggest that both the N-terminal and C-terminal domains of IFNγ play an important role in its antiviral and macrophage priming functions, possibly in a cooperative manner

  20. Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs.

    Science.gov (United States)

    Düvel, Juliane; Bense, Sarina; Möller, Stefan; Bertinetti, Daniela; Schwede, Frank; Morr, Michael; Eckweiler, Denitsa; Genieser, Hans-Gottfried; Jänsch, Lothar; Herberg, Friedrich W; Frank, Ronald; Häussler, Susanne

    2016-01-01

    High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein. In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in

  1. Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface*

    Science.gov (United States)

    Jastrzebska, Beata; Chen, Yuanyuan; Orban, Tivadar; Jin, Hui; Hofmann, Lukas; Palczewski, Krzysztof

    2015-01-01

    Although homo- and heterodimerizations of G protein-coupled receptors (GPCRs) are well documented, GPCR monomers are able to assemble in different ways, thus causing variations in the interactive interface between receptor monomers among different GPCRs. Moreover, the functional consequences of this phenomenon, which remain to be clarified, could be specific for different GPCRs. Synthetic peptides derived from transmembrane (TM) domains can interact with a full-length GPCR, blocking dimer formation and affecting its function. Here we used peptides corresponding to TM helices of bovine rhodopsin (Rho) to investigate the Rho dimer interface and functional consequences of its disruption. Incubation of Rho with TM1, TM2, TM4, and TM5 peptides in rod outer segment (ROS) membranes shifted the resulting detergent-solubilized protein migration through a gel filtration column toward smaller molecular masses with a reduced propensity for dimer formation in a cross-linking reaction. Binding of these TM peptides to Rho was characterized by both mass spectrometry and a label-free assay from which dissociation constants were calculated. A BRET (bioluminescence resonance energy transfer) assay revealed that the physical interaction between Rho molecules expressed in membranes of living cells was blocked by the same four TM peptides identified in our in vitro experiments. Although disruption of the Rho dimer/oligomer had no effect on the rates of G protein activation, binding of Gt to the activated receptor stabilized the dimer. However, TM peptide-induced disruption of dimer/oligomer decreased receptor stability, suggesting that Rho supramolecular organization could be essential for ROS stabilization and receptor trafficking. PMID:26330551

  2. Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface.

    Science.gov (United States)

    Jastrzebska, Beata; Chen, Yuanyuan; Orban, Tivadar; Jin, Hui; Hofmann, Lukas; Palczewski, Krzysztof

    2015-10-16

    Although homo- and heterodimerizations of G protein-coupled receptors (GPCRs) are well documented, GPCR monomers are able to assemble in different ways, thus causing variations in the interactive interface between receptor monomers among different GPCRs. Moreover, the functional consequences of this phenomenon, which remain to be clarified, could be specific for different GPCRs. Synthetic peptides derived from transmembrane (TM) domains can interact with a full-length GPCR, blocking dimer formation and affecting its function. Here we used peptides corresponding to TM helices of bovine rhodopsin (Rho) to investigate the Rho dimer interface and functional consequences of its disruption. Incubation of Rho with TM1, TM2, TM4, and TM5 peptides in rod outer segment (ROS) membranes shifted the resulting detergent-solubilized protein migration through a gel filtration column toward smaller molecular masses with a reduced propensity for dimer formation in a cross-linking reaction. Binding of these TM peptides to Rho was characterized by both mass spectrometry and a label-free assay from which dissociation constants were calculated. A BRET (bioluminescence resonance energy transfer) assay revealed that the physical interaction between Rho molecules expressed in membranes of living cells was blocked by the same four TM peptides identified in our in vitro experiments. Although disruption of the Rho dimer/oligomer had no effect on the rates of G protein activation, binding of Gt to the activated receptor stabilized the dimer. However, TM peptide-induced disruption of dimer/oligomer decreased receptor stability, suggesting that Rho supramolecular organization could be essential for ROS stabilization and receptor trafficking. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Synthetic peptides that cause F-actin bundling and block actin depolymerization

    Science.gov (United States)

    Sederoff, Heike [Raleigh, NC; Huber, Steven C [Savoy, IL; Larabell, Carolyn A [Berkeley, CA

    2011-10-18

    Synthetic peptides derived from sucrose synthase, and having homology to actin and actin-related proteins, sharing a common motif, useful for causing acting bundling and preventing actin depolymerization. Peptides exhibiting the common motif are described, as well as specific synthetic peptides which caused bundled actin and inhibit actin depolymerization. These peptides can be useful for treating a subject suffering from a disease characterized by cells having neoplastic growth, for anti-cancer therapeutics, delivered to subjects solely, or concomitantly or sequentially with other known cancer therapeutics. These peptides can also be used for stabilizing microfilaments in living cells and inhibiting growth of cells.

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

    NARCIS (Netherlands)

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

    1999-01-01

    To use peptides as synthetic vaccines, they have to be coupled to a carrier protein to make them more immunogenic. Coupling efficiency between a carrier protein and a peptide, however, is difficult to control with respect to loading density of the peptide. This makes these carrier proteins poorly

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

    Science.gov (United States)

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

    2017-01-01

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

  6. Preparation and isolation of antibodies to human MHC class II alpha chains by aid of synthetic peptides.

    Science.gov (United States)

    Chersi, A; Romano, T F; Chillemi, F

    1989-01-01

    Antibodies against HLA Class II alpha chains were prepared by using as immunogens synthetic peptides selected from the HLA-DQ1 alpha chains sequence. Antibodies raised against peptide E2, a 15-residue fragment of the polymorphic first domain, reacted preferentially with cells with the DQ1 phenotype; however, despite the low sequence homology of this fragment with corresponding segments in DQw2 and DQw3 alpha chains, a partial crossreactivity with cells not expressing the DQw1 specificity was detected. Antibodies to peptide H, selected from the monomorphic frame, might be specific for DQ alloantigens, and presumably do not react with DR antigens. The two peptides, in addition, bind anti-Class II antibodies from the serum of a rabbit immunized with human cells, and appear to represent immunogenic linear determinants in the native glycoprotein molecule.

  7. 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...... effect of the peptides confirming the target of the peptides....

  8. Efficacy of synthetic peptides RP-1 and AA-RP-1 against Leishmania species in vitro and in vivo.

    Science.gov (United States)

    Erfe, Marie Crisel B; David, Consuelo V; Huang, Cher; Lu, Victoria; Maretti-Mira, Ana Claudia; Haskell, Jacquelyn; Bruhn, Kevin W; Yeaman, Michael R; Craft, Noah

    2012-02-01

    Host defense peptides are naturally occurring molecules that play essential roles in innate immunity to infection. Based on prior structure-function knowledge, we tested two synthetic peptides (RP-1 and AA-RP-1) modeled on the conserved, microbicidal α-helical domain of mammalian CXCL4 platelet kinocidins. These peptides were evaluated for efficacy against Leishmania species, the causative agents of the group of diseases known as leishmaniasis. In vitro antileishmanial activity was assessed against three distinct Leishmania strains by measuring proliferation, metabolic activity and parasite viability after exposure to various concentrations of peptides. We demonstrate that micromolar concentrations of RP-1 and AA-RP-1 caused dose-dependent growth inhibition of Leishmania promastigotes. This antileishmanial activity correlated with rapid membrane disruption, as well as with a loss of mitochondrial transmembrane potential. In addition, RP-1 and AA-RP-1 demonstrated distinct and significant in vivo antileishmanial activities in a mouse model of experimental visceral leishmaniasis after intravenous administration. These results establish efficacy of RP-1 lineage synthetic peptides against Leishmania species in vitro and after intravenous administration in vivo and provide further validation of proof of concept for the development of these and related systemic anti-infective peptides targeting pathogens that are resistant to conventional antibiotics.

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

    Science.gov (United States)

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

    2002-03-15

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

  10. A CD36 synthetic peptide inhibits silica-induced lung fibrosis in the mice.

    Science.gov (United States)

    Wang, Xin; Lv, Lina; Chen, Ying; Chen, Jie

    2010-02-01

    Silicosis is a kind of pneumoconiosis caused by inhalation of silica dust, which is characterized by lung fibrosis. The biologically active form of transforming growth factor-beta1 (TGF-beta1) plays a key role in the development of lung fibrosis. CD36 is involved in the transformation of latent TGF-beta1 (L-TGF-beta1) to active TGF-beta1. The antagonistic effect of the synthetic peptide was analyzed by the administration of CD36 (93-110) synthetic peptide to the silicosis model of mice. The hydroxyproline content of the silica + CD36 (93-110) synthetic peptide group was significantly lower than that of the other experimental groups [silica and silica + CD36 (208-225) synthetic peptide groups] (p synthetic peptide group were less than those of the other experimental groups. The expressions of collagen I and III of the silica + CD36 (93-110) synthetic peptide group were significantly lower than those of the other experimental groups (p synthetic peptide reduced the tissue fibrotic pathologies and collagen accumulation in the silicosis model of mice, resulting in the decreased severity of silica-induced lung fibrosis.

  11. Eradication of Staphylococcus aureus Biofilm Infections Using Synthetic Antimicrobial Peptides.

    Science.gov (United States)

    Zapotoczna, Marta; Forde, Éanna; Hogan, Siobhan; Humphreys, Hilary; O'Gara, James P; Fitzgerald-Hughes, Deirdre; Devocelle, Marc; O'Neill, Eoghan

    2017-03-15

    Here, we demonstrate that antimicrobial peptides (AMPs) are an effective antibiofilm treatment when applied as catheter lock solutions (CLSs) against S. aureus biofilm infections. The activity of synthetic AMPs (Bac8c, HB43, P18, Omiganan, WMR, Ranalexin, and Polyphemusin) was measured against early and mature biofilms produced by methicillin-resistant S. aureus and methicillin-susceptible S. aureus isolates from patients with device-related infections grown under in vivo-relevant biofilm conditions. The cytotoxic and hemolytic activities of the AMPs against human cells and their immunomodulatory potential in human blood were also characterized. The D-Bac8c2,5Leu variant emerged as the most effective AMP during in vitro studies and was also highly effective in eradicating S. aureus biofilm infection when used in a CLS rat central venous catheter infection model. These data support the potential use of D-Bac8c2,5Leu, alone or in combination with other AMPs, in the treatment of S. aureus intravenous catheter infections. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  12. Antigenicity of peptides comprising the immunosuppressive domain of the retroviral envelope glycoprotein [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Bryony Jenkins

    2016-12-01

    Full Text Available To achieve persistent infection of the host, viruses often subvert or suppress host immunity through mechanisms that are not entirely understood. The envelope glycoprotein of several retroviruses is thought to possess potent immunosuppressive activity, mapped to a 17-amino acid residue conserved domain. Synthetic peptides corresponding to this immunosuppressive domain can inhibit lymphocyte activation, whereas mutation of key domain residues can increase the lymphocyte response to linked antigenic epitopes. Using three T cell receptors (TCRs of defined specificity, we examine the effect of the immunosuppressive domain on the T cell response to their respective antigenic peptides. We find that fusion of a T cell epitope to the immunosuppressive domain can greatly modulate its potency. However, the effects heavily depend on the particular combination of TCR and peptide-major histocompatibility complex class II (pMHC II, and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of TCR-pMHC interaction. These results offer an alternative explanation for the immunogenicity of T cell epitopes comprising the putative immunosuppressive domain, which is more consistent with an effect on peptide antigenicity than true immunosuppressive activity.

  13. Antigenicity of peptides comprising the immunosuppressive domain of the retroviral envelope glycoprotein [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Bryony Jenkins

    2017-02-01

    Full Text Available To achieve persistent infection of the host, viruses often subvert or suppress host immunity through mechanisms that are not entirely understood. The envelope glycoprotein of several retroviruses is thought to possess potent immunosuppressive activity, mapped to a 17-amino acid residue conserved domain. Synthetic peptides corresponding to this immunosuppressive domain can inhibit lymphocyte activation, whereas mutation of key domain residues can increase the lymphocyte response to linked antigenic epitopes. Using three T cell receptors (TCRs of defined specificity, we examine the effect of the immunosuppressive domain on the T cell response to their respective antigenic peptides. We find that fusion of a T cell epitope to the immunosuppressive domain can greatly modulate its potency. However, the effects heavily depend on the particular combination of TCR and peptide-major histocompatibility complex class II (pMHC II, and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of pMHC formation or TCR-pMHC interaction. These results offer an alternative explanation for the immunogenicity of T cell epitopes comprising the putative immunosuppressive domain, which is more consistent with an effect on peptide antigenicity than true immunosuppressive activity.

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

    Science.gov (United States)

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

    2017-01-01

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

  15. Helical synthetic peptides that stimulate cellular cholesterol efflux

    Science.gov (United States)

    Bielicki, John K.; Natarajan, Pradeep

    2010-04-06

    The present invention provides peptides comprising at least one amphipathic alpha helix and having an cholesterol mediating activity and a ABCA stabilization activity. The invention further provides methods of using such peptides.

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

    Science.gov (United States)

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

    2017-02-25

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

  17. In vitro determination of the short-chain synthetic peptide RP13 antimicrobial activity.

    Science.gov (United States)

    Sánchez, Adrián; Calderón, Ernesto; Castañón-Alonso, Sandra L; Santos, Araceli; Hernández, Beatriz; Vázquez, Alfredo

    2014-01-01

    The proliferation of antibiotic-resistant microorganisms, along with the lack of new drugs against them, has elicited the interest of the scientific community on the study and development of endogenous synthetic compounds with bacteriostatic or bactericidal activity. In recent years, several short-chain, low molecular weight peptides isolated from natural sources such as plants and animals have demonstrated an array of antimicrobial activities. Despite having structural characteristics similar to microbicidal peptides isolated from human platelets, peptide RP11 does not exhibit antimicrobial activity. In vitro determination of the antimicrobial activity of the synthetic peptide RP13. Peptide RP13 was prepared modifying the original amino acids sequence of peptide RP11, reversing the position of the amino acids lysine and tyrosine in order to modify the conformation of the original peptide. These amino acids are localized close to the N-terminus of the peptidic chain. Peptide RP13 was prepared in solution using conventional methods for peptide synthesis. The antimicrobial activity of RP13 was assessed against the microorganisms S. aureus, E. faecalis and E. coli in a test solution and later evaluated by cultivation of plates during the first 2 h after inoculation of bacteria. RP13 activity antimicrobial was compared against tetracycline, a broad-spectrum antibiotic. The new peptide RP13, resulting form the structural modification of the amino acid sequence of peptide RP11, displayed antimicrobial activity. RP13 demonstrated to be more efficient inhibiting the growth of gram-positive than gramnegative bacteria. The structural modification of peptide RP11, obtained from human platelets, resulted in a new peptide with improved antimicrobial activity. These results clearly demonstrate that peptides of natural origin, as well as their synthetic analogs, represent an attractive alternative against pathogenic agents.

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

    Science.gov (United States)

    Kao, Bunsho; Kadomatsu, Koichi; Hosaka, Yoshiaki

    2009-09-01

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

  19. Interaction of Serum Antibodies from Breast Cancer Patients with Synthetic Peptides.

    Science.gov (United States)

    Podlesnykh, S V; Kolosova, E A; Shcherbakov, D N; Shaidurov, A A; Anisimov, D S; Ryazanov, M A; Johnston, S A; Shoikhet, Ya N; Petrova, V D; Lazarev, A F; Chapoval, A I

    2016-10-01

    The blood serum of tumor patients contains antibodies recognizing tumor-associated antigens and other molecular products of tumor growth. We studied the interaction of blood antibodies from breast cancer patients with synthetic peptides that were applied on the microchip surface. The serum from healthy volunteers and breast cancer patients was shown to contain antibodies that interact with various peptides. Statistically significant between-group differences were observed in the level of binding with 122 informative peptides (0.01% of the total number of peptides on a microchip). Analysis of antibodies that interact with the peptide panel holds much promise for the diagnostics of breast cancer.

  20. Tips and Tricks for Exogenous Application of Synthetic Post-translationally Modified Peptides to Plants.

    Science.gov (United States)

    Czyzewicz, Nathan; Stes, Elisabeth; De Smet, Ive

    2017-01-01

    The first signaling peptide discovered and purified was insulin in 1921. However, it was not until 1991 that the first peptide signal, systemin, was discovered in plants. Since the discovery of systemin, peptides have emerged as a potent and diverse class of signaling molecules in plant systems. Peptides consist of small amino acid sequences, which often act as ligands of receptor kinases. However, not all peptides are created equal, and signaling peptides are grouped into several subgroups dependent on the type of post-translational processing they undergo. Here, we focus on the application of synthetic, post-translationally modified peptides (PTMPs) to plant systems, describing several methods appropriate for the use of peptides in Arabidopsis thaliana and crop models.

  1. Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

    Czech Academy of Sciences Publication Activity Database

    Ježek, Jan; El Ridi, R.; Salah, M.; Wagih, A.; Aziz, H. W.; Tallima, H.; El Shafie, M. H.; Khalek, T. A.; Ammou, F. F. A.; Strongylis, C.; Moussis, V.; Tsikaris, V.

    2008-01-01

    Roč. 90, č. 3 (2008), s. 349-357 ISSN 0006-3525 Institutional research plan: CEZ:AV0Z40550506 Keywords : cathepsin L proteinase * peptides * sequential oligopeptide carriers * synthetic peptide vaccine * Fasciiola gigantica Subject RIV: CC - Organic Chemistry Impact factor: 2.823, year: 2008

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

    DEFF Research Database (Denmark)

    Pedersen, Martin Holst Friborg; Baun, Michael

    2012-01-01

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

  3. Activity of Novel Synthetic Peptides against Candida albicans.

    Science.gov (United States)

    Lum, Kah Yean; Tay, Sun Tee; Le, Cheng Foh; Lee, Vannajan Sanghiran; Sabri, Nadia Hanim; Velayuthan, Rukumani Devi; Hassan, Hamimah; Sekaran, Shamala Devi

    2015-05-12

    Candida spp. are the most common causes of fungal infections worldwide. Among the Candida species, Candida albicans remains the predominant species that causes invasive candidiasis in most countries. In this study, we used two peptides, KABT-AMP and uperin 3.6 as templates to develop novel antifungal peptides. Their anticandidal activity was assessed using a combination of MIC, time-killing assay and biofilm reduction assay. Hybrid peptides, KU2 and KU3 containing a mixed backbone of KABT-AMP and Uperin 3.6 demonstrated the most potent anticandidal activity with MIC values ranging from 8-16 mg/L. The number of Trp residues and the amphipathic structure of peptides probably enhanced the anticandidal activity of peptides. Increasing the cationicity of the uperin 3.6 analogues resulted in reduced MIC from the range of 64-128 mg/L to 16-64 mg/L and this was also correlated with the antibiofilm activity and killing kinetics of the peptides. Peptides showed synergistic effects when used in combination with conventional antifungals. Peptides demonstrated low haemolytic activity but significant toxicity on two normal human epithelial cell lines. This study provides us with a better understanding on the structure-activity relationship and the balance between cationicity and hydrophobicity of the peptides although the therapeutic application of the peptides is limited.

  4. Diagnosis of tuberculosis infection based on synthetic peptides from Mycobacterium tuberculosis antigen 85 complex.

    Science.gov (United States)

    Kashyap, Rajpal S; Shekhawat, Seema D; Nayak, Amit R; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F

    2013-06-01

    The laboratory diagnosis of pulmonary tuberculosis (TB) and tuberculous meningitis (TBM) is particularly challenging. The aim of the present work is to develop an immunoassay for the diagnosis of TB infection, using synthetic peptides of antigen (Ag) 85 complex of M. tuberculosis (Mtb) H37Rv. Four peptides (7-10 amino acids long) corresponding to group-specific epitopes of Ag 85 complex of Mtb were synthesized. All peptides were evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoreactivity with sera and CSF samples of TB and TBM patients respectively. The diagnostic value of the four peptides was evaluated in both the samples. It was observed that Ag 85 peptide 1, 3 and 4 had the highest positive rates in the pulmonary patients; however, Ag 85 peptide 1 and 2 had shown good positivity in the TBM subjects. The synthetic peptide based ELISA using Ag 85 complex peptides is a sensitive, specific, rapid and cost effective immunoassay for early diagnosis of pulmonary and extrapulmonary TB. In addition, these synthetic peptides are comparatively easy to produce in a reproducible manner compared with the whole antigen. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. mRNA Display Based Selections Using Synthetic Peptide and Natural Protein Libraries

    Science.gov (United States)

    Cotten, Steve W.; Zou, Jianwei; Wang, Rong; Huang, Bao-cheng; Liu, Rihe

    2014-01-01

    mRNA display is a powerful in vitro selection technique that can be applied towards the identification of peptides or proteins with desired properties. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins in an RNase free environment. This protocol outlines the generation of synthetic peptide and natural proteome libraries as well as the steps required for generation of mRNA-protein fusion libraries, in vitro selection, and regeneration of the selected sequences. The selection procedures for the identification of Ca2+ dependent calmodulin binding proteins from synthetic peptide and natural proteome libraries are presented. PMID:22094812

  6. Class of cyclic ribosomal peptide synthetic genes in filamentous fungi.

    Science.gov (United States)

    Nagano, Nozomi; Umemura, Myco; Izumikawa, Miho; Kawano, Jin; Ishii, Tomoko; Kikuchi, Moto; Tomii, Kentaro; Kumagai, Toshitaka; Yoshimi, Akira; Machida, Masayuki; Abe, Keietsu; Shin-Ya, Kazuo; Asai, Kiyoshi

    2016-01-01

    Ustiloxins were found recently to be the first example of cyclic peptidyl secondary metabolites that are ribosomally synthesized in filamentous fungi. In this work, two function-unknown genes (ustYa/ustYb) in the gene cluster for ustiloxins from Aspergillus flavus were found experimentally to be involved in cyclization of the peptide. Their homologous genes are observed mainly in filamentous fungi and mushrooms. They have two "HXXHC" motifs that might form active sites. Computational genome analyses showed that these genes are frequently located near candidate genes for ribosomal peptide precursors, which have signal peptides at the N-termini and repeated sequences with core peptides for the cyclic portions, in the genomes of filamentous fungi, particularly Aspergilli, as observed in the ustiloxin gene cluster. Based on the combination of the ustYa/ustYb homologous genes and the nearby ribosomal peptide precursor candidate genes, 94 ribosomal peptide precursor candidates that were identified computationally from Aspergilli genome sequences were classified into more than 40 types including a wide variety of core peptide sequences. A set of the predicted ribosomal peptide biosynthetic genes was experimentally verified to synthesize a new cyclic peptide compound, designated as asperipin-2a, which comprises the amino acid sequence in the corresponding precursor gene, distinct from the ustiloxin precursors. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

    Science.gov (United States)

    Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

    2017-11-13

    A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

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

    Science.gov (United States)

    Sreejalekshmi, Kumaran G; Nair, Prabha D

    2011-02-01

    Biomimetic and bioactive biomaterials are desirable as tissue engineering scaffolds by virtue of their capability to mimic natural environments of the extracellular matrix. Biomimeticity has been achieved by the incorporation of synthetic short peptide sequences into suitable materials either by surface modification or by bulk incorporation. Research in this area has identified several novel synthetic peptide segments, some of them with cell-specific interactions, which may serve as potential candidates for use in explicit tissue applications. This review focuses on the developments and prospective directions of incorporating short synthetic peptide sequences onto scaffolds for tissue engineering, with emphasis on the chemistry of peptide immobilization and subsequent cell responses toward modified scaffolds. The article provides a decision-tree-type flow chart indicating the most probable cellular events on a given peptide-modified scaffold along with the consolidated list of synthetic peptide sequences, supports as well as cell types used in various tissue engineering studies, and aims to serve as a quick reference guide to peptide chemists and material scientists interested in the field. 2010 Wiley Periodicals, Inc.

  9. Design of Surfactant Protein B Peptide Mimics Based on the Saposin Fold for Synthetic Lung Surfactants.

    Science.gov (United States)

    Walther, Frans J; Gordon, Larry M; Waring, Alan J

    2016-01-01

    Surfactant protein (SP)-B is a 79-residue polypeptide crucial for the biophysical and physiological function of endogenous lung surfactant. SP-B is a member of the Saposin or Saposin-like proteins (SAPLIP) family of proteins that share an overall three-dimensional folding pattern based on secondary structures and disulfide connectivity and exhibit a wide diversity of biological functions. Here we review the synthesis, molecular biophysics and activity of synthetic analogs of Saposin proteins designed to mimic those interactions of the parent proteins with lipids that enhance interfacial activity. Saposin proteins generally interact with target lipids as either monomers or multimers via well-defined amphipathic helices, flexible hinge domains, and insertion sequences. Based on the known 3D-structural motif for the Saposin family, we show how bioengineering techniques may be used to develop minimal peptide constructs that maintain desirable structural properties and activities in biomedical applications. One important application is the molecular design, synthesis and activity of Saposin mimics based on the SP-B structure. Synthetic lung surfactants containing active SP-B analogs may be potentially useful in treating diseases of surfactant deficiency or dysfunction including the neonatal respiratory distress syndrome and acute lung injury/acute respiratory distress syndrome.

  10. Analysis of HIV-1 fusion peptide inhibition by synthetic peptides from E1 protein of GB virus C.

    Science.gov (United States)

    Sánchez-Martín, Maria Jesús; Hristova, Kalina; Pujol, Montserrat; Gómara, Maria J; Haro, Isabel; Alsina, M Asunción; Busquets, M Antònia

    2011-08-01

    The aim of this study was to identify proteins that could inhibit the activity of the peptide sequence representing the N-terminal of the surface protein gp41 of HIV, corresponding to the fusion peptide of the virus (HIV-1 FP). To do this we synthesized and studied 58 peptides corresponding to the envelope protein E1 of the hepatitis G virus (GBV-C). Five of the E1 synthetic peptides: NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18) and AQLVGELGSLYGPLSVSA (P22) were capable of inhibiting the leakage of vesicular contents caused by HIV-1 FP. A series of experiments were carried out to determine how these E1 peptides interact with HIV-1 FP. Our studies analyzed the interactions with and without the presence of lipid membranes. Isothermal titration calorimetry revealed that the binding of P7, P18 and P22 peptides to HIV-1 FP is strongly endothermic, and that binding is entropy-driven. Gibbs energy for the process indicates a spontaneous binding between E1 peptides and HIV-1 FP. Moreover, confocal microscopy of Giant Unilamellar Vesicles revealed that the disruption of the lipid bilayer by HIV-1 FP alone was inhibited by the presence of any of the five selected peptides. Our results highlight that these E1 synthetic peptides could be involved in preventing the entry of HIV-1 by binding to the HIV-1 FP. Therefore, the continued study into the interaction between GBV-C peptides and HIV-1 FP could lead to the development of new therapeutic agents for the treatment of AIDS. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    1991-11-01

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

  12. Specificity of rabbit antibodies elicited by related synthetic peptides.

    Science.gov (United States)

    Chersi, A; Houghten, R A; Chillemi, F; Zito, R; Centis, D

    1986-01-01

    Three 17-residue peptides, presenting from 65% to 70% sequence homology, and one endecapeptide, with no apparent homology with the first three, were chemically synthesized and investigated in their ability to elicit rabbit antipeptide antibodies. The complex cross reactivities of the antisera were investigated by testing the binding of the antibodies to the intact peptides, to their enzymatic fragments, and by the use of specific immunoadsorbents. Antipeptide antibodies may or may not crossreact with related "parent" peptides, this depending upon number, distribution, and localization of amino acid differences in low or high antigenicity regions of the immunogen. Related peptides may elicit antibodies that crossreact almost completely, and therefore not specific for one or the other "parent" peptide. Those antibodies may therefore be of little use for the selective recognition of closely related structures.

  13. Antifungal activity of synthetic peptides derived from Impatiens balsamina antimicrobial peptides Ib-AMP1 and Ib-AMP4

    NARCIS (Netherlands)

    Thevissen, K.; Francois, E.J.A.; Sijtsma, L.; Amerongen, van A.; Schaaper, W.M.M.; Meloen, R.; Posthuma-Trumpie, G.A.; Broekaert, W.F.; Cammue, B.P.A.

    2005-01-01

    Seeds of Impatiens balsamina contain a set of related antimicrobial peptides (Ib-AMPs). We have produced a synthetic variant of Ib-AMP1, oxidized to the bicyclic native conformation, which was fully active on yeast and fungal strains; and four linear 20-mer Ib-AMP variants, including two all-d

  14. Synthetic Peptides as Potential Antigens for Cutaneous Leishmaniosis Diagnosis.

    Science.gov (United States)

    Link, Juliana Seger; Alban, Silvana Maria; Soccol, Carlos Ricardo; Pereira, Gilberto Vinicius Melo; Thomaz Soccol, Vanete

    2017-01-01

    This work's goal was to research new candidate antigens for cutaneous leishmaniosis (CL). In order to reach the goal, we used random peptide phage display libraries screened using antibodies from Leishmania braziliensis patients. After selection, three peptides (P1, P2, and P3) were synthesized using Fmoc chemistry. The peptides individually or a mixture of them (MIX) was subsequently emulsified in complete and incomplete Freund's adjuvant and injected subcutaneously in golden hamsters. Sera from the hamsters administered with P1 presented antibodies that recognized proteins between 76 and 150 kDa from L. braziliensis . Sera from hamsters which had peptides P2 and P3, as well as the MIX, administered presented antibodies that recognized proteins between 52 and 76 kDa of L. braziliensis . The research on the similarity of the peptides' sequences in protein databases showed that they match a 63 kDa glycoprotein. The three peptides and the MIX were recognized by the sera from CL patients by immunoassay approach (ELISA). The peptides' MIX showed the best performance (79% sensitivity) followed by the P1 (72% sensitivity), and the AS presented 91% sensitivity. These results show a new route for discovering molecules for diagnosis or for immunoprotection against leishmaniosis.

  15. Identification of Staphylococcal Enterotoxin B Sequences Important for Induction of Lymphocyte Proliferation by Using Synthetic Peptide Fragments of the Toxin

    Science.gov (United States)

    1994-08-01

    Staphylococcal Enterotoxin B Sequences STO=T Important for induction of lymphocyte proliferatio WPP8, WPPM,by using synthetic peptide fragments of the...Lymphocyte Proliferation by Using Synthetic Peptide Fragments of the Toxin MARTI JETT.I* ROGER NEILL,’ CHRISTOPHER WELCH,’ THOMAS BOYLE,’ EDWARD BERNTON...fragment of SEC (41) and for an amino- ment of endotoxic shock (42), induction of immunosuppres- terminal synthetic peptide of SEA (36). Another study

  16. Design and Engineering Strategies for Synthetic Antimicrobial Peptides

    Science.gov (United States)

    Tossi, Alessandro

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

  17. Development of peptide-functionalized synthetic hydrogel microarrays for stem cell and tissue engineering applications.

    Science.gov (United States)

    Jia, Jia; Coyle, Robert C; Richards, Dylan J; Berry, Christopher Lloyd; Barrs, Ryan Walker; Biggs, Joshua; James Chou, C; Trusk, Thomas C; Mei, Ying

    2016-11-01

    Synthetic polymer microarray technology holds remarkable promise to rapidly identify suitable biomaterials for stem cell and tissue engineering applications. However, most of previous microarrayed synthetic polymers do not possess biological ligands (e.g., peptides) to directly engage cell surface receptors. Here, we report the development of peptide-functionalized hydrogel microarrays based on light-assisted copolymerization of poly(ethylene glycol) diacrylates (PEGDA) and methacrylated-peptides. Using solid-phase peptide/organic synthesis, we developed an efficient route to synthesize methacrylated-peptides. In parallel, we identified PEG hydrogels that effectively inhibit non-specific cell adhesion by using PEGDA-700 (M. W.=700) as a monomer. The combined use of these chemistries enables the development of a powerful platform to prepare peptide-functionalized PEG hydrogel microarrays. Additionally, we identified a linker composed of 4 glycines to ensure sufficient exposure of the peptide moieties from hydrogel surfaces. Further, we used this system to directly compare cell adhesion abilities of several related RGD peptides: RGD, RGDS, RGDSG and RGDSP. Finally, we combined the peptide-functionalized hydrogel technology with bioinformatics to construct a library composed of 12 different RGD peptides, including 6 unexplored RGD peptides, to develop culture substrates for hiPSC-derived cardiomyocytes (hiPSC-CMs), a cell type known for poor adhesion to synthetic substrates. 2 out of 6 unexplored RGD peptides showed substantial activities to support hiPSC-CMs. Among them, PMQKMRGDVFSP from laminin β4 subunit was found to support the highest adhesion and sarcomere formation of hiPSC-CMs. With bioinformatics, the peptide-functionalized hydrogel microarrays accelerate the discovery of novel biological ligands to develop biomaterials for stem cell and tissue engineering applications. In this manuscript, we described the development of a robust approach to prepare peptide

  18. Synthetic Peptide-Based Antibody Detection for Diagnosis of Chikungunya Infection with and without Neurological Complications.

    Science.gov (United States)

    Kashyap, Rajpal S; Bhullar, Shradha S; Chandak, Nitin H; Taori, Girdhar M

    2016-01-01

    Synthetic peptide-based diagnosis of Chikungunya can be an efficient and more accessible approach in immunodiagnostics. Here, we describe the identification of Chikungunya-specific 40 kD protein for development of synthetic peptide-based enzyme-linked immunosorbent assay for the detection of Chikungunya virus-specific antibodies in the patient's sample. The total sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile of the patient's sample can be done to identify specific protein bands. The identified proteins can be subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) for characterization. After characterization, immunogenic peptides can be designed using softwares and subsequently synthesized chemically. The peptides can be used to develop more specific, sensitive, and simpler diagnostic assay.

  19. An Overview on the Field of Micro- and Nanotechnologies for Synthetic Peptide-Based Vaccines

    Directory of Open Access Journals (Sweden)

    Aiala Salvador

    2011-01-01

    Full Text Available The development of synthetic peptide-based vaccines has many advantages in comparison with vaccines based on live attenuated organisms, inactivated or killed organism, or toxins. Peptide-based vaccines cannot revert to a virulent form, allow a better conservation, and are produced more easily and safely. However, they generate a weaker immune response than other vaccines, and the inclusion of adjuvants and/or the use of vaccine delivery systems is almost always needed. Among vaccine delivery systems, micro- and nanoparticulated ones are attractive, because their particulate nature can increase cross-presentation of the peptide. In addition, they can be passively or actively targeted to antigen presenting cells. Furthermore, particulate adjuvants are able to directly activate innate immune system in vivo. Here, we summarize micro- and nanoparticulated vaccine delivery systems used in the field of synthetic peptide-based vaccines as well as strategies to increase their immunogenicity.

  20. An Overview on the Field of Micro- and Nanotechnologies for Synthetic Peptide-Based Vaccines

    Science.gov (United States)

    Salvador, Aiala; Igartua, Manoli; Hernández, Rosa Maria; Pedraz, José Luis

    2011-01-01

    The development of synthetic peptide-based vaccines has many advantages in comparison with vaccines based on live attenuated organisms, inactivated or killed organism, or toxins. Peptide-based vaccines cannot revert to a virulent form, allow a better conservation, and are produced more easily and safely. However, they generate a weaker immune response than other vaccines, and the inclusion of adjuvants and/or the use of vaccine delivery systems is almost always needed. Among vaccine delivery systems, micro- and nanoparticulated ones are attractive, because their particulate nature can increase cross-presentation of the peptide. In addition, they can be passively or actively targeted to antigen presenting cells. Furthermore, particulate adjuvants are able to directly activate innate immune system in vivo. Here, we summarize micro- and nanoparticulated vaccine delivery systems used in the field of synthetic peptide-based vaccines as well as strategies to increase their immunogenicity. PMID:21773041

  1. In vitro digestive stability of complexes between gliadin and synthetic blocking peptides.

    Science.gov (United States)

    Hoffmann, Karolina; Carlsson, Nils-Gunnar; Alminger, Marie; Chen, Tingsu; Wold, Agnes; Olsson, Olof; Sandberg, Ann-Sofie

    2011-05-01

    Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

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

    Directory of Open Access Journals (Sweden)

    Hernando Curtidor

    2016-12-01

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

  3. Lipid membrane editing with peptide cargo linkers in cells and synthetic nanostructures

    OpenAIRE

    Pan, Hua; Myerson, Jacob W.; Ivashyna, Olena; Soman, Neelesh R.; Marsh, Jon N.; Hood, Joshua L.; Lanza, Gregory M.; Schlesinger, Paul H.; Wickline, Samuel A.

    2010-01-01

    Current strategies for deploying synthetic nanocarriers involve the creation of agents that incorporate targeting ligands, imaging agents, and/or therapeutic drugs into particles as an integral part of the formulation process. Here we report the development of an amphipathic peptide linker that enables postformulation editing of payloads without the need for reformulation to achieve multiplexing capability for lipidic nanocarriers. To exemplify the flexibility of this peptide linker strategy,...

  4. Radioimmunoassay for the middle region of human parathyroid hormone: comparison of two radioiodinated synthetic peptides

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, M.E.; Marx, S.J. (National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Bethesda, MD (USA))

    1985-01-01

    Two synthetic peptides were evaluated to develop radioligands for midregion-specific radioimmunoassay (RIA) of human parathyroid hormone (hPTH). Both radioligands were tested using three anti-PTH sera of proven clinical utility. While each of these midregion-directed antisera showed unique specificity, they all reacted with high affinity with both radioligands and none of them discriminated significantly between the two synthetic midregion peptides. Analysis of data on the relation of serum calcium and hPTH midregion immunoreactivity showed a useful separation of groups (all nonazotemic) with primary hyperparathyroidism, secondary hyperparathyroidism, primary hypoparathyroidism and secondary hypoparathyroidism.

  5. Radioimmunoassay for the middle region of human parathyroid hormone: comparison of two radioiodinated synthetic peptides

    International Nuclear Information System (INIS)

    Sharp, M.E.; Marx, S.J.

    1985-01-01

    Two synthetic peptides were evaluated to develop radioligands for midregion-specific radioimmunoassay (RIA) of human parathyroid hormone (hPTH). Both radioligands were tested using three anti-PTH sera of proven clinical utility. While each of these midregion-directed antisera showed unique specificity, they all reacted with high affinity with both radioligands and none of them discriminated significantly between the two synthetic midregion peptides. Analysis of data on the relation of serum calcium and hPTH midregion immunoreactivity showed a useful separation of groups (all nonazotemic) with primary hyperparathyroidism, secondary hyperparathyroidism, primary hypoparathyroidism and secondary hypoparathyroidism. (Auth.)

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  7. MS/MS of synthetic peptide is not sufficient to confirm new types of protein modifications.

    Science.gov (United States)

    Lee, Sangkyu; Tan, Minjia; Dai, Lunzhi; Kwon, Oh Kwang; Yang, Jeong Soo; Zhao, Yingming; Chen, Yue

    2013-02-01

    Protein post-translational modification (PTM) is one of the major regulatory mechanisms that fine-tune protein functions. Undescribed mass shifts, which may suggest novel types of PTMs, continue to be discovered because of the availabilities of more sensitive mass spectrometry technologies and more powerful sequence alignment algorithms. In this study, the histone extracted from HeLa cells was analyzed using an approach that takes advantages of in vitro propionylation, efficient peptide separation using isoelectric focusing fractionation, and the high sensitivity of the linear ion trap coupled with hybrid FT mass spectrometer. One modified peptide was identified with a new type of protein modification (+42 Da), which was assigned to acetylation of threonine 15 in histone2A. The modified peptide was verified by careful manual evaluation of the tandem mass spectrum and confirmed by high-resolution MS/MS analysis of the corresponding synthetic peptide. However, HPLC coelution and MS/MS/MS of key ions showed that the +42 Da mass shifts at threonine residue did not correspond to acetylation. The key fragment ion, y4, in the MS/MS/MS spectra (indicative of the modification site) differed between the in vivo and synthetic peptide. We showed that the misidentification was originated from sequence homologues and chemical derivitization during sample preparation. This result indicated that a more stringent procedure that includes MS/MS, MS/MS/MS, and HPLC coelution of synthetic peptides is required to identify a new PTM.

  8. Inhibition of emetic and superantigenic activities of staphylococcal enterotoxin A by synthetic peptides.

    Science.gov (United States)

    Maina, Edward K; Hu, Dong-Liang; Asano, Krisana; Nakane, Akio

    2012-11-01

    Staphylococcus aureus is a major human pathogen producing different types of toxins. Enterotoxin A (SEA) is the most common type among clinical and food-related strains. The aim of the present study was to estimate functional regions of SEA that are responsible for emetic and superantigenic activities using synthetic peptides. A series of 13 synthetic peptides corresponding to specific regions of SEA were synthesized, and the effect of these peptides on superantigenic activity of SEA including interferon γ (IFN-γ) production in mouse spleen cells, SEA-induced lethal shock in mice, spleen cell proliferation in house musk shrew, and emetic activity in shrews were assessed. Pre-treatment of spleen cells with synthetic peptides corresponding to the regions 21-40, 35-50, 81-100, or 161-180 of SEA significantly inhibited SEA-induced IFN-γ production and cell proliferation. These peptides also inhibited SEA-induced lethal shock. Interestingly, peptides corresponding to regions 21-40, 35-50 and 81-100 significantly inhibited SEA-induced emesis in house musk shrews, but region 161-180 did not. These findings indicated that regions 21-50 and 81-100 of SEA are important for both superantigenic and emetic activities of SEA molecule while region 161-180 is involved in superantigenic activity but not emetic activity of SEA. These regions could be important targets for therapeutic intervention against SEA exposure. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Synthetic peptides mimicking lipopolysaccharide as a potential vaccine candidates against Vibrio cholerae serogroup O1.

    Science.gov (United States)

    Ghazi, Fatemeh Mohammad Pour; Gargari, Seyed Latif Mousavi

    2017-08-01

    Cholera is a life-threatening diarrhea caused mainly by Gram-negative marine habitant Vibrio cholerae serogroup O1. Cholera vaccination is limited mainly to developed countries, due to the cumbersome and expensive task of vaccine production. In the present work, the aim was to study the immunogenicity of the synthetic mimotopes through two different routes of injection and oral administration. Lipopolysaccharide (LPS) is one of the immunogenic components in Gram-negative bacteria, which cannot be used as a vaccine candidate, due to its high toxic effect. Three phage-displayed selected peptides, with high affinity to anti-LPS VHH tested in our previous study, were chemically synthesized and used as a potential vaccine candidate. In order to enhance the antigenic properties and safe delivery, these peptides were conjugated to BSA as a carrier and encapsulated with PLGA. Peptides were injected intra-peritoneally or administered orally, alone or in combined form. Mice sera and feces were collected for assessment of humoral and mucosal antibody titers, respectively. ELISA plates were coated with mimotope conjugates and V. cholerae , Shigella sonnei and ETEC were used as target antigens. Antibody titer was measured by adding IgG and IgA as primary antibodies. Mice receiving three selected synthetic peptide conjugates (individually or in combination) showed higher antibody titer compared to control groups. The mice immunized with synthetic peptides were protected against more than 15 LD50 of V. cholerae. These peptides are mimicking LPS and can potentially act as vaccine candidates against V. cholerae.

  10. Filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide

    Science.gov (United States)

    van Houten, N.E.; Zwick, M.B.; Menendez, A.; Scott, J.K.

    2007-01-01

    Filamentous bacteriophage are widely used as immunogenic carriers for “phage-displayed” recombinant peptides. Here we report that they are an effective immunogenic carrier for synthetic peptides. The f1.K phage was engineered to have an additional Lys residue near the N-terminus of the major coat protein, pVIII, so as to enhance access to chemical cross-linking agents. The dimeric synthetic peptide, B2.1, was conjugated to f1.K (f1.K/B2.1) in high copy number and compared as an immunogen to B2.1 conjugated to ovalbumin (OVA/B2.1) and to phage-displayed, recombinant B2.1 peptide. All immunogens were administered without adjuvant. The serum antibody titers were measured against: the peptide, the carrier, and, if appropriate, the cross-linker. All immunogens elicited anti-peptide antibody titers, with those elicited by OVA/B2.1 exceeding those by f1.K/B2.1; both titers were greater than that elicited by recombinant B2.1 phage. Comparison of the anti-peptide and anti-carrier antibody responses showed that f1.K/B2.1 elicited a more focused anti-peptide antibody response than OVA/B2.1. The anti-peptide antibody response against f1.K/B2.1 was optimized for the injection route, dose and adjuvant. Dose and adjuvant did not have a significant effect on anti-peptide antibody titers, but a change in injection route from intraperitoneal (IP) to subcutaneous (SC) enhanced anti-peptide antibody titers after seven immunizations. The optimized anti-peptide antibody response exceeded the anti-carrier one by 21-fold, compared to 0.07-fold elicited by OVA/B2.1. This indicates that phage as a carrier can focus the antibody response against the peptide. The results are discussed with respect to the advantages of phage as an alternative to traditional carrier proteins for synthetic peptides, carbohydrates and haptens, and to further improvements in phage as immunogenic carriers. PMID:16488517

  11. Effect of enamel matrix derivative and of proline-rich synthetic peptides on the differentiation of human mesenchymal stem cells toward the osteogenic lineage.

    Science.gov (United States)

    Ramis, Joana Maria; Rubert, Marina; Vondrasek, Jiri; Gayà, Antoni; Lyngstadaas, Staale Petter; Monjo, Marta

    2012-06-01

    With the aim of discovering new molecules for induction of bone formation and biomineralization, combination of bioinformatics and simulation methods were used to design the structure of artificial peptides based on proline-rich domains of enamel matrix proteins. In this study, the effect of such peptides on the differentiation toward the osteogenic lineage of human umbilical cord mesenchymal stem cells (hUCMSCs) was evaluated with or without osteogenic supplements (hydrocortisone, β-glycerol phosphate, and ascorbic acid) and compared to the effect of the commercially available enamel matrix derivative (EMD). It was hypothesized that the differentiation toward the osteogenic lineage of hUCMSCs would be promoted by the treatment with the synthetic peptides when combined with differentiation media, or it could even be directed exclusively by the synthetic peptides. Osteoinductivity was assessed by cell proliferation, bone morphogenetic protein-2 secretion, and gene expression of osteogenic markers after 1, 3, and 14 days of treatment. All peptides were safe with the dosages used, showing lower cell toxicity. P2, P4, and P6 reduced cell proliferation with growing media by 10%-15%. Higher expression of early osteoblast markers was found after 3 days of treatment with EMD in combination with osteogenic supplements, while after 14 days of treatment, cells treated by the different synthetic peptides in combination with osteogenic supplements showed higher osteocalcin mRNA levels. We can conclude that osteogenic differentiation of hUCMSCs is promoted by short-term EMD treatment in combination with osteogenic supplements and by long-term treatment by the synthetic peptides in combination with osteogenic supplements, showing similar results for all the peptide variants analyzed in this study.

  12. Synthetic Peptide-Based ELISA and ELISpot Assay for Identifying Autoantibody Epitopes.

    Science.gov (United States)

    Pozsgay, Judit; Szarka, Eszter; Huber, Krisztina; Babos, Fruzsina; Magyar, Anna; Hudecz, Ferenc; Sarmay, Gabriella

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) is an invaluable diagnostic tool to detect serum autoantibody binding to target antigen. To map the autoantigenic epitope(s), overlapping synthetic peptides covering the total sequence of a protein antigen are used. A large set of peptides synthesized on the crown of pins can be tested by Multipin ELISA for fast screening. Next, to validate the results, the candidate epitope peptides are resynthesized by solid-phase synthesis, coupled to ELISA plate directly, or in a biotinylated form, bound to neutravidin-coated surface and the binding of autoantibodies from patients' sera is tested by indirect ELISA. Further, selected epitope peptides can be applied in enzyme-linked immunospot assay to distinguish individual, citrullinated peptide-specific autoreactive B cells in a pre-stimulated culture of patients' lymphocytes.

  13. Yeast-Based Synthetic Biology Platform for Antimicrobial Peptide Production.

    Science.gov (United States)

    Cao, Jicong; de la Fuente-Nunez, Cesar; Ou, Rui Wen; Torres, Marcelo Der Torossian; Pande, Santosh G; Sinskey, Anthony J; Lu, Timothy K

    2018-03-16

    Antibiotic resistance is one of the most challenging global health threats in our society. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics for the treatment of drug-resistant infections. However, they are limited by their high manufacturing cost. Engineering living organisms represents a promising approach to produce such molecules in an inexpensive manner. Here, we genetically modified the yeast Pichia pastoris to produce the prototypical AMP apidaecin Ia using a fusion protein approach that leverages the beneficial properties ( e.g., stability) of human serum albumin. The peptide was successfully isolated from the fusion protein construct, purified, and demonstrated to have bioactivity against Escherichia coli. To demonstrate this approach as a manufacturing solution to AMPs, we scaled-up production in bioreactors to generate high AMP yields. We envision that this system could lead to improved AMP biomanufacturing platforms.

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

    Science.gov (United States)

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

    2014-05-01

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

  15. Ligand selectivity of a synthetic CXCR4 mimetic peptide.

    Science.gov (United States)

    Groß, Andrea; Brox, Regine; Damm, Dominik; Tschammer, Nuška; Schmidt, Barbara; Eichler, Jutta

    2015-07-15

    The chemokine receptor CXCR4 belongs to the family of seven-transmembrane G-protein coupled receptors (GPCRs). It is activated by its natural ligand SDF-1α. In addition, CXCR4, along with CCR5, serve as coreceptors during HIV-1 entry into its target cell. Recently, we introduced a CXCR4 mimetic peptide, termed CX4-M1, which presents the three extracellular loops (ECLs) of the receptor. CX4-M1 was shown to selectively bind to gp120 of X4-tropic, that is, CXCR4 using, HIV-1, as well as to peptides that present the V3-loops of these gp120 proteins. Furthermore, CX4-M1 selectively inhibits infection of cells with X4-tropic HIV-1. We have now adapted the sequence of the ECLs presented by CX4-M1 to the recently published crystal structure of CXCR4. The binding behavior, as well as the effect on HIV-1 infection, of the resulting peptide (CX4-Mc) was very similar to CX4-M1, validating retrospectively the original design of CX4-M1. A peptide presenting the ECLs of CCR5 (CR5-M), on the other hand, did neither bind to gp120 from X4-tropic HIV-1, nor did it inhibit infection of cells with X4-tropic HIV-1. Furthermore, we could show that CX4-M1, as well as CX4-Mc, but not CR5-M, are selectively recognized by anti-CXCR4 antibodies, bind to SDF-1α, and also inhibit SDF-1α signaling, extending the scope of selective functional CXCR4 mimicry through CX4-M1. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Stabilization of peptides against proteolysis through disulfide-bridged conjugation with synthetic aromatics.

    Science.gov (United States)

    Chen, Yaqi; Li, Tao; Li, Jianguo; Cheng, Shiyan; Wang, Jinghui; Verma, Chandra; Zhao, Yibing; Wu, Chuanliu

    2017-02-22

    Peptides have been promising molecular scaffolds for the development of potential therapeutics with high affinity and specificity to biomacromolecules. However, their inherent proteolytic instability significantly hampers their biological applications. Strategies that can stabilize peptides against proteolytic digestion on the basis of noncovalent interactions-without extensive manipulation of the sequence or use of unnatural residues-are greatly desired. In this work, we developed a general, convenient, and efficient strategy for the stabilization of peptides against proteolysis, which involves noncovalent π-π interactions between aromatic amino acid residues in peptides and synthetic electron-deficient aromatics (NDI), together with the implication of steric hindrance (from the bulky NDI moiety), and the enhancement of peptide α-helicity. This strategy is complementary in concept to the conventional well-established covalent approaches for peptide stabilization, and is thus promising for being utilized, in combination with the latter ones, to circumvent the problem of proteolytic instability of peptides. We envisioned that this study should provide invaluable guidelines to the design and synthesis of organic molecule-peptide hybrids with significantly improved proteolytic resistance, and benefit the development of peptide therapeutics and probes.

  17. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

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

  18. Synthetic Peptides as cGMP-Independent Activators of cGMP-Dependent Protein Kinase Iα.

    Science.gov (United States)

    Moon, Thomas M; Tykocki, Nathan R; Sheehe, Jessica L; Osborne, Brent W; Tegge, Werner; Brayden, Joseph E; Dostmann, Wolfgang R

    2015-12-17

    PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix, was found to bind to and activate PKG Iα in a cyclic guanosine monophosphate (cGMP)-independent manner. The most potent S-tide derivative (S1.5) increased the open probability of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Synthetic molecular evolution of pore–forming peptides by Iterative combinatorial library screening

    Science.gov (United States)

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

    2013-01-01

    We previously reported the de novo design of a combinatorial peptide library that was subjected to high–throughput screening to identify membrane permeabilizing antimicrobial peptides that have β–sheet–like secondary structure. Those peptides do not form discreet pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24–26–residue, 16,200 member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non–helical secondary structure. We used a new high–throughput assay to identify members that self–assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore–formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore–forming peptides known. Furthermore, they are not α–helical, which makes them unusual, as most of the highly potent pore–forming peptides are amphipathic α–helices. Here we demonstrate that this synthetic molecular evolution–based approach, taken together with the new high–throughput tools we have developed, enables the identification, refinement and optimization of unique membrane active peptides. PMID:23394375

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

    Science.gov (United States)

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

    2013-04-19

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

  1. SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides for in vitro applications.

    Science.gov (United States)

    Ulrich, Veronika; Cryle, Max J

    2017-01-01

    Understanding the structure and function of protein complexes and multi-domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps - sortase-mediated and native chemical ligation - together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  2. Amphipathic tail-anchoring peptide and Bcl-2 homology domain-3 (BH3) peptides from Bcl-2 family proteins induce apoptosis through different mechanisms.

    Science.gov (United States)

    Ko, Jae-Kyun; Choi, Kyoung-Han; Peng, Jun; He, Feng; Zhang, Zhi; Weisleder, Noah; Lin, Jialing; Ma, Jianjie

    2011-03-18

    Bcl-2 homology domain-3 (BH3) peptides are potent cancer therapeutic reagents that target regulators of apoptotic cell death in cancer cells. However, their cytotoxic effects are affected by different expression levels of Bcl-2 family proteins. We recently found that the amphipathic tail-anchoring peptide (ATAP) from Bfl-1, a bifunctional Bcl-2 family member, produced strong pro-apoptotic activity by permeabilizing the mitochondrial outer membrane. Here, we test whether the activity of ATAP requires other cellular factors and whether ATAP has an advantage over the BH3 peptides in targeting cancer cells. Confocal microscopic imaging illustrates specific targeting of ATAP to mitochondria, whereas BH3 peptides show diffuse patterns of cytosolic distribution. Although the pro-apoptotic activities of BH3 peptides are largely inhibited by either overexpression of anti-apoptotic Bcl-2 or Bcl-xL or nullification of pro-apoptotic Bax and Bak in cells, the pro-apoptotic function of ATAP is not affected by these cellular factors. Reconstitution of synthetic ATAP into liposomal membranes results in release of fluorescent molecules of the size of cytochrome c from the liposomes, suggesting that the membrane permeabilizing activity of ATAP does not require additional protein factors. Because ATAP can target to the mitochondrial membrane and its pro-apoptotic activity does not depend on the content of Bcl-2 family proteins, it represents a promising candidate for anti-cancer drugs that can potentially overcome the intrinsic apoptosis-resistant nature of cancer cells.

  3. Antimicrobial peptide inhibition of Porphyromonas gingivalis 381-induced hemagglutination is improved with a synthetic decapeptide.

    Science.gov (United States)

    Dixon, Douglas R; Jeffrey, Nicole R; Dubey, Vinod S; Leung, Kai P

    2009-12-01

    The effects of various antimicrobial peptides (AMPs) on disrupting the hemagglutinating ability of cellular components of the putative oral pathogen Porphyromonas gingivalis were examined. AMP inhibition of P. gingivalis 381-induced hemagglutination using vesicles (VES) or outer membrane (OM) preparations was determined within standardized hemagglutination assays using various mammalian erythrocytes. A synthetic decapeptide (KSL-W) and its truncated peptide analogs were evaluated and compared with selected classes of AMPs derived from naturally occurring innate defense peptides. All tested AMPs were effective in disrupting P. gingivalis-induced hemagglutination among tested erythrocytes, with the exception of magainin I and the truncated KSL-W analogs. LL-37 was generally the most potent followed by histatin 5. The synthetic decapeptide (KSL-W) was found to be similar to the histatin 8 peptide in terms of inhibitory effect. In addition, co-application assays (with selected oral-related AMPs+/-KSL-W) were employed to determine if co-application procedures would improve hemagglutination abrogation above that of oral-related AMPs alone. These experiments revealed that the KSL-W peptide improved hemagglutination inhibition above that of each of the oral-related peptides (histatin 5 and 8, LL-37) alone. Among mammalian erythrocytes, significant peptide-induced hemagglutination was observed for the cathelicidin class AMPs, LL-37 and indolicidin (>or=25 and >or=100 microM respectively). In contrast, KSL-W did not induce erythrocyte agglutination throughout any concentration range tested (0.1-1000 microM). Our results suggest that several AMPs are effective in disrupting P. gingivalis 381-induced hemagglutination and that the co-application of a small, synthetically derived peptide may serve to augment the role of local host AMPs engaged in innate defense.

  4. Synthetic modifications of the antibiotic peptide gramicidin S : conformational and biological aspects

    NARCIS (Netherlands)

    Knijnenburg, Annemiek Dorien

    2011-01-01

    The research described in this thesis focuses on synthetic modifications of the antibiotic peptide gramicidin S (GS). The aim of the research is the development of non–toxic analogs of GS using conformational and amphipathic changes induced by sugar amino acids (SAAs) and/or non–proteinogenic amino

  5. Strategies for controlling plant diseases and mycotoxin contamination using antimicrobial synthetic peptides

    Science.gov (United States)

    Development of disease-resistant transgenic crops is very difficult due to the fact that host plant-pathogen interaction is a very complex phenomenon and it is often crop/variety or pathogen/strain-specific. Synthetic peptides are useful in controlling a broad spectrum of plant pathogens including ...

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

    NARCIS (Netherlands)

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

    2002-01-01

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

  7. Syntheticpeptides acquire prion-like properties in the brain.

    Science.gov (United States)

    Xiao, Xiangzhu; Cali, Ignazio; Yuan, Jue; Cracco, Laura; Curtiss, Paul; Zeng, Liang; Abouelsaad, Mai; Gazgalis, Dimitris; Wang, Gong-Xian; Kong, Qingzhong; Fujioka, Hisashi; Puoti, Gianfranco; Zou, Wen-Quan

    2015-01-20

    In transmission studies with Alzheimer's disease (AD) animal models, the formation of Aβ plaques is proposed to be initiated by seeding the inoculated amyloid β (Aβ) peptides in the brain. Like the misfolded scrapie prion protein (PrPSc) in prion diseases, Aβ in AD shows a certain degree of resistance to protease digestion while the biochemical basis for protease resistance of Aβ remains poorly understood. Using in vitro assays, histoblotting, and electron microscopy, we characterize the biochemical and morphological features of syntheticpeptides and Aβ isolated from AD brain tissues. Consistent with previous observations, monomeric and oligomeric Aβ species extracted from AD brains are insoluble in detergent buffers and resistant to digestions with proteinase K (PK). Histoblotting of AD brain tissue sections exhibits an increased Aβ immunoreactivity after digestion with PK. In contrast, synthetic Aβ40 and Aβ42 are soluble in detergent buffers and fully digested by PK. Electron microscopy of Aβ40 and Aβ42 synthetic peptides shows that both species of Aβ form mature fibrils. Those generated from Aβ40 are longer but less numerous than those made of Aβ42. When spiked into human brain homogenates, both Aβ40 and Aβ42 acquire insolubility in detergent and resistance to PK. Our study favors the hypothesis that the human brain may contain cofactor(s) that confers the syntheticpeptides PrPSc-like physicochemical properties.

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

    Science.gov (United States)

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

    2015-03-01

    Fungal keratitis is a leading cause of ocular morbidity. It is frequently misdiagnosed as bacterial keratitis, causing a delay in proper treatment. Furthermore, due to the lack of safe and effective anti-fungal agents for clinical use, treatment of fugal keratitis remains a challenge. In recent years, antimicrobial peptides (AMPs) have received considerable attention as potent and broad-spectrum antimicrobial agents with the potential to overcome antibiotics resistance. We previously reported the design of short synthetic β-sheet forming peptides (IKIK)2-NH2 and (IRIK)2-NH2 with excellent antimicrobial activities and selectivities against various clinically relevant microorganisms, including Gram-positive Staphylococcus epidermidis and Staphylococcus aureus, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and yeast Candida albicans (C. albicans). In this study, we evaluated the application of the two most promising synthetic β-sheet forming peptide candidates for in vivo fungal keratitis treatment in comparison with the commercially available amphotericin B. It was found that topical solutions of the designed peptides are safe, and as effective as the clinically used amphotericin B. Compared to the costly and unstable amphotericin B, (IKIK)2-NH2 and (IRIK)2-NH2 are water-soluble, less expensive and stable. Thus, the synthetic β-sheet forming peptides are presented as promising candidates for the treatment of fungal keratitis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Immunoinformatic Analysis of Crimean Congo Hemorrhagic Fever Virus Glycoproteins and Epitope Prediction for Synthetic Peptide Vaccine.

    Science.gov (United States)

    Tipu, Hamid Nawaz

    2016-02-01

    To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Cross-sectional study. Combined Military Hospital, Khuzdar Cantt, in May 2015. Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLAclass I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. HLAA*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. Atotal of 35 nanomers from GP1, and 3 from GP2 were identified. HLAB*0702 bound maximum number of peptides (6), while HLAB*4001 showed strongest binding affinity. HLAspecific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates.

  10. Identification of the primary peptide contaminant that inhibits fibrillation and toxicity in synthetic amyloid-β42.

    Science.gov (United States)

    Adams, Daniel J; Nemkov, Travis G; Mayer, John P; Old, William M; Stowell, Michael H B

    2017-01-01

    Understanding the pathophysiology of Alzheimer disease has relied upon the use of amyloid peptides from a variety of sources, but most predominantly synthetic peptides produced using t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. These synthetic methods can lead to minor impurities which can have profound effects on the biological activity of amyloid peptides. Here we used a combination of cytotoxicity assays, fibrillation assays and high resolution mass spectrometry (MS) to identify impurities in synthetic amyloid preparations that inhibit both cytotoxicity and aggregation. We identify the Aβ42Δ39 species as the major peptide contaminant responsible for limiting both cytotoxicity and fibrillation of the amyloid peptide. In addition, we demonstrate that the presence of this minor impurity inhibits the formation of a stable Aβ42 dimer observable by MS in very pure peptide samples. These results highlight the critical importance of purity and provenance of amyloid peptides in Alzheimer's research in particular, and biological research in general.

  11. A synthetic peptide selectively kills only virulent Paracoccidioides brasiliensis yeasts.

    Science.gov (United States)

    Kioshima, Erika Seki; Aliperti, Fabiana; Maricato, Juliana Terzi; Mortara, Renato Arruda; Bagagli, Eduardo; Mariano, Mario; Lopes, José Daniel

    2011-03-01

    This work was conducted to identify virulence biomarkers for Paracoccidioides brasiliensis (Pb), the fungus responsible for Paracoccidioidomycosis (PCM), a systemic disease endemic in Latin America. Measurement of mortality showed that all B10.A mice were killed after 250 days by the virulent Pb18 isolate while only one of the mice that received the attenuated counterpart died. Also, number of lung CFUs from virulent Pb18 inoculated mice were much higher when these isolates were compared. Phage display methodology allowed selection of three phages that specifically bound to virulent Pb18. Variability of p04 phage binding to different Pb isolates were examples of variability of expression by the fungus of its binding molecule, strongly suggesting p04 as a biomarker of virulence. In vitro, its derived peptide pep04 killed only virulent fungi, and confocal microscopy showed that it was internalized only by the virulent isolate. Pep04 blocked establishment of Pb infection in mice and virulent Pb18 pre-incubated with p04 showed significantly inhibited lung infection. Furthermore, infected mice treated with p04 showed highly significant reduction in lung CFUs. These findings firmly establish p04 as a biomarker of Pb virulence. Therefore, after proper peptide engineering, p04 may become a useful adjuvant for the distressing treatment of PCM. Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

  12. Evaluation of online carbon isotope dilution mass spectrometry for the purity assessment of synthetic peptide standards.

    Science.gov (United States)

    Cueto Díaz, Sergio; Ruiz Encinar, Jorge; García Alonso, J Ignacio

    2014-09-24

    We present a novel method for the purity assessment of peptide standards which is applicable to any water soluble peptide. The method is based on the online (13)C isotope dilution approach in which the peptide is separated from its related impurities by liquid chromatography (LC) and the eluent is mixed post-column with a continuous flow of (13)C-enriched sodium bicarbonate. An online oxidation step using sodium persulfate in acidic media at 99°C provides quantitative oxidation to (12)CO2 and (13)CO2 respectively which is extracted to a gaseous phase with the help of a gas permeable membrane. The measurement of the isotope ratio 44/45 in the mass spectrometer allows the construction of the mass flow chromatogram. As the only species that is finally measured in the mass spectrometer is CO2, the peptide content in the standard can be quantified, on the base of its carbon content, using a generic primary standard such as potassium hydrogen phthalate. The approach was validated by the analysis of a reference material (NIST 8327), and applied to the quantification of two commercial synthetic peptide standards. In that case, the results obtained were compared with those obtained using alternative methods, such as amino acid analysis and ICP-MS. The results obtained proved the value of the method for the fast, accurate and precise mass purity assignment of synthetic peptide standards. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Conformational analysis of a synthetic fish kisspeptin 1 peptide in membrane mimicking environments.

    Science.gov (United States)

    Thakuria, Dimpal; Shahi, Neetu; Singh, Atul Kumar; Khangembam, Victoria Chanu; Singh, Arvind Kumar; Kumar, Satish

    2017-01-01

    Kisspeptin 1 is a neuropeptide hormone of the RFamide family, which act as an upstream regulator of brain-pituitary-gonad (BPG) axis in most vertebrates including teleosts. In the present study, a 16 amino acid long putative mature bioactive peptide (kiss 1) from preprokisspeptin 1 of golden mahseer, Tor putitora (Hamilton, 1822), was synthesized and characterized using an integrated (experimental and in silico) approach. The far-UV circular dichroism (CD) spectrum of this peptide was evaluated both in aqueous and membrane mimicking solvents (TFE, HFIP and Dioxane). The results indicate that kiss 1 peptide adopted helical, turn and β conformations in membrane like environments. The near-UV CD spectroscopy was also carried out to examine the tertiary packing around aromatic residues of kiss 1 peptide and the peptide-membrane complex. The kiss 1 peptide exhibited little signal in water, but a prominent negative band was observed at around 275 nm when membrane mimetic solution was added. The observed ordered conformations of kiss 1 peptide in the different solvents indicated its potential biological activity which could enhance the secretion of gonadotropin-releasing hormone (GnRH) at BPG axis. The conformational information generated from the present study reinforces the application prospects of bioactive synthetic peptide analogs of kisspeptin 1 in improving the reproductive performances of important cultivable fish species.

  14. Multiple antigenic peptide (MAP): a synthetic peptide dendrimer for diagnostic, antiviral and vaccine strategies for emerging and re-emerging viral diseases.

    Science.gov (United States)

    Joshi, Vinay Ganeshrao; Dighe, Vikas D; Thakuria, Dimpal; Malik, Yashpal Singh; Kumar, Satish

    2013-12-01

    The peptide dendrimer provides novel strategies for various biological applications. Assembling of peptide in macromolecular structure is expected to give rational models as drugs, their delivery and diagnostic reagents. Improved understanding of virus structure and their molecular interactions with ligands have paved the way for treatment and control of emerging and re-emerging viral diseases. This review presents a brief account of a synthetic peptide dendrimer used for diagnostic, therapeutic and prophylactic applications. The designs comprise of multiple antigenic peptides which are being used as alternate synthetic antigens for different viruses.

  15. Enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin.

    Science.gov (United States)

    Sánchez-Vásquez, Lorenzo; Silva-Sanchez, Jesus; Jiménez-Vargas, Juana Maria; Rodríguez-Romero, Adela; Muñoz-Garay, Carlos; Rodríguez, Maria C; Gurrola, Georgina B; Possani, Lourival D

    2013-06-01

    Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity. Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured. A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26-Α29), and K4N Δ(K12-Q18; Ν26-Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25μM and 5 to 25μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900μM for their EC50, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells. These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections. Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Production of Epitope-Specific Antibodies by Immunization with Synthetic Epitope Peptide Formulated with CpG-DNA-Liposome Complex Without Carriers.

    Science.gov (United States)

    Kim, Dongbum; Lee, Younghee; Kwon, Hyung-Joo

    2015-01-01

    Antibody production using synthetic peptides has been investigated extensively to develop therapeutic antibodies and prophylactic vaccines. Previously, we reported that a complex of CpG-DNA and synthetic peptides corresponding to B cell epitopes, encapsulated in a phosphatidyl-β-oleoyl-γ-palmitoyl ethanolamine (DOPE):cholesterol hemisuccinate (CHEMS) complex, significantly enhanced the synthetic peptide-specific IgG production. Here, we describe synthetic peptide-based epitope screening and antibody production without conventional carriers.

  17. Selective solid phase extraction of JWH synthetic cannabinoids by using computationally designed peptides.

    Science.gov (United States)

    Mascini, Marcello; Montesano, Camilla; Perez, German; Wang, Joseph; Compagnone, Dario; Sergi, Manuel

    2017-05-15

    The objective of the present work is to demonstrate a rational way to prepare selective sorbents able to extract simultaneously several structural analogs. For this purpose the binding specificity of two hexapeptides computationally designed (VYWLVW and YYIGGF) versus four synthetic cannabinoids Naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH 018), naphthalen-1-yl-(1-butylindol-3-yl)methanone (JWH 073), (R)-(1-((1-methylpiperidin-2-yl)methyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone (AM 1220) and (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55) was computationally studied and then experimentally tested by solid-phase extraction (SPE) clean-up and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The two peptides were chosen using a semi combinatorial virtual technique by generating 4 cycles of peptide libraries (around 2.3×10 4 elements). To select the two peptides, the simulated binding scores between synthetic cannabinoids and peptides was used by maximizing the recognition properties of amino acid motif between the two JWH and the other synthetic cannabinoids. In particular, the peptide YYIGGF, having also affinity for AM 120, was selected as control because it was the only one without tryptophan residues within the best peptides obtained from simulation. Experimentally, the two hexapeptides were tested as SPE sorbent using nanomolar solutions of the four drugs. After optimization of best retentions the binding constants were calculated by loading synthetic cannabinoids solutions at different concentrations. The results indicated a strong interaction between hexapeptide VYWLVW and JWH 018 (15.58±2.03×10 6 M -1 ), 3-fold and 40-fold larger compared to the analog JWH 073 and both AM 1220 and the WIN 55. Similar trend was observed for the hexapeptide YYIGGF but the binding constants were at least three times lower highlighting the key role of

  18. The Cysteine S-Alkylation Reaction as a Synthetic Method to Covalently Modify Peptide Sequences.

    Science.gov (United States)

    Calce, Enrica; De Luca, Stefania

    2017-01-05

    Synthetic methodologies to chemically modify peptide molecules have long been investigated for their impact in the field of chemical biology. They allow the introduction of biochemical probes useful for studying protein functions, for manipulating peptides with therapeutic potential, and for structure-activity relationship investigations. The commonly used approach was the derivatization of an amino acid side chain. In this regard, the cysteine, for its unique reactivity, has been widely employed as the substrate for such modifications. Herein, we report on methodologies developed to modify the cysteine thiol group through the S-alkylation reaction. Some procedures perform the alkylation of cysteine derivatives, in order to prepare building blocks to be used during the peptide synthesis, whilst some others selectively modify peptide sequences containing a cysteine residue with a free thiol group, both in solution and in the solid phase. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Air oxidation method employed for the disulfide bond formation of natural and synthetic peptides.

    Science.gov (United States)

    Calce, Enrica; Vitale, Rosa Maria; Scaloni, Andrea; Amodeo, Pietro; De Luca, Stefania

    2015-08-01

    Among the available protocols, chemically driven approaches to oxidize cysteine may not be required for molecules that, under the native-like conditions, naturally fold in conformations ensuring an effective pairing of the right disulfide bridge pattern. In this contest, we successfully prepared the distinctin, a natural heterodimeric peptide, and some synthetic cyclic peptides that are inhibitors of the CXCR4 receptor. In the first case, the air oxidation reaction allowed to connect two peptide chains via disulfide bridge, while in the second case allowed the cyclization of rationally designed peptides by an intramolecular disulfide bridge. Computational approaches helped to either drive de-novo design or suggest structural modifications and optimal oxidization protocols for disulfide-containing molecules. They are able to both predict and to rationalize the propensity of molecules to spontaneously fold in suitable conformations to achieve the right disulfide bridges.

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

    Science.gov (United States)

    Trier, Nicole Hartwig; Holm, Bettina Eide; Slot, Ole; Locht, Henning; Lindegaard, Hanne; Svendsen, Anders; Nielsen, Christoffer Tandrup; Jacobsen, Søren; Theander, Elke; Houen, Gunnar

    2016-02-01

    Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n=60), healthy controls (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 correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity. Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Evaluation of online carbon isotope dilution mass spectrometry for the purity assessment of synthetic peptide standards

    Energy Technology Data Exchange (ETDEWEB)

    Díaz, Sergio Cueto; Ruiz Encinar, Jorge, E-mail: ruizjorge@uniovi.es; García Alonso, J. Ignacio, E-mail: jiga@uniovi.es

    2014-09-24

    Highlights: • Purity assessment of peptide standards applicable to any water soluble peptide. • Online {sup 13}C isotope dilution mass spectrometry. • Mass flow chromatogram from measured 44/45 isotope ratios. • Validation by the analysis of NIST 8327. - Abstract: We present a novel method for the purity assessment of peptide standards which is applicable to any water soluble peptide. The method is based on the online {sup 13}C isotope dilution approach in which the peptide is separated from its related impurities by liquid chromatography (LC) and the eluent is mixed post-column with a continuous flow of {sup 13}C-enriched sodium bicarbonate. An online oxidation step using sodium persulfate in acidic media at 99 °C provides quantitative oxidation to {sup 12}CO{sub 2} and {sup 13}CO{sub 2} respectively which is extracted to a gaseous phase with the help of a gas permeable membrane. The measurement of the isotope ratio 44/45 in the mass spectrometer allows the construction of the mass flow chromatogram. As the only species that is finally measured in the mass spectrometer is CO{sub 2}, the peptide content in the standard can be quantified, on the base of its carbon content, using a generic primary standard such as potassium hydrogen phthalate. The approach was validated by the analysis of a reference material (NIST 8327), and applied to the quantification of two commercial synthetic peptide standards. In that case, the results obtained were compared with those obtained using alternative methods, such as amino acid analysis and ICP-MS. The results obtained proved the value of the method for the fast, accurate and precise mass purity assignment of synthetic peptide standards.

  2. Protective Role of PEDF-Derived Synthetic Peptide Against Experimental Diabetic Nephropathy.

    Science.gov (United States)

    Ishibashi, Y; Matsui, T; Taira, J; Higashimoto, Y; Yamagishi, S

    2016-09-01

    Pigment epithelium-derived factor (PEDF) is a glycoprotein with complex neuroprotective, anti-angiogenic, and anti-inflammatory properties, all of which could potentially be exploited as a therapeutic option for vascular complications in diabetes. We have previously shown that PEDF-derived synthetic peptide, P5-3 (FIFVLRD) has a comparable ability with full PEDF protein to inhibit rat corneal neovascularization induced by chemical cauterization. However, the effects of PEDF peptide on experimental diabetic nephropathy remain unknown. To address the issue, we modified P5-3 to stabilize and administered the modified peptide (d-Lys-d-Lys-d-Lys-Gln-d-Pro-P5-3-Cys-amide, 0.2 nmol/day) or vehicle to streptozotocin-induced diabetic rats (STZ-rats) intraperitoneally by an osmotic mini pump for 2 weeks. We further examined the effects of modified peptide on human proximal tubular cells. Renal PEDF expression was decreased in STZ-rats. Although the peptide administration did not affect blood glucose or blood pressure, it decreased urinary excretion levels of 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker, and reduced plasminogen activator inhibitor-1 (PAI-1) gene expression, and suppressed glomerular expansion in the diabetic kidneys. High glucose or advanced glycation end products stimulated oxidative stress generation and PAI-1 gene expression in tubular cells, all of which were significantly suppressed by 10 nM modified P5-3 peptide. Our present study suggests that PEDF-derived synthetic modified peptide could protect against experimental diabetic nephropathy and inhibit tubular cell damage under diabetes-like conditions through its anti-oxidative properties. Supplementation of modified P5-3 peptide may be a novel therapeutic strategy for diabetic nephropathy. © Georg Thieme Verlag KG Stuttgart · New York.

  3. Aggregation and Its Influence on the Immunomodulatory Activity of Synthetic Innate Defense Regulator Peptides.

    Science.gov (United States)

    Haney, Evan F; Wu, Bing Catherine; Lee, Kelsey; Hilchie, Ashley L; Hancock, Robert E W

    2017-08-17

    There is increasing interest in developing cationic host defense peptides (HDPs) and their synthetic derivatives as antimicrobial, immunomodulatory, and anti-biofilm agents. These activities are often evaluated without considering biologically relevant concentrations of salts or serum; furthermore certain HDPs have been shown to aggregate in vitro. Here we examined the effect of aggregation on the immunomodulatory activity of a synthetic innate defense regulator peptide, 1018 (VRLIVAVRIWRR-NH 2 ). A variety of salts and solutes were screened to determine their influence on 1018 aggregation, revealing that this peptide "salts out" of solution in an anion-specific and concentration-dependent manner. Furthermore, the immunomodulatory activity of 1018 was found to be inhibited under aggregation-promoting conditions. A series of 1018 derivatives were synthesized with the goal of disrupting this self-assembly process. Indeed, some derivatives exhibited reduced aggregation while maintaining certain immunomodulatory functions, demonstrating that it is possible to engineer optimized synthetic HDPs to avoid unwanted peptide aggregation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor.

    Science.gov (United States)

    Felizzola, Ornella; Martínez, Juan Carlos; Zerpa, Noraida; Malavé, Caridad

    2013-09-01

    Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFbeta isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFbeta1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

  5. Slc15a1 is involved in the transport of synthetic F5-peptide into the seminiferous epithelium in adult rat testes.

    Science.gov (United States)

    Su, Linlin; Zhang, Yufei; Cheng, Yan C; Lee, Will M; Ye, Keping; Hu, Dahai

    2015-11-05

    Spermiation and BTB restructuring, two critical cellular events that occur across seminiferous epithelium in mammalian testis during spermatogenesis, are tightly coordinated by biologically active peptides released from laminin chains. Our earlier study reported that F5-peptide, synthesized based on a stretch of 50 amino acids within laminin-γ3 domain IV, could reversibly induce the impairment of spermatogenesis, disruption of BTB integrity, and germ cell loss, and thus is a promising male contraceptive. However, how F5-peptide when administered intratesticularly enters seminiferous tubules and exerts effects beyond BTB is currently unknown. Here we demonstrated that Slc15a1, a peptide transporter also known as Pept1, was predominantly present in peritubular myoid cells, interstitial Leydig cells, vascular endothelial cells and germ cells, while absent in Sertoli cells or BTB site. The steady-state protein level of Slc15a1 in adult rat testis was not affected by F5-peptide treatment. Knockdown of Slc15a1 by in vivo RNAi in rat testis was shown to prevent F5-peptide induced disruptive effects on spermatogenesis. This study suggests that Slc15a1 is involved in the transport of synthetic F5-peptide into seminiferous epithelium, and thus Slc15a1 is a novel target in testis that could be genetically modified to improve the bioavailability of F5-peptide as a prospective male contraceptive.

  6. Towards the Development of Synthetic Antibiotics: Designs Inspired by Natural Antimicrobial Peptides.

    Science.gov (United States)

    Azmi, Fazren; Skwarczynski, Mariusz; Toth, Istvan

    2016-01-01

    Virtually every living organism produces gene-encoded antimicrobial peptides (AMPs) that provide an immediate defence against pathogen invasion. Many AMPs have been isolated and used as antibiotics that are effective against multidrug-resistant bacteria. Although encouraging, AMPs have such poor drug-like properties that their application for clinical use is restricted. In turn, this has diverted research to the development of synthetic molecules that retain the therapeutic efficacy of AMPs but are endowed with greater biological stability and safety profiles. Most of the synthetic molecules, either based on a peptidic or non-peptidic scaffold, have been designed to mimic the amphiphilic properties of native AMPs, which are widely believed to be the key determinant of their antibacterial activity. In this review, the structural, chemical and biophysical features that govern the biological activities of various synthetic designs are discussed extensively. Recent innovative approaches from the literature that exhibit novel concepts towards the development of new synthetic antibacterial agents, including the engineered delivery platform incorporated with AMP mimetics, are also emphasised.

  7. Studies of Cell-Mediated Immunity Against Immune Disorders Using Synthetic Peptides and Rotating Bioreactor System

    Science.gov (United States)

    Sastry, Jagannadha K.

    1997-01-01

    Our proposed experiments included: (1) immunzing mice with synthetic peptides; (2) preparing spleen and lymph node cells; (3) growing them under conventional conditions as well as in the rotatory vessel in appropriate medium reconstituting with synthetic peptides and/or cytokines as needed; and (4) comparing at regular time intervals the specific CTL activity as well as helper T-cell activity (in terms of both proliferative responses and cytokine production) using established procedures in my laboratory. We further proposed that once we demonstrated the merit of rotatory vessel technology to achieve desired results, these studies would be expanded to include immune cells from non-human primates (rhesus monkeys and chimpanzees) and also humans. We conducted a number of experiments to determine CTL induction by the synthetic peptides corresponding to antigenic proteins in HIV and HPV in different mouse strains that express MHC haplotypes H-2b or H-2d. We immunized mice with 100 ug of the synthetic peptide, suspended in sterile water, and emulsified in CFA (1:1). The immune lymph node cells obtained after 7 days were restimulated by culturing in T25 flask, HARV-10, or STLV-50, in the presence of the peptide at 20 ug/ml. The results from the 5'Cr-release assay consistently revealed complete abrogation of CTL activity of cells grown in the bioreactors (both HARV and STLV), while significant antigen-specific CTL activity was observed with cells cultured in tissue culture flasks. Thus, overall the data we generated in this study proved the usefulness of the NASA-developed developed technology for understanding the known immune deficiency during space travel. Additionally, this ex vivo microgravity technology since it mimics effectively the in vivo situation, it is also useful in understanding immune disorders in general. Thus, our proposed studies in TMC-NASA contract round II application benefit from data generated in this TMC-NASA contract round I study.

  8. Anti-mycobacterial activities of synthetic cationic α-helical peptides and their synergism with rifampicin.

    Science.gov (United States)

    Khara, Jasmeet S; Wang, Ying; Ke, Xi-Yu; Liu, Shaoqiong; Newton, Sandra M; Langford, Paul R; Yang, Yi Yan; Ee, Pui Lai Rachel

    2014-02-01

    The rapid emergence of multi-drug resistant tuberculosis (TB) and the lack of effective therapies have prompted the development of compounds with novel mechanisms of action to tackle this growing public health concern. In this study, a series of synthetic cationic α-helical antimicrobial peptides (AMPs) modified with different hydrophobic amino acids was investigated for their anti-mycobacterial activity, both alone and in synergistic combinations with the frontline anti-tuberculosis drug rifampicin. The addition of thiol groups by incorporating cysteine residues in the AMPs did not improve anti-mycobacterial activity against drug-susceptible and drug-resistant Mycobacterium tuberculosis, while the enhancement of peptide hydrophobicity by adding methionine residues increased the efficacy of the primary peptide against all strains tested, including clinically isolated multidrug-resistant mycobacteria. The peptide with the optimal composition M(LLKK)2M was bactericidal, and eradicated mycobacteria via a membrane-lytic mechanism as demonstrated by confocal microscopic studies. Mycobacteria did not develop resistance after multiple exposures to sub-lethal doses of the peptide. In addition, the peptide displayed synergism with rifampicin against both Mycobacterium smegmatis and Mycobacterium bovis BCG and additivity against M. tuberculosis. Moreover, such combination therapy is effective in delaying the emergence of rifampicin resistance. The ability to potentiate anti-TB drug activity, kill drug-resistant bacteria and prevent drug resistance highlights the potential utility of the peptide in combating multidrug-resistant TB. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. An anti-infective synthetic peptide with dual antimicrobial and immunomodulatory activities.

    Science.gov (United States)

    Silva, O N; de la Fuente-Núñez, C; Haney, E F; Fensterseifer, I C M; Ribeiro, S M; Porto, W F; Brown, P; Faria-Junior, C; Rezende, T M B; Moreno, S E; Lu, T K; Hancock, R E W; Franco, O L

    2016-11-02

    Antibiotic-resistant infections are predicted to kill 10 million people per year by 2050, costing the global economy $100 trillion. Therefore, there is an urgent need to develop alternative technologies. We have engineered a synthetic peptide called clavanin-MO, derived from a marine tunicate antimicrobial peptide, which exhibits potent antimicrobial and immunomodulatory properties both in vitro and in vivo. The peptide effectively killed a panel of representative bacterial strains, including multidrug-resistant hospital isolates. Antimicrobial activity of the peptide was demonstrated in animal models, reducing bacterial counts by six orders of magnitude, and contributing to infection clearance. In addition, clavanin-MO was capable of modulating innate immunity by stimulating leukocyte recruitment to the site of infection, and production of immune mediators GM-CSF, IFN-γ and MCP-1, while suppressing an excessive and potentially harmful inflammatory response by increasing synthesis of anti-inflammatory cytokines such as IL-10 and repressing the levels of pro-inflammatory cytokines IL-12 and TNF-α. Finally, treatment with the peptide protected mice against otherwise lethal infections caused by both Gram-negative and -positive drug-resistant strains. The peptide presented here directly kills bacteria and further helps resolve infections through its immune modulatory properties. Peptide anti-infective therapeutics with combined antimicrobial and immunomodulatory properties represent a new approach to treat antibiotic-resistant infections.

  10. Antimicrobial activity of the indolicidin-derived novel synthetic peptide In-58.

    Science.gov (United States)

    Vasilchenko, A S; Vasilchenko, A V; Pashkova, T M; Smirnova, M P; Kolodkin, N I; Manukhov, I V; Zavilgelsky, G B; Sizova, E A; Kartashova, O L; Simbirtsev, A S; Rogozhin, E A; Duskaev, G K; Sycheva, M V

    2017-12-01

    Natural peptides with antimicrobial activity are extremely diverse, and peptide synthesis technologies make it possible to significantly improve their properties for specific tasks. Here, we investigate the biological properties of the natural peptide indolicidin and the indolicidin-derived novel synthetic peptide In-58. In-58 was generated by replacing all tryptophan residues on phenylalanine in D-configuration; the α-amino group in the main chain also was modified by unsaturated fatty acid. Compared with indolicidin, In-58 is more bactericidal, more resistant to proteinase K, and less toxic to mammalian cells. Using molecular physics approaches, we characterized the action of In-58 on bacterial cells at the cellular level. Also, we have found that studied peptides damage bacterial membranes. Using the Escherichia coli luminescent biosensor strain MG1655 (pcolD'::lux), we investigated the action of indolicidin and In-58 at the subcellular level. At subinhibitory concentrations, indolicidin and In-58 induced an SOS response. Our data suggest that indolicidin damages the DNA, but bacterial membrane perturbation is its principal mode of action. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

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

    Directory of Open Access Journals (Sweden)

    Rampratap S. Kushwaha

    2004-01-01

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

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

    Science.gov (United States)

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

    2009-04-01

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

  13. New opportunities for allergen immunotherapy using synthetic peptide immuno-regulatory epitopes (SPIREs).

    Science.gov (United States)

    Klimek, Ludger; Pfaar, Oliver; Worm, Margitta

    2016-10-01

    Allergen immunotherapy (AIT) reduces allergic rhinoconjunctivitis (ARC) symptoms, but long-term efficacy requires treatment for 3-5 years. Synthetic peptide immuno-regulatory epitopes, a new class of AIT, are allergen peptides with a shorter, more convenient treatment regimen that could potentially have benefits on adherence and outcomes. Phase 2 trials of therapies derived from cat, house dust mite, grass, and ragweed allergen peptides demonstrated significant reduction in ARC symptoms after short-course treatment; improvement was sustained for 18-24 months posttreatment. We conducted a PubMed literature search for clinical publications using the search terms AIT; allergen peptides; ARC; cat, grass, house dust mite, and ragweed allergy; SCIT; SLIT; and synthetic peptides. Expert commentary: Long-term disease modification is a realistic goal of AIT. The inconvenience of conventional AIT regimens negatively impacts long-term persistence and, thus, efficacy. In comparison, SPIREs have a more convenient treatment regimen that could potentially have benefits on adherence and outcomes.

  14. Immune response to synthetic peptides representing antigenic sites on the glycoprotein of infectious hematopoietic necrosis virus

    Science.gov (United States)

    Emmenegger, Eveline J.; Huang, C.; LaPatra, S.; Winton, James R.

    1995-01-01

    Summary ― Monoclonal antibodies against infectious hematopoietic necrosis virus have been used to react with recombinant expression products in immunoblots and to select neutralization-resistant mutants for sequence analysis. These strategies identified neutralizing and non-neutralizing antigenic sites on the viral glycoprotein. Synthetic peptides based upon the amino acid sequences of these antigenic sites were synthesized and were injected together with an adjuvant into rainbow trout. The constructs generally failed to stimulate neutralizing antibodies in the fish. These results indicate that we need to understand more about the ability of peptide antigens to stimulate fish immune systems.

  15. Synthetic Proteins and Peptides for the Direct Interrogation of α-Synuclein Posttranslational Modifications.

    Science.gov (United States)

    Pratt, Matthew R; Abeywardana, Tharindumala; Marotta, Nicholas P

    2015-06-25

    α-Synuclein is the aggregation-prone protein associated with Parkinson's disease (PD) and related neurodegenerative diseases. Complicating both its biological functions and toxic aggregation are a variety of posttranslational modifications. These modifications have the potential to either positively or negatively affect α-synuclein aggregation, raising the possibility that the enzymes that add or remove these modifications could be therapeutic targets in PD. Synthetic protein chemistry is uniquely positioned to generate site-specifically and homogeneously modified proteins for biochemical study. Here, we review the application of synthetic peptides and proteins towards understanding the effects of α-synuclein posttranslational modifications.

  16. Isolation of antibodies specific to sickle hemoglobin by affinity chromatography using a synthetic peptide

    International Nuclear Information System (INIS)

    Young, N.S.; Curd, J.G.; Eastlake, A.; Furie, B.; Schechter, A.N.

    1975-01-01

    Antibodies to hemoglobin have been studied with a radioimmunoassay which employs [ 14 C]carbamylated (= carbamoylated) hemoglobin S. An antiserum raised against hemoglobin S, which initially discriminated poorly between hemoglobins S and A, was fractionated by absorption to a column of Sepharose to which a synthetic peptide corresponding to the first 13 amino-acid residues of the β chain of sickle hemoglobin had been covalently bound. A subpopulation of the antiserum was eluted from this column with 4 M guanidine . HCl. These antibodies showed binding to hemoglobin S but not to hemoglobin A and this interaction could be inhibited by the synthetic peptide. These antibodies, of demonstrated fine structural specificity, may be useful in the detection of sickle hemoglobin and in the study of its structure in solution

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  18. Modeling human response errors in synthetic flight simulator domain

    Science.gov (United States)

    Ntuen, Celestine A.

    1992-01-01

    This paper presents a control theoretic approach to modeling human response errors (HRE) in the flight simulation domain. The human pilot is modeled as a supervisor of a highly automated system. The synthesis uses the theory of optimal control pilot modeling for integrating the pilot's observation error and the error due to the simulation model (experimental error). Methods for solving the HRE problem are suggested. Experimental verification of the models will be tested in a flight quality handling simulation.

  19. Electron transfer dissociation (ETD) of synthetic and natural peptides containing lanthionine/methyllanthionine bridges.

    Science.gov (United States)

    Dolle, Ashwini; Jagadeesh, Narasimhappagari; Bhaumik, Suman; Prakash, Sunita; Biswal, Himansu S; Gowd, Konkallu Hanumae

    2018-03-08

    The modes of cleavages of lanthionine/methyllanthionine bridges under electron transfer dissociation (ETD) was investigated using synthetic and natural lantipeptides. Knowledge on the mass spectrometric fragmentation of lanthionine/methyllanthionine bridges may assist in the development of analytical methods for rapid discovery of new lantibiotics. Present study strengthens the advantage of ETD in the characterization of posttranslational modifications of peptides and proteins. Synthetic and natural lantipeptides were obtained by desulfurization of peptide disulfide and cyanogen bromide digestion of lantibiotic nisin, respectively. These peptides were subjected for electrospray ionization CID-MS/MS and ETD-MS/MS using HCT ultra ETDII ion trap mass spectrometer. MS 3 CID was performed on desire product ions to prove cleavage of lanthionine/methyllanthionine bridge during ETD-MS/MS. ETD has advantage over CID in the cleavage of side chain of lanthionine/methyllanthionine bridges. The cleavage of N-Cα backbone peptide bond followed by C-terminal side chain of lanthionine bridge results in formation of c •+ and z + ions. Cleavage at preceding peptide bond to the C-terminal side chain of lanthionine/methyllanthionine bridge yield specific fragments with cysteine/methylcysteine thiyl radical and dehydroalanine. ETD successfully cleaves the lanthionine/methyllanthionine bridges of synthetic and natural lantipeptides. Diagnostic fragment ions of ETD cleavage of lanthionine/methyllanthionine bridges are the N-terminal cysteine/methylcysteine thiyl radical and C-terminal dehydroalanine. Detection of cysteine/methylcysteine thiyl radical and dehydroalanine in combined ETD-CID-MS may be used for rapid identification of lantipeptide natural products. This article is protected by copyright. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    S. V. Korobova

    2016-01-01

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

  1. Imaging of metastatic melanoma utilising a technetium-99m labelled RGD-containing synthetic peptide

    Energy Technology Data Exchange (ETDEWEB)

    Sivolapenko, G.B. [Encephalos Research and Therapeutic Inst., Athens (Greece)]|[Agii Anargiri Cancer Hospital, Athens (Greece); Skarlos, D. [Agii Anargiri Cancer Hospital, Athens (Greece); Pectasides, D. [Metaxa Cancer Hospital, Athens (Greece); Stathopoulou, E.; Milonakis, A.; Sirmalis, G.; Konstantinides, K. [Encephalos Research and Therapeutic Inst., Athens (Greece); Stuttle, A.; Courtenay-Luck, N.S.; Epenetos, A.A. [Antisoma plc., London (United Kingdom)

    1998-10-01

    Integrins are cell-surface glycoproteins found in different forms on all cells except erythrocytes. Integrins bind to cell adhesion molecules and to proteins found in the extracellular matrix. A tripeptidic sequence Arg-Gly-Asp (RGD) is often the primary site of recognition by integrins which are expressed on tumour cells and are responsible for tumour invasion and metastasis. A synthetic decapeptide designated {alpha}P2 containing two RGD sequences radiolabelled with technetium-99m was used to image malignant melanoma in vivo. Fourteen patients previously diagnosed with metastatic melanoma underwent gamma camera imaging 20-180 min following intravenous administration of the radiolabelled synthetic decapeptide {alpha}P2. Six out of eight (6/8) of the lymph node metastases (75%) and all other neoplastic sites (11 sites) were successfully imaged, with the exception of three sites in the mediastinal area which were not positively imaged. In two cases there was false positive uptake in the rounded pigmented areolar/nipple area. In three cases (seven sites) the peptide scan confirmed the absence of disease in suspected lesions (true-negative). The synthetic peptide was rapidly removed from the circulation by filtration through the kidneys and excretion in the urine. No toxicity or adverse events were recorded. Radiolabelled {alpha}P2 peptide, which binds specifically to adhesion molecules on tumours, can be used for the in vivo detection of neoplastic metastases. (orig.) With 5 figs., 2 tabs., 24 refs.

  2. Analysis of binding centers in nicotinic receptors with the aid of synthetic peptides.

    Science.gov (United States)

    Kasheverov, I E; Kryukova, E V; Kudryavtsev, D S; Ivanov, I A; Egorova, N V; Zhmak, M N; Spirova, E N; Shelukhina, I V; Odinokov, A V; Alfimov, M V; Tsetlin, V I

    2016-09-01

    We studies the receptor-binding specificity of the synthetic peptide HAP (High Affinity Peptide) and its analogues, which are regarded as a model of the orthosteric site nicotinic acetylcholine receptors (nAChR). Using radioligand analysis, electrophysiology tests, and calcium imaging, we assessed the ability of HAP to interact with nAChR antagonists: long α-neurotoxins and α-conotoxins. A high affinity of HAP for α-bungarotoxin and the absence of its interaction with α-cobratoxin and α-conotoxins was found. The synthesized analogues of HAP in general retained the properties of the original peptide. Thus, HAP cannot be a model of a ligand-binding site.

  3. Patchwork protein chemistry: a practitioner's treatise on the advances in synthetic peptide stitchery.

    Science.gov (United States)

    Verzele, Dieter; Madder, Annemieke

    2013-06-17

    With the study of peptides and proteins at the heart of many scientific endeavors, the omics era heralded a multitude of opportunities for chemists and biologists alike. Across the interface with life sciences, peptide chemistry plays an indispensable role, and progress made over the past decades now allows proteins to be treated as molecular patchworks stitched together through synthetic tailoring. The continuous elaboration of sophisticated strategies notwithstanding, Merrifield's solid-phase methodology remains a cornerstone of chemical protein design. Although the non-practitioner might misjudge peptide synthesis as trivial, routine, or dull given its long history, we comment here on its many advances, obstacles, and prospects from a practitioner's point of view. While sharing our perspectives through thematic highlights across the literature, this treatise provides an interpretive overview as a guide to novices, and a recap for specialists. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Directory of Open Access Journals (Sweden)

    Nazila Amini

    2014-06-01

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

  5. A novel synthetic peptide from a tomato defensin exhibits antibacterial activities against Helicobacter pylori.

    Science.gov (United States)

    Rigano, M M; Romanelli, A; Fulgione, A; Nocerino, N; D'Agostino, N; Avitabile, C; Frusciante, L; Barone, A; Capuano, F; Capparelli, R

    2012-12-01

    Defensins are a class of cysteine-rich proteins, which exert broad spectrum antimicrobial activity. In this work, we used a bioinformatic approach to identify putative defensins in the tomato genome. Fifteen proteins had a mature peptide that includes the well-conserved tetradisulfide array. We selected a representative member of the tomato defensin family; we chemically synthesized its γ-motif and tested its antimicrobial activity. Here, we demonstrate that the synthetic peptide exhibits potent antibacterial activity against Gram-positive bacteria, such as Staphylococcus aureus A170, Staphylococcus epidermidis, and Listeria monocytogenes, and Gram-negative bacteria, including Salmonella enterica serovar Paratyphi, Escherichia coli, and Helicobacter pylori. In addition, the synthetic peptide shows minimal (<5%) hemolytic activity and absence of cytotoxic effects against THP-1 cells. Finally, SolyC exerts an anti-inflammatory activity in vitro, as it downregulates the level of the proinflammatory cytokines TNF-α and IFN-γ. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

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

    Science.gov (United States)

    Amini, Nazila; Vishteh, Mohadeseh Naghi; Zarei, Omid; Hadavi, Reza; Ahmadvand, Negah; Rabbani, Hodjattallah; Jeddi-Tehrani, Mahmood

    2014-06-01

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

  7. Immunomodulatory potential of partially hydrolyzed β-lactoglobulin and large synthetic peptides.

    Science.gov (United States)

    Adel-Patient, Karine; Nutten, Sophie; Bernard, Hervé; Fritsché, Rodolphe; Ah-Leung, Sandrine; Meziti, Narimane; Prioult, Guénolée; Mercenier, Annick; Wal, Jean-Michel

    2012-10-31

    The immunomodulatory potential of fragments derived from the cow's milk allergen bovine β-lactoglobulin (BLG) was assessed in a mouse model of oral tolerance (OT) [Adel-Patient, K.; Wavrin, S.; Bernard, H.; Meziti, N.; Ah-Leung, S.; Wal, J. M. Oral tolerance and Treg cells are induced in BALB/c mice after gavage with bovine β-lactoglobulin. Allergy 2011, 66 (10), 1312-1321]. Native BLG (nBLG) and chemically denatured BLG (lacking S-S bridges, dBLG), products resulting from their hydrolysis using cyanogen bromide (CNBr) and some synthetic peptides, were produced and precisely characterized. CNBr hydrolysates correspond to pools of peptides of various sizes that are still associated by S-S bridges when derived from nBLG. nBLG, dBLG, and CNBr hydrolysate of nBLG efficiently prevented further sensitization. CNBr hydrolysate of dBLG was less efficient, suggesting that the association by S-S bridges of peptides increased their immunomodulatory potential. Conversely, synthetic peptides were inefficient even if covering 50% of the BLG sequence, demonstrating that the immunomodulatory potential requires the presence of all derived fragments of BLG and further supporting the use of partially hydrolyzed milk proteins to favor OT induction in infants with a risk of atopy.

  8. Antimicrobial activity of the synthetic peptide Lys-a1 against oral streptococci.

    Science.gov (United States)

    da Silva, Bruno Rocha; de Freitas, Victor Aragão Abreu; Carneiro, Victor Alves; Arruda, Francisco Vassiliepe Sousa; Lorenzón, Esteban Nicolás; de Aguiar, Andréa Silvia Walter; Cilli, Eduardo Maffud; Cavada, Benildo Sousa; Teixeira, Edson Holanda

    2013-04-01

    The peptide LYS-[TRP(6)]-Hy-A1 (Lys-a1) is a synthetic derivative of the peptide Hy-A1, initially isolated from the frog species Hypsiboas albopunctatus. According to previous research, it is a molecule with broad antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of the synthetic peptide Lys-a1 (KIFGAIWPLALGALKNLIK-NH2) on the planktonic and biofilm growth of oral bacteria. The methods used to evaluate antimicrobial activity include the following: determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in microtiter plates for growth in suspension and quantification of biomass by crystal violet staining and counting of colony forming units for biofilm growth. The microorganisms Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus mutans and Streptococcus sobrinus were grown in Brain Heart Infusion broth at 37°C under atmospheric pressure with 10% CO2. The peptide was solubilized in 0.1% acetic acid (v/v) at various concentrations (500-1.9 μg mL(-1)). Chlorhexidine gluconate 0.12% was used as the positive control, and BHI culture medium was used as the negative control. The tested peptide demonstrated a remarkable antimicrobial effect, inhibiting the planktonic and biofilm growth of all strains tested, even at low concentrations. Thus, the peptide Lys-a1 is an important source for potential antimicrobial agents, especially for the control and prevention of microbial biofilms, which is one of the most important factors in cariogenic processes. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Infrared study of synthetic peptide analogues of the calcium-binding site III of troponin C: The role of helix F of an EF-hand motif.

    Science.gov (United States)

    Nara, Masayuki; Morii, Hisayuki; Tanokura, Masaru

    2013-05-01

    The EF-hand motif (helix-loop-helix) is a Ca(2+)-binding domain that is common among many intracellular Ca(2+)-binding proteins. We applied Fourier-transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E-loop-helix F). The 17-residue peptides corresponding to loop-helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D-type amino acid, were investigated to disturb the α-helical conformation of helix F systematically. These D-type-substituted peptides showed no band at about 1555 cm(-1) even in the Ca(2+)-loaded state although the native peptide (L-type only) showed a band at about 1555 cm(-1) in the Ca(2+)-loaded state, which is assigned to the side-chain COO(-) group of Glu at the 12th position, serving as the ligand for Ca(2+) in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca(2+) and the side-chain COO(-) group of Glu at the 12th position. Implications of the COO(-) antisymmetric stretch and the amide-I' of the synthetic peptide analogues of the Ca(2+)-binding sites are discussed. Copyright © 2012 Wiley Periodicals, Inc.

  10. Toward Personalized Peptide-Based Cancer Nanovaccines: A Facile and Versatile Synthetic Approach.

    Science.gov (United States)

    Kakwere, Hamilton; Ingham, Elizabeth S; Allen, Riley; Mahakian, Lisa M; Tam, Sarah M; Zhang, Hua; Silvestrini, Matthew T; Lewis, Jamal S; Ferrara, Katherine W

    2017-11-15

    Personalized cancer vaccines (PCVs) are receiving attention as an avenue for cancer immunotherapy. PCVs employ immunogenic peptide epitopes capable of stimulating the immune system to destroy cancer cells with great specificity. Challenges associated with effective delivery of these peptides include poor solubility of hydrophobic sequences, rapid clearance, and poor immunogenicity, among others. The incorporation of peptides into nanoparticles has the potential to overcome these challenges, but the broad range of functionalities found in amino acids presents a challenge to conjugation due to possible interferences and lack of reaction specificity. Herein, a facile and versatile approach to generating nanosized PCVs under mild nonstringent conditions is reported. Following a simple two-step semibatch synthetic approach, amphiphilic hyperbranched polymer-peptide conjugates were prepared by the conjugation of melanoma antigen peptides, either TRP2 (hydrophobic) or MUT30 (hydrophilic), to an alkyne functionalized core via strain-promoted azide-alkyne click chemistry. Self-assembly of the amphiphiles gave spherical nanovaccines (by transmission electron microscopy) with sizes in the range of 10-30 nm (by dynamic light scattering). Fluorescently labeled nanovaccines were prepared to investigate the cellular uptake by antigen presenting cells (dendritic cells), and uptake was confirmed by flow cytometry and microscopy. The TRP2 nanovaccine was taken up the most followed by MUT30 nanoparticles and, finally, nanoparticles without peptide. The nanovaccines showed good biocompatibility against B16-F10 cells, yet the TRP2 peptide showed signs of toxicity, possibly due to its hydrophobicity. A test for immunogenicity revealed that the nanovaccines were poorly immunogenic, implying the need for an adjuvant when administered in vivo. Treatment of mice with melanoma tumors showed that in combination with adjuvant, CpG, groups with the peptide nanovaccines slowed tumor growth and

  11. Site-specific labeling of synthetic peptide using the chemoselective reaction between N-methoxyamino acid and isothiocyanate.

    Science.gov (United States)

    Hara, Toshiaki; Purwati, Euis Maras; Tainosyo, Akira; Kawakami, Toru; Hojo, Hironobu; Aimoto, Saburo

    2015-10-01

    Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N-terminus, leaving side chain amino group intact. The synthetic human β-defensin-2 carrying MeOGly at its N-terminus or the side chain amino group of Lys(10) reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  12. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma

    Science.gov (United States)

    Rosenberg, Steven A.; Yang, James C.; Schwartzentruber, Douglas J.; Hwu, Patrick; Marincola, Francesco M.; Topalian, Suzanne L.; Restifo, Nicholas P.; Dudley, Mark E.; Schwarz, Susan L.; Spiess, Paul J.; Wunderlich, John R.; Parkhurst, Maria R.; Kawakami, Yutaka; Seipp, Claudia A.; Einhorn, Jan H.; White, Donald E.

    2007-01-01

    The cloning of the genes encoding cancer antigens has opened new possibilities for the treatment of patients with cancer. In this study, immunodominant peptides from the gp100 melanoma-associated antigen were identified, and a synthetic peptide, designed to increase binding to HLA-A2 molecules, was used as a cancer vaccine to treat patients with metastatic melanoma. On the basis of immunologic assays, 91% of patients could be successfully immunized with this synthetic peptide, and 13 of 31 patients (42%) receiving the peptide vaccine plus IL-2 had objective cancer responses, and four additional patients had mixed or minor responses. Synthetic peptide vaccines based on the genes encoding cancer antigens hold promise for the development of novel cancer immunotherapies. PMID:9500606

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

    Science.gov (United States)

    Srinivasan, Alagiri

    2012-01-01

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

  14. AWRK6, A Synthetic Cationic Peptide Derived from Antimicrobial Peptide Dybowskin-2CDYa, Inhibits Lipopolysaccharide-Induced Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Qiuyu Wang

    2018-02-01

    Full Text Available Lipopolysaccharides (LPS are major outer membrane components of Gram-negative bacteria and produce strong inflammatory responses in animals. Most antibiotics have shown little clinical anti-endotoxin activity while some antimicrobial peptides have proved to be effective in blocking LPS. Here, the anti-LPS activity of the synthetic peptide AWRK6, which is derived from antimicrobial peptide dybowskin-2CDYa, has been investigated in vitro and in vivo. The positively charged α-helical AWRK6 was found to be effective in blocking the binding of LBP (LPS binding protein with LPS in vitro using ELISA. In a murine endotoxemia model, AWRK6 offered satisfactory protection efficiency against endotoxemia death, and the serum levels of LPS, IL-1β, IL-6, and TNF-α were found to be attenuated using ELISA. Further, histopathological analysis suggested that AWRK6 could improve the healing of liver and lung injury in endotoxemia mice. The results of real-time PCR and Western blotting showed that AWRK6 significantly reversed LPS-induced TLR4 overexpression and IκB depression, as well as the enhanced IκB phosphorylation. Additionally, AWRK6 did not produce any significant toxicity in vivo and in vitro. In summary, AWRK6 showed efficacious protection from LPS challenges in vivo and in vitro, by blocking LPS binding to LBP, without obvious toxicity, providing a promising strategy against LPS-induced inflammatory responses.

  15. Conserved C-terminal nascent peptide binding domain of HYPK ...

    Indian Academy of Sciences (India)

    2014-07-09

    Jul 9, 2014 ... viability and decreases caspase activities in Huntington's disease (HD) cell culture model. This domain is found to be required ... Huntington's disease (HD), this domain reduces cellular toxicity. We also find that ..... the adaptive functional value conferred by the NPAA domain of. HYPK is quite higher in case ...

  16. Lipoproteins/peptides are sepsis-inducing toxins from bacteria that can be neutralized by synthetic anti-endotoxin peptides.

    Science.gov (United States)

    Martinez de Tejada, Guillermo; Heinbockel, Lena; Ferrer-Espada, Raquel; Heine, Holger; Alexander, Christian; Bárcena-Varela, Sergio; Goldmann, Torsten; Correa, Wilmar; Wiesmüller, Karl-Heinz; Gisch, Nicolas; Sánchez-Gómez, Susana; Fukuoka, Satoshi; Schürholz, Tobias; Gutsmann, Thomas; Brandenburg, Klaus

    2015-09-22

    Sepsis, a life-threatening syndrome with increasing incidence worldwide, is triggered by an overwhelming inflammation induced by microbial toxins released into the bloodstream during infection. A well-known sepsis-inducing factor is the membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS), signalling via Toll-like receptor-4. Although sepsis is caused in more than 50% cases by Gram-positive and mycoplasma cells, the causative compounds are still poorly described. In contradicting investigations lipoproteins/-peptides (LP), lipoteichoic acids (LTA), and peptidoglycans (PGN), were made responsible for eliciting this pathology. Here, we used human mononuclear cells from healthy donors to determine the cytokine-inducing activity of various LPs from different bacterial origin, synthetic and natural, and compared their activity with that of natural LTA and PGN. We demonstrate that LP are the most potent non-LPS pro-inflammatory toxins of the bacterial cell walls, signalling via Toll-like receptor-2, not only in vitro, but also when inoculated into mice: A synthetic LP caused sepsis-related pathological symptoms in a dose-response manner. Additionally, these mice produced pro-inflammatory cytokines characteristic of a septic reaction. Importantly, the recently designed polypeptide Aspidasept(®) which has been proven to efficiently neutralize LPS in vivo, inhibited cytokines induced by the various non-LPS compounds protecting animals from the pro-inflammatory activity of synthetic LP.

  17. Application of atomic force microscopy to protein anatomy:. Imaging of supramolecular structures of self-assemblies formed from synthetic peptides

    Science.gov (United States)

    Shibata-Seki, T.; Masai, J.; Ogawa, Y.; Sato, K.; Yanagawa, H.

    This paper reports morphological studies of structures of self-assemblies from synthetic peptide fragments with the use of atomic force microscope (AFM) and transmission electron microscope (TEM). Two systems of synthetic peptides have been examined: one is peptides from barnase (a ribonuclease) and the other is those from tau protein (Alzheimer's disease-related protein). The AFM observation was carried out by using a commercially available AFM operated in the tapping mode in air. The general appearance in shape and size of the peptide assemblies in AFM images was essentially similar to that in TEM images, except that the AFM images provide us with fruitful three-dimensional information about the assemblies. For assemblies from barnase peptides, possible formation processes of the supramolecular structures from the corresponding peptide fragment have been proposed on the basis of the AFM images.

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

    DEFF Research Database (Denmark)

    Rønn, L C; Doherty, P; Holm, A

    2000-01-01

    identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons...... from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1......, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears...

  19. Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery.

    Science.gov (United States)

    Collins, Louise; Kaszuba, Michael; Fabre, John W

    2004-04-07

    The physical properties of non-viral vector/DNA nanoparticles in physiological aqueous solution are poorly understood. A Fluid Particle Image Analyser (FPIA), normally used for analysis of industrial and environmental fluids, was used to visualise individual (Lys)(16)-containing peptide/DNA particles. Eight (Lys)(16)-containing synthetic peptides were used to generate peptide/DNA particles at a constant + to - charge ratio of 2.8:1 with 10 microg/ml of plasmid DNA in phosphate buffered saline. Dynamic Light Scattering (DLS) and gene delivery studies were also performed. We present the first images of non-viral vector/DNA nanoparticles in physiological aqueous solution, together with precise measurements of individual particle size and shape in solution and, for the first time, an accurate measure of particle number. Particle size and shape, particle number, and efficiency for gene delivery varied markedly with different peptides. Under standard conditions for in vitro gene delivery, we estimate approximately 60 peptide/DNA nanoparticles per target cell, each containing approximately 70,000 plasmids. This novel capacity to image individual vector/DNA nanoparticles in solution and to count them accurately will enable a more precise assessment of non-viral gene delivery systems, and a more quantitative interpretation of gene delivery experiments.

  20. Peptides containing antigenic and cationic domains have enhanced, multivalent immunogenicity when bound to DNA vaccines.

    Science.gov (United States)

    Riedl, Petra; Reimann, Jörg; Schirmbeck, Reinhold

    2004-02-01

    We explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8(+) T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide. Complexes containing high (but not low) molar ratios of cationic peptide to DNA facilitate transfection (DNA uptake and expression of the plasmid-encoded product) of cells. In contrast, complexes containing low (but not high) molar ratios of cationic peptide to DNA prime potent multispecific T cell responses after a single intramuscular injection of the complexes. The general validity of this observation was confirmed mixing different antigenic/cationic peptides with different DNA vaccines. In these vaccine formulations, multispecific CD8(+) T cell responses specific for epitopes of the peptide- as well as the DNA-based vaccine were efficiently coprimed, together with humoral antibody responses to conformational determinants of large viral antigens encoded by the DNA vaccine. The data indicate that mixtures of DNA vaccines with antigenic, cationic peptides are immunogenic vaccine formulations particularly suited for the induction of multispecific T cell responses.

  1. Effects of synthetic peptides on the inflammatory response and their therapeutic potential.

    Science.gov (United States)

    Selvatici, Rita; Rita, Selvatici; Siniscalchi, Anna; Anna, Siniscalchi; Spisani, Susanna; Susanna, Spisani

    2013-04-01

    Recently, interest in small peptide molecules as potential drug candidates has revived. In this review, two series of synthetic peptides and their selective effects on the inflammatory response have been described, focusing on the intracellular pathways involved and on their therapeutic potential. A series of F(D)LF(D)LF analogs has been synthesized, including either N- t-Boc or different N-ureido substituents. The free acid derivatives as they are good candidates as antiinflammatory drugs are able to antagonize the multiple neutrophil functions evoked by N-formyl-L-methionyl-L-leucyl-Lphenylalanine (fMLF), i.e. chemotaxis, superoxide anion production and lysozyme release. Their methyl-ester derivatives are ineffective. The second series of peptides derives from the endogenous protein kinase C (PKC) inhibitor PKI55, a 55-amino acid protein, whose synthesis is induced by PKC activation, so that a feedback loop of inhibition is established. In vitro experiments showed that PKI55 inhibits recombinant PKC isoforms α, β1, β2, γ, δ, ζ, ; to identify the minimal amino acid sequence of PKI55 protein maintaining the inhibitory effects on PKC, peptides derived from both C- and N-terminal sequences have been synthesized. The N-terminal peptides 5 (MLYKLHDVCRQLWFSC), 8 (CRQLWFSC) and 9 (CRQLW), that in human neutrophils retain the inhibitory activity on PKC, decrease the chemotaxis, and, in mice, display anti-inflammatory and analgesic action, after both central and peripheral administration of very low doses. Furthermore, the peptide 5 shows neuroprotective activity in a model of cerebral ischemia in vitro, favouring the recovery of synaptic function. These findings suggest interesting possible therapeutic applications for these peptides.

  2. Antimicrobial efficacy of granulysin-derived synthetic peptides in acne vulgaris.

    Science.gov (United States)

    Lim, Hee-Sun; Chun, Seung-Min; Soung, Min-Gyu; Kim, Jenny; Kim, Seong-Jin

    2015-07-01

    Antimicrobial peptides are considered as a potential alternative to antibiotic treatment in acne vulgaris because the development of a resistant strain of Propionibacterium acnes is problematic. Granulysin can be regarded as an ideal substance with which to treat acne because it has antimicrobial and anti-inflammatory effects. This study was performed to explore the effectiveness of granulysin-derived peptides (GDPs) in killing P. acnes in vitro under a standard microbiologic assay and to evaluate their potential use in a topical agent for the treatment of acne vulgaris. Twenty different peptides based on the known sequence of a GDP were synthesized and tested in vitro for antimicrobial activity. Thirty patients with facial acne vulgaris were instructed to apply a topical formulation containing synthetic GDP to acne lesions twice per day for 12 weeks. A newly synthesized peptide in which aspartic acid was substituted with arginine, and methionine was substituted with cysteine, showed the highest antimicrobial activity against P. acnes. Moreover, it was effective against both Gram-positive and Gram-negative bacteria in vitro. After treatment with the topical formulation containing 50 ppm of synthetic peptide for 12 weeks, a significant reduction in the number of pustules was observed, regardless of the increase in the number of comedones. In addition, a significant reduction in the clinical grade of acne based on the Korean Acne Grading System (KAGS) was evident. Synthesized GDP shows strong antimicrobial activity against P. acnes in vitro. The clinical improvement observed suggests a topical formulation containing the GDP has therapeutic potential for the improvement of inflammatory-type acne vulgaris by its antimicrobial activity. © 2015 The International Society of Dermatology.

  3. Lipid membrane editing with peptide cargo linkers in cells and synthetic nanostructures

    Science.gov (United States)

    Pan, Hua; Myerson, Jacob W.; Ivashyna, Olena; Soman, Neelesh R.; Marsh, Jon N.; Hood, Joshua L.; Lanza, Gregory M.; Schlesinger, Paul H.; Wickline, Samuel A.

    2010-01-01

    Current strategies for deploying synthetic nanocarriers involve the creation of agents that incorporate targeting ligands, imaging agents, and/or therapeutic drugs into particles as an integral part of the formulation process. Here we report the development of an amphipathic peptide linker that enables postformulation editing of payloads without the need for reformulation to achieve multiplexing capability for lipidic nanocarriers. To exemplify the flexibility of this peptide linker strategy, 3 applications were demonstrated: converting nontargeted nanoparticles into targeting vehicles; adding cargo to preformulated targeted nanoparticles for in vivo site-specific delivery; and labeling living cells for in vivo tracking. This strategy is expected to enhance the clinical application of molecular imaging and/or targeted therapeutic agents by offering extended flexibility for multiplexing targeting ligands and/or drug payloads that can be selected after base nanocarrier formulation.—Pan, H., Myerson, J. W., Ivashyna, O., Soman, N. R., Marsh, J. N., Hood, J. L., Lanza, G. M., Schlesinger, P. H., Wickline, S. A.. Lipid membrane editing with peptide cargo linkers in cells and synthetic nanostructures. PMID:20335225

  4. Biochemical Characterisation of Phage Pseudomurein Endoisopeptidases PeiW and PeiP Using Synthetic Peptides

    Science.gov (United States)

    Schofield, Linley R.; Beattie, Amy K.; Tootill, Catherine M.; Dey, Debjit; Ronimus, Ron S.

    2015-01-01

    Pseudomurein endoisopeptidases cause lysis of the cell walls of methanogens by cleaving the isopeptide bond Ala-ε-Lys in the peptide chain of pseudomurein. PeiW and PeiP are two thermostable pseudomurein endoisopeptidases encoded by phage ΨM100 of Methanothermobacter wolfei and phages ΨM1 and ΨM2 of Methanothermobacter marburgensis, respectively. A continuous assay using synthetic peptide substrates was developed and used in the biochemical characterisation of recombinant PeiW and PeiP. The advantages of these synthetic peptide substrates over natural substrates are sensitivity, high purity, and characterisation and the fact that they are more easily obtained than natural substrates. In the presence of a reducing agent, purified PeiW and PeiP each showed similar activity under aerobic and anaerobic conditions. Both enzymes required a divalent metal for activity and showed greater thermostability in the presence of Ca2+. PeiW and PeiP involve a cysteine residue in catalysis and have a monomeric native conformation. The kinetic parameters, K M and k cat, were determined, and the ε-isopeptide bond between alanine and lysine was confirmed as the bond lysed by these enzymes in pseudomurein. The new assay may have wider applications for the general study of peptidases and the identification of specific methanogens susceptible to lysis by specific pseudomurein endoisopeptidases. PMID:26483615

  5. Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography.

    Science.gov (United States)

    Amarouche, Nassima; Giraud, Matthieu; Forni, Luciano; Butte, Alessandro; Edwards, F; Borie, Nicolas; Renault, Jean-Hugues

    2014-04-11

    Protected synthetic peptide intermediates are often hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), usually used for industrial production. It is then challenging to develop alternative chromatographic purification processes. Support-free liquid-liquid chromatographic techniques, including both hydrostatic (centrifugal partition chromatography or CPC) and hydrodynamic (counter-current chromatography or CCC) devices, are mainly involved in phytochemical studies but have also been applied to synthetic peptide purification. In this framework, two new biphasic solvent system compositions covering a wide range of polarity were developed to overcome solubility problems mentioned above. The new systems composed of heptane/tetrahydrofuran/acetonitrile/dimethylsulfoxide/water and heptane/methyl-tetrahydrofuran/N-methylpyrrolidone/water were efficiently used for the CPC purification of a 39-mer protected exenatide (Byetta®) and a 8-mer protected peptide intermediate of bivalirudin (Angiox®) synthesis. Phase compositions of the different biphasic solvent systems were determined by (1)H nuclear magnetic resonance. Physico-chemical properties including viscosity, density and interfacial tension of these biphasic systems are also described. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Immunoinformatic Analysis of Crimean Congo Hemorrhagic Fever Virus Glycoproteins and Epitope Prediction for Synthetic Peptide Vaccine

    International Nuclear Information System (INIS)

    Tipu, H. N.

    2016-01-01

    Objective: To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Study Design: Cross-sectional study. Place and Duration of Study: Combined Military Hospital, Khuzdar Cantt, in May 2015. Methodology: Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLA class I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. Results: HLA A*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. A total of 35 nanomers from GP1, and 3 from GP2 were identified. HLA B*0702 bound maximum number of peptides (6), while HLA B*4001 showed strongest binding affinity. Conclusion: HLA specific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates. (author)

  7. A synthetic peptide corresponding to the extracellular loop 2 region of claudin-4 protects against Clostridium perfringens enterotoxin in vitro and in vivo.

    Science.gov (United States)

    Shrestha, Archana; Robertson, Susan L; Garcia, Jorge; Beingasser, Juliann; McClane, Bruce A; Uzal, Francisco A

    2014-11-01

    Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since rclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain

    DEFF Research Database (Denmark)

    Oh, E S; Couchman, J R; Woods, A

    1997-01-01

    sequence. We investigated phosphorylation of syndecan-2 cytoplasmic domain by PKC, using purified GST-syndecan-2 fusion proteins and synthetic peptides corresponding to regions of the cytoplasmic domain. A synthetic peptide encompassing the entire cytoplasmic domain of syndecan-2 was phosphorylated by PKC...

  9. Mechanism of membrane permeation induced by synthetic β-hairpin peptides.

    Science.gov (United States)

    Gupta, Kshitij; Jang, Hyunbum; Harlen, Kevin; Puri, Anu; Nussinov, Ruth; Schneider, Joel P; Blumenthal, Robert

    2013-11-05

    We have investigated the membrane destabilizing properties of synthetic amphiphilic cationic peptides, MAX1 and MAX35, which have the propensity to form β-hairpin structures under certain conditions, and a control non-β-hairpin-forming peptide MAX8V16E. All three peptides bind to liposomes containing a mixture of zwitterionic POPC and negatively charged POPS lipids as determined by Zeta potential measurements. Circular dichroism measurements indicated folding of MAX1 and MAX35 in the presence of the POPC/POPS liposomes, whereas no such folding was observed with MAX8V16E. There was no binding or folding of these peptides to liposomes containing only POPC. MAX1 and MAX35 induced release of contents from negatively charged liposomes, whereas MAX8V16E failed to promote solute release under identical conditions. Thus, MAX1 and MAX35 bind to, and fold at the surface of negatively charged liposomes adopting a lytic conformation. We ruled out leaky fusion as a mechanism of release by including 2 mol % PEG-PE in the liposomes, which inhibits aggregation/fusion but not folding of MAX or MAX-induced leakage. Using a concentration-dependent quenching probe (calcein), we determined that MAX-induced leakage of liposome contents was an all-or-none process. At MAX1 concentrations, which cause release of ~50% of the liposomes that contain small (R(h) peptides are relatively more stable than MAX8V16E barrels in the bilayer, suggesting that barrels of this size are responsible for the peptides lytic action. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Antibodies, synthetic peptides and related constructs for planetary health based on green chemistry in the Anthropocene.

    Science.gov (United States)

    C Caoili, Salvador Eugenio

    2018-03-01

    The contemporary Anthropocene is characterized by rapidly evolving complex global challenges to planetary health vis-a-vis sustainable development, yet innovation is constrained under the prevailing precautionary regime that regulates technological change. Small-molecule xenobiotic drugs are amenable to efficient large-scale industrial synthesis; but their pharmacokinetics, pharmacodynamics, interactions and ultimate ecological impact are difficult to predict, raising concerns over initial testing and environmental contamination. Antibodies and similar agents can serve as antidotes and drug buffers or vehicles to address patient safety and decrease dosing requirements. More generally, peptidic agents including synthetic peptide-based constructs exemplified by vaccines can be used together with or instead of nonpeptidic xenobiotics, thus enabling advances in planetary health based on principles of green chemistry from manufacturing through final disposition.

  11. Specificity of anti-MHC class II antibody binding to synthetic peptides.

    Science.gov (United States)

    Chersi, A; Romano, T F; Ruocco, E

    1989-01-01

    This study indicates that antibodies raised against a DR4,w6; DQw1,3 positive cell line may bind to synthetic peptides selected from the polymorphic amino acid sequences 51-59 and 63-79 on the DQw2 beta chain. This cross-reaction may be explained by the relatively high sequence homology of these sequences in the beta chains of class II histocompatibility antigens, and suggests that antibody binding to small peptides may be scarsely selective. Based on the observations of the reactivity of the antibodies with several cell lines, and comparison of the amino acid sequences of beta chains of DR and DQ molecules, an attempt to identify the cross-reacting epitope is presented.

  12. Postinfection Activity of Synthetic Antimicrobial Peptides Against Stemphylium vesicarium in Pear.

    Science.gov (United States)

    Puig, M; Moragrega, C; Ruz, L; Montesinos, E; Llorente, I

    2014-11-01

    Brown spot of pear is a fungal disease of economic importance caused by Stemphylium vesicarium that affects the pear crops in Europe. Due to the characteristics of this disease and the moderate efficacy of available fungicides, the effectiveness of control measures is very limited; however, synthetic antimicrobial peptides (AMPs) may be a complement to these fungicides. In the present study, 12 AMPs of the CECMEL11 library were screened for fungicidal activity against S. vesicarium. In vitro experiments showed that eight AMPs significantly reduced the germination of conidia. The most effective peptides, BP15, BP22, and BP25, reduced fungal growth and sporulation at concentrations below 50 μM. Leaf assays showed that preventive application of BP15 and BP22 did not reduce infection; however, when the peptides were applied curatively, infection was significantly reduced. The use of a BP15 fluorescein 5-isothiocyanate conjugate revealed that the peptide binds to hyphae and germ tubes and produces malformations that irreversibly stop their development.

  13. Inhibition of dengue virus entry into target cells using synthetic antiviral peptides.

    Science.gov (United States)

    Alhoot, Mohammed Abdelfatah; Rathinam, Alwin Kumar; Wang, Seok Mui; Manikam, Rishya; Sekaran, Shamala Devi

    2013-01-01

    Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6% and 40.6% respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection.

  14. TIP peptide inhalation in experimental acute lung injury: effect of repetitive dosage and different synthetic variants.

    Science.gov (United States)

    Hartmann, Erik K; Thomas, Rainer; Liu, Tanghua; Stefaniak, Joanna; Ziebart, Alexander; Duenges, Bastian; Eckle, Daniel; Markstaller, Klaus; David, Matthias

    2014-01-01

    Inhalation of TIP peptides that mimic the lectin-like domain of TNF-α is a novel approach to attenuate pulmonary oedema on the threshold to clinical application. A placebo-controlled porcine model of acute respiratory distress syndrome (ARDS) demonstrated a reduced thermodilution-derived extravascular lung water index (EVLWI) and improved gas exchange through TIP peptide inhalation within three hours. Based on these findings, the present study compares a single versus a repetitive inhalation of a TIP peptide (TIP-A) and two alternate peptide versions (TIP-A, TIP-B). Following animal care committee approval ARDS was induced by bronchoalveolar lavage followed by injurious ventilation in 21 anaesthetized pigs. A randomised-blinded three-group setting compared the single-dosed peptide variants TIP-A and TIP-B as well as single versus repetitive inhalation of TIP-A (n = 7 per group). Over two three-hour intervals parameters of gas exchange, transpulmonary thermodilution, calculated alveolar fluid clearance, and ventilation/perfusion-distribution were assessed. Post-mortem measurements included pulmonary wet/dry ratio and haemorrhage/congestion scoring. The repetitive TIP-A inhalation led to a significantly lower wet/dry ratio than a single dose and a small but significantly lower EVLWI. However, EVLWI changes over time and the derived alveolar fluid clearance did not differ significantly. The comparison of TIP-A and B showed no relevant differences. Gas exchange and ventilation/perfusion-distribution significantly improved in all groups without intergroup differences. No differences were found in haemorrhage/congestion scoring. In comparison to a single application the repetitive inhalation of a TIP peptide in three-hour intervals may lead to a small additional reduction the lung water content. Two alternate TIP peptide versions showed interchangeable characteristics.

  15. Inhibitory Activity of Synthetic Peptide Antibiotics on Feline Immunodeficiency Virus Infectivity In Vitro

    Science.gov (United States)

    Ma, Jia; Kennedy-Stoskopf, Suzanne; Jaynes, Jesse M.; Thurmond, Linda M.; Tompkins, Wayne A.

    2002-01-01

    Natural peptide antibiotics are part of host innate immunity against a wide range of microbes, including some viruses. Synthetic peptides modeled after natural peptide antibiotics interfere with microbial membranes and are termed peptidyl membrane-interactive molecules (peptidyl-MIM [Demegen Inc, Pittsburgh, Pa.]). Sixteen peptidyl-MIM candidates were tested for activity against feline immunodeficiency virus (FIV) on infected CrFK cells. Three of them (D4E1, DC1, and D1D6) showed potent anti-FIV activity in chronically infected CrFK cells as measured by decreased reverse transcriptase (RT) activity, having 50% inhibitory concentrations of 0.46, 0.75, and 0.94 μM, respectively, which were approximately 10 times lower than their direct cytotoxic concentrations. Treatment of chronically infected CrFK cells with 2 μM D4E1 for 3 days completely reversed virus-induced cytopathic effect. Immunofluorescence revealed reduced p26 staining in these cells. Treatment of chronically infected CrFK cells with 2 μM D4E1 suppressed virus production (∼50%) for up to 7 days, The virions from the D4E1-treated culture had impaired infectivity, as measured by the 50% tissue culture infectious dose and nested PCR analysis of proviral DNA. However, these noninfectious virions were able to bind and internalize, suggesting a defect at some postentry step. After chronically infected CrFK cells were treated with D4E1 for 24 h, increased cell-associated mature p26 Gag and decreased extracellular virus-associated p26 Gag were observed by Western blot analysis, suggesting that virus assembly and/or release may be blocked by D4E1 treatment, whereas virus binding, penetration, RNA synthesis, and protein synthesis appear to be unaffected. Synthetic peptide antibiotics may be useful tools in the search for antiviral drugs having a wide therapeutic window for host cells. PMID:12208971

  16. Infectivity inhibition by overlapping synthetic peptides derived from the gH/gL heterodimer of herpes simplex virus type 1.

    Science.gov (United States)

    Franci, Gianluigi; Falanga, Annarita; Zannella, Carla; Folliero, Veronica; Martora, Francesca; Galdiero, Marilena; Galdiero, Stefania; Morelli, Giancarlo; Galdiero, Massimiliano

    2017-04-01

    Herpes simplex virus (HSV) is a human pathogen that infects epithelial cells. The cutaneous lesions, caused by the virus, spread to the nervous system creating several complications. Fusion of host membranes with the viral envelope is mandatory and mediated by a group of glycoproteins conserved in all Herpesviridae subfamilies, such as the glycoproteins B (gB), H (gH), L (gL) and D (gD). We investigated the inhibitory activity mediated by synthetic overlapping peptides spanning the entire ectodomains of gH and gL glycoproteins. We have performed a brute analysis of the complete gH/gL heterodimer in order to explore the inhibitory activity of peptides modelled on these glycoproteins against HSV-1 infection. Twenty-four of the gH peptides at a concentration of 150 μM reached the 50% of inhibition cut-off. Interestingly, they are mainly located in the gH carboxy-terminal domain. None of the gL peptides had a clear inhibiting effect. No peptide toxicity was observed by lactate dehydrogenase assay at the concentrations used in our experimental conditions. HSV-1 therapy is based on acyclovir treatment, but some resistant strains are emerging. In this scenario, innovative approaches for HSV-1 treatment are necessary. Our data support the direct involvement of the described domains in the process of virus penetration; therefore, these results are of relevance to the potential development of novel therapeutic compounds to prevent HSV-1 infections. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  17. Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide-Oligonucleotide Conjugates.

    Science.gov (United States)

    Taskova, Maria; Mantsiou, Anna; Astakhova, Kira

    2017-09-05

    The main objective of this work is to provide an update on synthetic nucleic acid analogues and nanoassemblies as tools in gene therapy. In particular, the synthesis and properties of peptide-oligonucleotide conjugates (POCs), which have high potential in research and as therapeutics, are described in detail. The exploration of POCs has already led to fruitful results in the treatment of neurological diseases, lung disorders, cancer, leukemia, viral, and bacterial infections. However, delivery and in vivo stability are the major barriers to the clinical application of POCs and other analogues that still have to be overcome. This review summarizes recent achievements in the delivery and in vivo administration of synthetic nucleic acid analogues, focusing on POCs, and compares their efficiency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Conformational photoswitching of a synthetic peptide foldamer bound within a phospholipid bilayer.

    Science.gov (United States)

    De Poli, Matteo; Zawodny, Wojciech; Quinonero, Ophélie; Lorch, Mark; Webb, Simon J; Clayden, Jonathan

    2016-04-29

    The dynamic properties of foldamers, synthetic molecules that mimic folded biomolecules, have mainly been explored in free solution. We report on the design, synthesis, and conformational behavior of photoresponsive foldamers bound in a phospholipid bilayer akin to a biological membrane phase. These molecules contain a chromophore, which can be switched between two configurations by different wavelengths of light, attached to a helical synthetic peptide that both promotes membrane insertion and communicates conformational change along its length. Light-induced structural changes in the chromophore are translated into global conformational changes, which are detected by monitoring the solid-state (19)F nuclear magnetic resonance signals of a remote fluorine-containing residue located 1 to 2 nanometers away. The behavior of the foldamers in the membrane phase is similar to that of analogous compounds in organic solvents. Copyright © 2016, American Association for the Advancement of Science.

  19. A Synthetic Peptide with the Putative Iron Binding Motif of Amyloid Precursor Protein (APP) Does Not Catalytically Oxidize Iron

    Science.gov (United States)

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

    2012-01-01

    The β-amyloid precursor protein (APP), which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II) binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III) product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II) just as in ferritin. We measured the ferroxidase activity indirectly (i) by the incorporation of the Fe(III) product of the ferroxidase reaction into transferrin and directly (ii) by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II) oxidation by molecular oxygen. Zn(II) binds to transferrin and diminishes its Fe(III) incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and – by implication – of APP should be re-evaluated. PMID:22916096

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

    Directory of Open Access Journals (Sweden)

    Kourosh Honarmand Ebrahimi

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

  1. Cholesterol-Enriched Domain Formation Induced by Viral-Encoded, Membrane-Active Amphipathic Peptide.

    Science.gov (United States)

    Hanson, Joshua M; Gettel, Douglas L; Tabaei, Seyed R; Jackman, Joshua; Kim, Min Chul; Sasaki, Darryl Y; Groves, Jay T; Liedberg, Bo; Cho, Nam-Joon; Parikh, Atul N

    2016-01-05

    The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of ∼100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. Efficient generation of dopamine neuron-like cells from skin-derived precursors with a synthetic peptide derived from von Hippel-Lindau protein.

    Science.gov (United States)

    Kubo, Atsuhiko; Yoshida, Tetsuhiko; Kobayashi, Nahoko; Yokoyama, Takaakira; Mimura, Toshiro; Nishiguchi, Takao; Higashida, Tetsuhiro; Yamamoto, Isao; Kanno, Hiroshi

    2009-12-01

    Skin-derived precursors (SKPs) from mammalian dermis represent neural crest-related stem cells capable of differentiating into both neural and mesodermal progency. SKPs are of clinical interest because they serve as accessible autologous donor cells for neuronal repair for neuronal intractable diseases. However, little is known about the efficient generation of neurons from SKPs, and phenotypes of neurons generated from SKPs have been restricted. In addition, the neuronal repair using their generated neurons as donor cells has not been achieved. The von Hippel-Lindau protein (pVHL) is one of the proteins that play an important role during neuronal differentiation, and recently neuronal differentiation of neural progenitor cells by intracellular delivery of a synthetic VHL peptide derived from elongin BC-binding site has been demonstrated. In the present study, a synthetic VHL peptide derived from elongin BC-binding site was conjugated to the protein transduction domain (PTD) of HIV-TAT protein (TATVHL peptide) to facilitate entry into cells, and we demonstrate the efficient generation of cells with dopaminergic phenotype from SKPs with the intracellular delivery of TATVHL peptide, and characterized the generated cells. The TATVHL peptide-treated SKPs expressed neuronal marker proteins, particularly dopamine neuron markers, and also up-regulated mRNA levels of proneural basic helix-loop-helix factors. After the TATVHL peptide treatment, transplanted SKPs into Parkinson's disease (PD) model rats sufficiently differentiated into dopamine neuron-like cells in PD model rats, and partially but significantly corrected behavior of PD model rats. The generated dopamine neuron-like cells are expected to serve as donor cells for neuronal repair for PD.

  3. Synthetic Peptide Drugs for Targeting Skin Cancer: Malignant Melanoma and Melanotic Lesions.

    Science.gov (United States)

    Eberle, Alex N; Rout, Bhimsen; Qi, Mei Bigliardi; Bigliardi, Paul L

    2017-01-01

    Peptides play decisive roles in the skin, ranging from host defense responses to various forms of neuroendocrine regulation of cell and organelle function. Synthetic peptides conjugated to radionuclides or photosensitizers may serve to identify and treat skin tumors and their metastatic forms in other organs of the body. In the introductory part of this review, the role and interplay of the different peptides in the skin are briefly summarized, including their potential application for the management of frequently occurring skin cancers. Special emphasis is given to different targeting options for the treatment of melanoma and melanotic lesions. Radionuclide Targeting: α-Melanocyte-stimulating hormone (α-MSH) is the most prominent peptide for targeting of melanoma tumors via the G protein-coupled melanocortin-1 receptor that is (over-)expressed by melanoma cells and melanocytes. More than 100 different linear and cyclic analogs of α-MSH containing chelators for 111In, 67/68Ga, 64Cu, 90Y, 212Pb, 99mTc, 188Re were synthesized and examined with experimental animals and in a few clinical studies. Linear Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH2 (NAP-amide) and Re-cyclized Cys- Cys-Glu-His-D-Phe-Arg-Trp-Cys-Arg-Pro-Val-NH2 (Re[Arg11]CCMSH) containing different chelators at the N- or C-terminus served as lead compounds for peptide drugs with further optimized characteristics. Alternatively, melanoma may be targeted with radiopeptides that bind to melanin granules occurring extracellularly in these tumors. Photosensitizer targeting: A more recent approach is the application of photosensitizers attached to the MSH molecule for targeted photodynamic therapy using LED or coherent laser light that specifically activates the photosensitizer. Experimental studies have demonstrated the feasibility of this approach as a more gentle and convenient alternative compared to radionuclides. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. [Evaluation of synthetic peptide vaccines against foot-and-mouth disease type A].

    Science.gov (United States)

    Tang, Hua; Liu, Xinsheng; Fang, Yuzhen; Jiang, Shoutian; Pan, Li; Lv, Jianliang; Zhang, Zhongwang; Zhou, Peng; Zhang, Yongguang; Wang, Yonglu

    2013-06-04

    We developed a synthetic vaccine against foot-and-mouth disease type A. We studied two peptide-based vaccines containing residues 131 to 159 of VP1, 20 to 35 of VP4, 21 to 35 of 3A and 29 to 42 of 3B of the AF/72 strain of foot-and-mouth disease virus (FMDV) coupled with a CpG oligodeoxynucleotide (5'-TCGCGAACGTTCGCCCGATCGTCGGTA-3') in guinea pigs. We assayed the FMDV-specific IgG level, serum neutralizing antibody titer, splenic lymphocytes proliferative capacity and peripheral blood T lymphocyte CD4-CD8 subsets distribution. The data show that high dose did not ensure a good immunity. In our study, 8% (4/5) of peptide 364-2.5-inoculated guinea pigs (2.5 microg of peptide 364 per animal) were protected against AF/72 strain challenge, while the protection ratio from other peptide-immunized groups was lower except the inactivated vaccine-inoculated group which showed a full protection. Our results also indicated that the stimulatory ability of CD4+ T lymphocyte response played a key role in evaluating effective FMDV vaccine. The highest percentage of CD4+ T lymphocyte was 36.6% appeared in inactivated vaccine-immunized guinea pigs, the second was 33.7% in peptide 364-2.5-vaccinated group, whereas the remaining ranged from 18.1% to 27.7%. There was no obvious relation between CD8+ T cells and anti-FMDV infection; our data showed that the CD4/CD8 ratio was not always appropriate for assessing the immune system status. In general, we not only designed an effective vaccine against FMDV type A, but also discovered some useful information of humoral and cellular responses induced by foot-and-mouth disease vaccines.

  5. Short, Synthetic Cationic Peptides Have Antibacterial Activity against Mycobacterium smegmatis by Forming Pores in Membrane and Synergizing with Antibiotics.

    Science.gov (United States)

    Gupta, Kajal; Singh, Sameer; van Hoek, Monique L

    2015-08-24

    Multicellular organisms are constantly exposed to a multitude of pathogenic microbes. Infection is inhibited in vivo by the innate and adaptive immune system. Mycobacterium species have emerged that are resistant to most antibiotics. We identified several naturally occurring cationic antimicrobial peptides that were active at low micromolar concentrations against Mycobacterium smegmatis. Human-derived cathelicidin LL-37 is well characterized and studied against M. smegmatis; we compared LL-37 with Chinese cobra-derived cathelicidin NA-CATH and mouse cathelicidin (mCRAMP). Two synthetic 11-residue peptides (ATRA-1A and ATRA-2) containing variations of a repeated motif within NA-CATH were tested for their activity against M. smegmatis along with a short synthetic peptide derivative from the human beta-defensin hBD3 (hBD3-Pep4). We hypothesized that these smaller synthetic peptides may demonstrate antimicrobial effectiveness with shorter length (and at less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds or use in combination with antibiotics. These peptides have antimicrobial activity with EC50 ranging from 0.05 to 1.88 μg/mL against Mycobacterium smegmatis. The ATRA-1A short peptide was found to be the most effective antimicrobial peptide (AMP) (EC50 = 0.05 μg/mL). High bactericidal activity correlated with bacterial membrane depolarization and permeabilization activities. The efficacy of the peptides was further analyzed through Minimal Inhibitory Concentration (MIC) assays. The MICs were determined by the microdilution method. The peptide mCRAMP showed the best MIC activity at 15.6 μg/mL. Neither of the effective short synthetic peptides demonstrated synergy with the antibiotic rifampicin, although both demonstrated synergy with the cyclic peptide antibiotic polymyxin B. The peptides LL-37 and mCRAMP displayed synergism with rifampicin in MIC assays, whereas antibiotic polymyxin B displayed synergism

  6. Short, Synthetic Cationic Peptides Have Antibacterial Activity against Mycobacterium smegmatis by Forming Pores in Membrane and Synergizing with Antibiotics

    Directory of Open Access Journals (Sweden)

    Kajal Gupta

    2015-08-01

    Full Text Available Multicellular organisms are constantly exposed to a multitude of pathogenic microbes. Infection is inhibited in vivo by the innate and adaptive immune system. Mycobacterium species have emerged that are resistant to most antibiotics. We identified several naturally occurring cationic antimicrobial peptides that were active at low micromolar concentrations against Mycobacterium smegmatis. Human-derived cathelicidin LL-37 is well characterized and studied against M. smegmatis; we compared LL-37 with Chinese cobra-derived cathelicidin NA-CATH and mouse cathelicidin (mCRAMP. Two synthetic 11-residue peptides (ATRA-1A and ATRA-2 containing variations of a repeated motif within NA-CATH were tested for their activity against M. smegmatis along with a short synthetic peptide derivative from the human beta-defensin hBD3 (hBD3-Pep4. We hypothesized that these smaller synthetic peptides may demonstrate antimicrobial effectiveness with shorter length (and at less cost, making them strong potential candidates for development into broad-spectrum antimicrobial compounds or use in combination with antibiotics. These peptides have antimicrobial activity with EC50 ranging from 0.05 to 1.88 μg/mL against Mycobacterium smegmatis. The ATRA-1A short peptide was found to be the most effective antimicrobial peptide (AMP (EC50 = 0.05 μg/mL. High bactericidal activity correlated with bacterial membrane depolarization and permeabilization activities. The efficacy of the peptides was further analyzed through Minimal Inhibitory Concentration (MIC assays. The MICs were determined by the microdilution method. The peptide mCRAMP showed the best MIC activity at 15.6 μg/mL. Neither of the effective short synthetic peptides demonstrated synergy with the antibiotic rifampicin, although both demonstrated synergy with the cyclic peptide antibiotic polymyxin B. The peptides LL-37 and mCRAMP displayed synergism with rifampicin in MIC assays, whereas antibiotic polymyxin B displayed

  7. THERMAL STABILITY OF SYNTHETIC PEPTIDES MIMICKING THE SEQUENCE OF THE REGION CONTAINING THE SKIP RESIDUES IN SQUID MYOSIN ROD

    Directory of Open Access Journals (Sweden)

    Yoshihiro Ochiai

    2017-08-01

    Full Text Available Myosin is the major protein in skeletal muscles including those of fish and shellfish. The characteristics of this protein are closely related to the biological function and the quality and physical properties of musclefood. In the myosin rod (the coiled-coil region of myosin, several amino acid residues, known as skip residues, seem to destabilize the ordered structure (heptad repeat. These residues might be responsible for reducing thermal stability. Attempts were thus made to examine the role of these residues in the rod of squid myosin, based on the thermodynamic properties of synthetic peptides which have been designed to mimic the partial sequence of myosin heavy chain from the squid Todarodes pacificus mantle muscle. Five peptides, namely, with the sequence of Trp1343 -Ala1372  having the skip residue Glu1357 at the center (Peptide WT, without the skip residue (Peptide Δ, with the replacements of the skip residue (Glu by Ile, Gln and Pro (Peptides E/I, E/Q, and E/P, respectively to modify the helix forming propensity, were synthesized. The results obtained showed that the stability of the peptides as measured by circular dichroism spectrometry was in the order of Peptide Δ > Peptide WT > Peptide E/Q > Peptide E/P > Peptide E/I. It is suggested that the presence of the skip residues dexterously tunes the stability or flexibility of the coiled-coil structure, thus possibly regulating thick filament formation and further gel formation ability of myosin.

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

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    Garry Robert F

    2007-11-01

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

  9. Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

    International Nuclear Information System (INIS)

    Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

    1991-01-01

    Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

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

    Science.gov (United States)

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

    2018-02-01

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

  11. Antimicrobial activity predictors benchmarking analysis using shuffled and designed synthetic peptides.

    Science.gov (United States)

    Porto, William F; Pires, Állan S; Franco, Octavio L

    2017-08-07

    The antimicrobial activity prediction tools aim to help the novel antimicrobial peptides (AMP) sequences discovery, utilizing machine learning methods. Such approaches have gained increasing importance in the generation of novel synthetic peptides by means of rational design techniques. This study focused on predictive ability of such approaches to determine the antimicrobial sequence activities, which were previously characterized at the protein level by in vitro studies. Using four web servers and one standalone software, we evaluated 78 sequences generated by the so-called linguistic model, being 40 designed and 38 shuffled sequences, with ∼60 and ∼25% of identity to AMPs, respectively. The ab initio molecular modelling of such sequences indicated that the structure does not affect the predictions, as both sets present similar structures. Overall, the systems failed on predicting shuffled versions of designed peptides, as they are identical in AMPs composition, which implies in accuracies below 30%. The prediction accuracy is negatively affected by the low specificity of all systems here evaluated, as they, on the other hand, reached 100% of sensitivity. Our results suggest that complementary approaches with high specificity, not necessarily high accuracy, should be developed to be used together with the current systems, overcoming their limitations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Komodo dragon-inspired synthetic peptide DRGN-1 promotes wound-healing of a mixed-biofilm infected wound.

    Science.gov (United States)

    M C Chung, Ezra; Dean, Scott N; Propst, Crystal N; Bishop, Barney M; van Hoek, Monique L

    2017-01-01

    Cationic antimicrobial peptides are multifunctional molecules that have a high potential as therapeutic agents. We have identified a histone H1-derived peptide from the Komodo dragon ( Varanus komodoensis) , called VK25. Using this peptide as inspiration, we designed a synthetic peptide called DRGN-1. We evaluated the antimicrobial and anti-biofilm activity of both peptides against Pseudomonas aeruginosa and Staphylococcus aureus . DRGN-1, more than VK25, exhibited potent antimicrobial and anti-biofilm activity, and permeabilized bacterial membranes. Wound healing was significantly enhanced by DRGN-1 in both uninfected and mixed biofilm ( Pseudomonas aeruginosa and Staphylococcus aureus )-infected murine wounds. In a scratch wound closure assay used to elucidate the wound healing mechanism, the peptide promoted the migration of HEKa keratinocyte cells, which was inhibited by mitomycin C (proliferation inhibitor) and AG1478 (epidermal growth factor receptor inhibitor). DRGN-1 also activated the EGFR-STAT1/3 pathway. Thus, DRGN-1 is a candidate for use as a topical wound treatment. Wound infections are a major concern; made increasingly complicated by the emerging, rapid spread of bacterial resistance. The novel synthetic peptide DRGN-1 (inspired by a peptide identified from Komodo dragon) exhibits pathogen-directed and host-directed activities in promoting the clearance and healing of polymicrobial ( Pseudomonas aeruginosa & Staphylococcus aureus ) biofilm infected wounds. The effectiveness of this peptide cannot be attributed solely to its ability to act upon the bacteria and disrupt the biofilm, but also reflects the peptide's ability to promsote keratinocyte migration. When applied in a murine model, infected wounds treated with DRGN-1 healed significantly faster than did untreated wounds, or wounds treated with other peptides. The host-directed mechanism of action was determined to be via the EGFR-STAT1/3 pathway. The pathogen-directed mechanism of action was

  13. Efficiency and mechanism of antigen-specific CD8+ T-cell activation using synthetic long peptides.

    Science.gov (United States)

    Zandvliet, Maarten L; Kester, Michel G D; van Liempt, Ellis; de Ru, Arnoud H; van Veelen, Peter A; Griffioen, Marieke; Guchelaar, Henk-Jan; Falkenburg, J H Frederik; Meij, Pauline

    2012-01-01

    Synthetic long peptides that contain immunogenic T-cell epitopes have been used to induce activation of antigen-specific CD8 T cells in vitro for immune monitoring or adoptive transfer, or in vivo after peptide vaccination. However, the efficiency and mechanisms of presentation of exogenous long peptides in human leukocyte antigen (HLA) class I remain to be elucidated. In this study, we demonstrated that the efficiency of antigen-specific CD8 T-cell activation using extended peptide variants of common viral epitopes is variable. We demonstrated that processing and HLA class I presentation of the long peptides were not dependent on the proteasome and transporter associated with antigen processing, illustrating that the classic route of HLA class I presentation was not required for activation of specific CD8 T cells by exogenous synthetic long peptides. Although long peptides were shown to bind to the relevant HLA class I molecules, peptide trimming was likely to be essential for optimal HLA class I presentation and T-cell activation. As the proteasome was not required for processing of exogenous peptides, it is very likely that peptide trimming was mediated by peptidases, which may be located extracellularly at the cell surface, in the cytosol, endoplasmic reticulum, or in endosomal and lysosomal compartments. Furthermore, the results suggested that processing of the correct minimal peptides was facilitated by binding in HLA class I molecules. This mechanism of HLA-guided processing may be important in HLA class I presentation of exogenous long peptides to induce activation of specific CD8 T cells.

  14. A rapid and clean synthetic approach to cyclic peptides via micro-flow peptide chain elongation and photochemical cyclization: synthesis of a cyclic RGD peptide.

    Science.gov (United States)

    Mifune, Yuto; Nakamura, Hiroyuki; Fuse, Shinichiro

    2016-11-29

    A cyclic RGD peptide was efficiently synthesized based on micro-flow, triphosgene-mediated peptide chain elongation and micro-flow photochemical macrolactamization. Our approach enabled a rapid (amidation for peptide chain elongation peptide.

  15. Evaluation of the Hydrolysis Specificity of an Aminopeptidase from Bacillus licheniformis SWJS33 Using Synthetic Peptides and Soybean Protein Isolate.

    Science.gov (United States)

    Lei, Fenfen; Zhao, Qiangzhong; Lin, Lianzhu; Sun, Baoguo; Zhao, Mouming

    2017-01-11

    The substrate specificity of aminopeptidases has often been determined against aminoacyl-p-nitroanilide; thus, its specificity toward synthetic peptides and complex substrates remained unclear. The hydrolysis specificity of an aminopeptidase from Bacillus licheniformis SWJS33 (BLAM) was evaluated using a series of synthetic peptides and soybean protein isolate. The aminopeptidase showed high specificity for dipeptides with Leu, Val, Ala, Gly, and Phe at the N-terminus, and the specificity was significantly affected by the nature of the penultimate residue. In the hydrolysis of soy protein isolate, BLAM preferred peptides with Leu, Glu, Gly, and Ala at the N-terminus by free amino acid analysis and preferred peptides with Leu, Ala, Ser, Trp, and Tyr at the N-terminus by UPLC-MS/MS. The introduction of complex substrates provides a deeper understanding of the aminopeptidase's specificity, which can instruct the application of the enzyme in protein hydrolysis.

  16. Efficacy of synthetic peptide candidate vaccines against serotype-A foot-and-mouth disease virus in cattle.

    Science.gov (United States)

    Zhang, Zhongwang; Pan, Li; Ding, Yaozhong; Zhou, Peng; Lv, Jianliang; Chen, Haotai; Fang, Yuzhen; Liu, Xinsheng; Chang, Huiyun; Zhang, Jie; Shao, Junjun; Lin, Tong; Zhao, Furong; Zhang, Yongguang; Wang, Yonglu

    2015-02-01

    Foot-and-mouth disease (FMD) remains a major threat to livestock worldwide, especially in developing countries. To improve the efficacy of vaccination against FMD, various types of vaccines have been developed, including synthetic peptide vaccines. We designed three synthetic peptide vaccines, 59 to 87 aa in size, based on immunogenic epitopes in the VP1, 3A, and 3D proteins of the A/HuBWH/CHA/2009 strain of the foot-and-mouth disease virus (FMDV), corresponding to amino acid positions 129 to 169 of VP1, 21 to 35 of 3A, and 346 to 370 of 3D. The efficacies of the vaccines were evaluated in cattle and guinea pigs challenged with serotype-A FMDV. All of the vaccines elicited the production of virus-neutralizing antibodies. The PB peptide, which contained sequences corresponding to positions 129 to 169 of V P1 and 346 to 370 of 3D, demonstrated the highest levels of immunogenicity and immunoprotection against FMDV. Two doses of 50 μg of the synthetic PB peptide vaccine provided 100% protection against FMDV infection in guinea pigs, and a single dose of 100 μg provided 60% protection in cattle. These findings provide empirical data for facilitating the development of synthetic peptide vaccines against FMD.

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

    Science.gov (United States)

    Wang, Bing; Swaminathan, Sivakumar; Bhattacharyya, Madan K

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Bing Wang

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

  19. Conserved C-terminal nascent peptide binding domain of HYPK ...

    Indian Academy of Sciences (India)

    energies (in kcal/mol) (marked in black circles) for each amino acid residues of human HYPK protein. NPAA domain region has been ... Aromatic (H, F, W, Y). 1.17. 0.88. 0.77. 0.32. 0.99. 0.73 0.82. 0.51. 0.69. 0.47. 0.55. 0.18 polar (R, N, D, E, Q, H, K, S, T) 5.97. 1.50. 6.74. 1.22. 6.47. 1.59 6.58. 1.52. 6.32. 1.16. 6.43. 0.77.

  20. Molecular cloning, over expression, and activity studies of a peptidic HIV-1 protease inhibitor: designed synthetic gene to functional recombinant peptide.

    Science.gov (United States)

    Vathipadiekal, Vinod; Umasankar, Perunthottathu K; Patole, Milind S; Rao, Mala

    2010-01-01

    The aspartic protease inhibitor (ATBI) purified from a Bacillus sp. is a potent inhibitor of several proteases including recombinant HIV-1 protease, pepsin, and fungal aspartic protease. In this study, we report the cloning, and over expression of a synthetic gene coding for ATBI in Escherichia coli and establish a purification protocol. The ATBI molecule consists of eleven amino acids and is peptidic in nature. We used the peptide sequence data of ATBI to synthesize complementary oligonucleotides, which were annealed and subsequently cloned in-frame with the gene for glutathione-S-transferase (GST). The expression of the resulting fusion protein was induced in E. coli BL21-A1 cells using arabinose. The recombinant peptide was purified using a reduced glutathione column, and cleaved with Factor Xa to remove the GST tag. The resultant product was further purified to homogeneity using RP-HPLC. Mass spectroscopy analysis revealed that the purified peptide had a molecular weight of 1186Da which matches the theoretical molecular weight of the amino acids present in the synthetic gene. The recombinant peptide was found to be active in vitro against HIV-1 protease, pepsin, and fungal aspartic protease. The protocol described in this study may be used to clone pharmaceutically important peptide molecules.

  1. Defining intrinsic hydrophobicity of amino acids' side chains in random coil conformation. Reversed-phase liquid chromatography of designed synthetic peptides vs. random peptide data sets.

    Science.gov (United States)

    Shamshurin, Dmitry; Spicer, Vic; Krokhin, Oleg V

    2011-09-16

    The two leading RP-HPLC approaches for deriving hydrophobicity values of amino acids utilize either sets of designed synthetic peptides or extended random datasets often extracted from proteomics experiments. We find that the best examples of these two methods provide virtually identical results--with exception of Lys, Arg, and His. The intrinsic hydrophobicity values of the remaining residues as determined by Kovacs et al. (Biopolymers 84 (2006) 283) correlates with an R(2)-value of 0.995+ against amino acid retention coefficients from our Sequence Specific Retention Calculator model (Anal. Chem. 78 (2006) 7785). This novel finding lays the foundation for establishing consensus amino acids hydrophobicity scales as determined by RP-HPLC. Simultaneously, we find the assignment of hydrophobicity values for charged residues (Lys, Arg and His at pH 2) is ambiguous; their retention contribution is strongly affected by the overall peptide hydrophobicity. The unique behavior of the basic residues is related to the dualistic character of the RP peptide retention mechanism, where both hydrophobic and ion-pairing interactions are involved. We envision the introduction of "sliding" hydrophobicity scales for charged residues as a new element in peptide retention prediction models. We also show that when using a simple additive retention prediction model, the "correct" coefficient value optimization (0.98+ correlation against values determined by synthetic peptide approach) requires a training set of at least 100 randomly selected peptides. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Predicting binding within disordered protein regions to structurally characterised peptide-binding domains.

    Directory of Open Access Journals (Sweden)

    Waqasuddin Khan

    Full Text Available Disordered regions of proteins often bind to structured domains, mediating interactions within and between proteins. However, it is difficult to identify a priori the short disordered regions involved in binding. We set out to determine if docking such peptide regions to peptide binding domains would assist in these predictions.We assembled a redundancy reduced dataset of SLiM (Short Linear Motif containing proteins from the ELM database. We selected 84 sequences which had an associated PDB structures showing the SLiM bound to a protein receptor, where the SLiM was found within a 50 residue region of the protein sequence which was predicted to be disordered. First, we investigated the Vina docking scores of overlapping tripeptides from the 50 residue SLiM containing disordered regions of the protein sequence to the corresponding PDB domain. We found only weak discrimination of docking scores between peptides involved in binding and adjacent non-binding peptides in this context (AUC 0.58.Next, we trained a bidirectional recurrent neural network (BRNN using as input the protein sequence, predicted secondary structure, Vina docking score and predicted disorder score. The results were very promising (AUC 0.72 showing that multiple sources of information can be combined to produce results which are clearly superior to any single source.We conclude that the Vina docking score alone has only modest power to define the location of a peptide within a larger protein region known to contain it. However, combining this information with other knowledge (using machine learning methods clearly improves the identification of peptide binding regions within a protein sequence. This approach combining docking with machine learning is primarily a predictor of binding to peptide-binding sites, and is not intended as a predictor of specificity of binding to particular receptors.

  3. Achieving peptide binding specificity and promiscuity by loops: case of the forkhead-associated domain.

    Science.gov (United States)

    Huang, Yu-Ming M; Chang, Chia-En A

    2014-01-01

    The regulation of a series of cellular events requires specific protein-protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, α helix and β sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design.

  4. Synthetic Peptide Ligands of the Antigen Binding Receptor Induce Programmed Cell Death in a Human B-Cell Lymphoma

    Science.gov (United States)

    Renschler, Markus F.; Bhatt, Ramesh R.; Dower, William J.; Levy, Ronald

    1994-04-01

    Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.

  5. N-terminal region of human ameloblastin synthetic peptide promotes bone formation.

    Science.gov (United States)

    Kitagawa, Masae; Ando, Toshinori; Subarnbhesaj, Ajiravudh; Uchida, Takashi; Miyauchi, Mutsumi; Takata, Takashi

    2017-01-01

    The aim of this study was to examine the effect of 16 amino acids of the N-terminal region of human ameloblastin (16N-AMBN) synthetic peptide, on the proliferation and differentiation of MC3T3-E1 cells and bone regeneration. While 16N-AMBN did not affect the proliferation, it induced mRNA expression of type I collagen, alkaline phosphatase (ALP), bone sialoprotein, and osteocalcin. 16N-AMBN also stimulated ALP activity and promoted mineralized nodule formation. On the other hand, these activities were inhibited by anti-16N-AMBN antibody. Treatment of rat calvarial bone defects with 16N-AMBN resulted in almost complete healing compared to that of the control treatments. These findings suggest that 16N-AMBN may be applicable for regeneration therapy of bone defects.

  6. Inhibition of adjuvant-induced arthritis by nasal administration of novel synthetic peptides from heat shock protein 65.

    Science.gov (United States)

    Shi, Xiao-Lian; Wang, Li-Ping; Feng, Xuan; Fan, Dan-Dan; Zang, Wei-Jin; Wang, Bing; Zhou, Jun

    2014-07-25

    Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease mediated by T cells. The aim of the present study was to investigate the therapeutic efficacy of synthetic peptides (HP-R1, HP-R2 and HP-R3), derived from the sequence of 65-kD mycobacterial heat shock protein (HSP), in the treatment of RA using adjuvant-induced arthritis (AA) animal model. AA was induced by a single intradermal injection Freund's complete adjuvant in male Lewis rats. At the first clinical sign of disease, rats were administered nasally by micropipette of peptides or phosphate buffer saline (PBS). Disease progression was monitored by measurement of body weight, arthritis score and paw swelling. The changes of histopathology were assessed by hematoxylin eosin staining. The serum levels of tumor necrosis factor (TNF) - alpha and interleukin (IL)-4 were measured by enzyme-linked immunosorbent assay (ELISA). The peptides efficiently inhibited the footpad swelling and arthritic symptoms in AA rats. The synthetic peptides displayed significantly less inflammatory cellular infiltration and synovium hyperplasia than model controls. This effect was associated with a suppression of pro-inflammatory cytokine TNF-alpha production and an increase of anti-inflammatory cytokine IL-4 production after peptides treatment. These results suggest that the synthetic peptides derived from HSP65 induce highly effective protection against AA, which is mediated in part by down-regulation of inflammatory cytokines, and support the view that the synthetic peptides is a potential therapy for RA that may help to diminish both joint inflammation and destruction.

  7. A short synthetic peptide fragment of human C2ORF40 has therapeutic potential in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Chaoyang [Shandong Univ., Jinan (China); Zhang, Pengju [Shandong Univ., Jinan (China); Jiang, Anli [Shandong Univ., Jinan (China); Mao, Jian-Hua [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Wei, Guangwei [Shandong Univ. School of Medicine, Jinan (China)

    2017-03-30

    C2ORF40 encodes a secreted protein which is cleaved to generate soluble peptides by proteolytic processing and this process is believed to be necessary for C2ORF40 to exert cell type specific biological activity. Here, we reported a short mimic peptide of human C2ORF40 acts potential therapeutic efficacy in human cancer cells in vitro and in vivo. We synthesized a short peptide of human C2ORF40, named C2ORF40 mimic peptide fragment and assessed its biological function on cancer cell growth, migration and tumorigenesis. Cell growth assay showed that C2ORF40 mimic peptide fragment significantly suppressed cell proliferation of breast and lung cancer cells. Moreover, C2ORF40 mimic peptide fragment significantly inhibited the migration and invasion of breast cancer cells. Furthermore, we showed that this peptide suppressed tumorigenesis in breast tumor xenograft model. Cell cycle assay indicated that the C2ORF40 mimic peptide fragment suppressed the growth of tumor cells through inducing mitotic phase arrest. In conclusion, our results firstly suggested that this short synthetic peptide of human C2ORF40 may be a candidate tumor therapeutic agent.

  8. A short synthetic peptide fragment of human C2ORF40 has therapeutic potential in breast cancer.

    Science.gov (United States)

    Li, Chaoyang; Zhang, Pengju; Jiang, Anli; Mao, Jian-Hua; Wei, Guangwei

    2017-06-27

    C2ORF40 encodes a secreted protein which is cleaved to generate soluble peptides by proteolytic processing and this process is believed to be necessary for C2ORF40 to exert cell type specific biological activity. Here, we reported a short mimic peptide of human C2ORF40 acts potential therapeutic efficacy in human cancer cells in vitro and in vivo. We synthesized a short peptide of human C2ORF40, named C2ORF40 mimic peptide fragment and assessed its biological function on cancer cell growth, migration and tumorigenesis. Cell growth assay showed that C2ORF40 mimic peptide fragment significantly suppressed cell proliferation of breast and lung cancer cells. Moreover, C2ORF40 mimic peptide fragment significantly inhibited the migration and invasion of breast cancer cells. Furthermore, we showed that this peptide suppressed tumorigenesis in breast tumor xenograft model. Cell cycle assay indicated that the C2ORF40 mimic peptide fragment suppressed the growth of tumor cells through inducing mitotic phase arrest. In conclusion, our results firstly suggested that this short synthetic peptide of human C2ORF40 may be a candidate tumor therapeutic agent.

  9. Induction of human immunodeficiency virus (HIV-1 envelope specific cell-mediated immunity by a non-homologous synthetic peptide.

    Directory of Open Access Journals (Sweden)

    Ammar Achour

    2007-11-01

    Full Text Available Cell mediated immunity, including efficient CTL response, is required to prevent HIV-1 from cell-to-cell transmission. In previous investigations, we have shown that B1 peptide derived by Fourier transformation of HIV-1 primary structures and sharing no sequence homology with the parent proteins was able to generate antiserum which recognizes envelope and Tat proteins. Here we have investigated cellular immune response towards a novel non-homologous peptide, referred to as cA1 peptide.The 20 amino acid sequence of cA1 peptide was predicted using the notion of peptide hydropathic properties; the peptide is encoded by the complementary anti-sense DNA strand to the sense strand of previously described non-homologous A1 peptide. In this report we demonstrate that the cA1 peptide can be a target for major histocompatibility complex (MHC class I-restricted cytotoxic T lymphocytes in HIV-1-infected or envelope-immunized individuals. The cA1 peptide is recognized in association with different MHC class I allotypes and could prime in vitro CTLs, derived from gp160-immunized individuals capable to recognize virus variants.For the first time a theoretically designed immunogen involved in broad-based cell-immune memory activation is described. Our findings may thus contribute to the advance in vaccine research by describing a novel strategy to develop a synthetic AIDS vaccine.

  10. A Conserved 19-Amino Acid Synthetic Peptide from the Carboxy Terminus of Phosphoenolpyruvate Carboxylase Inhibits the in Vitro Phosphorylation of the Enzyme by the Calcium-Independent Phosphoenolpyruvate Carboxylase Kinase1

    Science.gov (United States)

    Alvarez, Rosario; García-Mauriño, Sofía; Feria, Ana-Belén; Vidal, Jean; Echevarría, Cristina

    2003-01-01

    Higher plant phosphoenolpyruvate carboxylase (PEPC) is subject to in vivo phosphorylation of a regulatory serine located in the N-terminal domain of the protein. Studies using synthetic peptide substrates and mutated phosphorylation domain photosynthetic PEPC (C4 PEPC) suggested that the interaction of phosphoenolpyruvate carboxylase kinase (PEPCk) with its target was not restricted to this domain. However, no further information was available as to where PEPCk-C4 PEPC interactions take place. In this work, we have studied the possible interaction of the conserved 19-amino acid C-terminal sequence of sorghum (Sorghum vulgare Pers cv Tamaran) C4 PEPC with PEPCk. In reconstituted assays, a C-terminal synthetic peptide containing this sequence (peptide C19) was found to inhibit the phosphorylation reaction by the partially purified Ca2+-independent PEPCk (50% inhibition of initial activity = 230 μm). This effect was highly specific because peptide C19 did not alter C4 PEPC phosphorylation by either a partially purified sorghum leaf Ca2+-dependent protein kinase or the catalytic subunit of mammalian protein kinase A. In addition, the Ca2+-independent PEPCk was partially but significantly retained in affinity chromatography using a peptide C19 agarose column. Because peptide C15 (peptide C19 lacking the last four amino acids, QNTG) also inhibited C4 PEPC phosphorylation, it was concluded that the amino acid sequence downstream from the QNTG motif was responsible for the inhibitory effect. Specific antibodies raised against peptide C19 revealed that native C4 PEPC could be in two different conformational states. The results are discussed in relation with the reported crystal structure of the bacterial (Escherichia coli) and plant (maize [Zea mays]) enzymes. PMID:12805637

  11. Parasiticidal activity of a novel synthetic peptide from the core α-helical region of NK-lysin.

    Science.gov (United States)

    Lee, Sung Hyen; Lillehoj, Hyun S; Tuo, Wenbin; Murphy, Charles A; Hong, Yeong H; Lillehoj, Erik P

    2013-10-18

    NK-lysin is an anti-microbial peptide that plays a critical role in innate immunity against infectious pathogens through its selective membrane disruptive property. We previously expressed and purified a full-length chicken NK-lysin (cNKL) recombinant protein, and demonstrated its in vitro anti-parasitic activity against the apicomplexan protozoan, Eimeria, the etiologic agent of avian coccidiosis. This study evaluated the in vitro and in vivo anti-parasitic properties of a synthetic peptide (cNK-2) incorporating a predicted membrane-permeating, amphipathic α-helix of the full-length cNKL protein. The cNK-2 peptide exhibited dose- and time-dependent in vitro cytotoxic activity against E. acervulina and E. tenella sporozoites. The cytotoxic activity of 1.5 μM of cNK-2 peptide against E. acervulina following 6h incubation was equal to that of 2.5 μM of melittin, the principal active component of apitoxin (bee venom) that also exhibits anti-microbial activity. Even greater activity was detected against E. tenella, where 0.3 μM of cNK-2 peptide was equivalent to 2.5 μM of melittin. Against Neospora caninum tacyzoites, however, the cytotoxic activity of cNK-2 peptide was inferior to that of melittin. Transmission electron microscopy of peptide-treated E. tenella sporozoites revealed disruption of the outer plasma membrane and loss of intracellular contents. In vivo administration of 1.5 μM of cNK-2 peptide increased protection against experimental E. acervulina infection, as measured by greater body weight gain and reduced fecal oocyst shedding, compared with saline controls. These results suggest that the cNK-2 synthetic peptide is a novel anti-infective peptide that can be used for protection against avian coccidiosis during commercial poultry production. Published by Elsevier B.V.

  12. Functional C-TERMINALLY ENCODED PEPTIDE (CEP) plant hormone domains evolved de novo in the plant parasite Rotylenchulus reniformis.

    Science.gov (United States)

    Eves-Van Den Akker, Sebastian; Lilley, Catherine J; Yusup, Hazijah B; Jones, John T; Urwin, Peter E

    2016-10-01

    Sedentary plant-parasitic nematodes (PPNs) induce and maintain an intimate relationship with their host, stimulating cells adjacent to root vascular tissue to re-differentiate into unique and metabolically active 'feeding sites'. The interaction between PPNs and their host is mediated by nematode effectors. We describe the discovery of a large and diverse family of effector genes, encoding C-TERMINALLY ENCODED PEPTIDE (CEP) plant hormone mimics (RrCEPs), in the syncytia-forming plant parasite Rotylenchulus reniformis. The particular attributes of RrCEPs distinguish them from all other CEPs, regardless of origin. Together with the distant phylogenetic relationship of R. reniformis to the only other CEP-encoding nematode genus identified to date (Meloidogyne), this suggests that CEPs probably evolved de novo in R. reniformis. We have characterized the first member of this large gene family (RrCEP1), demonstrating its significant up-regulation during the plant-nematode interaction and expression in the effector-producing pharyngeal gland cell. All internal CEP domains of multi-domain RrCEPs are followed by di-basic residues, suggesting a mechanism for cleavage. A synthetic peptide corresponding to RrCEP1 domain 1 is biologically active and capable of up-regulating plant nitrate transporter (AtNRT2.1) expression, whilst simultaneously reducing primary root elongation. When a non-CEP-containing, syncytia-forming PPN species (Heterodera schachtii) infects Arabidopsis in a CEP-rich environment, a smaller feeding site is produced. We hypothesize that CEPs of R. reniformis represent a two-fold adaptation to sustained biotrophy in this species: (i) increasing host nitrate uptake, whilst (ii) limiting the size of the syncytial feeding site produced. © 2016 The Authors. Molecular Plant Pathology Published by British Society for Plant Pathology and John Wiley & Sons Ltd.

  13. Transferrin trojan horses as a rational approach for the biological delivery of therapeutic peptide domains.

    Science.gov (United States)

    Ali, S A; Joao, H C; Hammerschmid, F; Eder, J; Steinkasserer, A

    1999-08-20

    One novel approach for the biological delivery of peptide drugs is to incorporate the sequence of the peptide into the structure of a natural transport protein, such as human serum transferrin. To examine whether this is feasible, a peptide sequence cleavable by the human immunodeficiency virus type 1 protease (VSQNYPIVL) was inserted into various regions of human serum transferrin, and the resultant proteins were tested for function. Experimentally, molecular modeling was used to identify five candidate insertion sites in surface exposed loops of human serum transferrin that were distant from biologically active domains. These insertions were cloned using polymerase chain reaction mutagenesis, and the proteins were expressed using a baculovirus expression vector system. Analysis of the mutant proteins provided a number of important findings: (a) they retained native human serum transferrin function, (b) the inserted peptide sequence was surface exposed, and most importantly, (c) two of these mutants could be cleaved by human immunodeficiency virus-1 protease. In conclusion, this investigation has validated the use of human serum transferrin as a carrier protein for functional peptide domains introduced into its structure using protein engineering. These findings will be useful for developing a novel class of therapeutic agents for a broad spectrum of diseases.

  14. Synthetic fermentation of bioactive non-ribosomal peptides without organisms, enzymes or reagents

    Science.gov (United States)

    Huang, Yi-Lin; Bode, Jeffrey W.

    2014-10-01

    Microbial fermentation can rapidly provide potent compounds that can be easily screened for biological activity, and the active components can be isolated. Its success in drug discovery has inspired extensive efforts to modulate and control the products. In this Article, we document a ‘synthetic fermentation’ of bioactive, unnatural peptides ‘grown’ from small building blocks in water using amide-forming ligations. No organisms, enzymes or reagents are needed. The sequences, structures and compositions of the products can be modulated by adjusting the building blocks and conditions. No specialized knowledge of organic chemistry or handling of toxic material is required to produce complex organic molecules. The ‘fermentations’ can be conducted in arrays and screened for biological activity without isolation or workup. As a proof-of-concept, about 6,000 unnatural peptides were produced from just 23 building blocks, from which a hepatitis C virus NS3/4A protease inhibitor with a half-maximum inhibitory concentration of 1.0 μM was identified and characterized.

  15. Immobilization of a bone and cartilage stimulating peptide to a synthetic bone graft.

    Science.gov (United States)

    Wang, Vivian; Misra, Gauri; Amsden, Brian

    2008-05-01

    A synthetic peptide fragment of human collagen type I (BCSP-1) was linked to the surface of a commercially available ceramic in an effort to improve the properties of the bone graft substitute to accelerate local healing. BCSP-1 was covalently immobilized on the surface of the ceramic via the linkers 3-aminopropyl-triethoxysilane (APTES) and suberic acid bis-N-hydroxysuccinimide ester (DSS). The chosen chemistry was non-cytotoxic. A rat calvaria cell assay using alkaline phosphatase (ALP) as an osteoblast differentiation marker, showed that modifying the surface of the ceramic was enough to enhance ALP activity, although the total cell population on the surface decreased. A significant increase in ALP activity/cell was noted with serum albumin bound to the surface, however, the BCSP-1 bound surface exhibited an even greater ALP activity that showed a surface concentration dependent trend. An optimal BCSP-1 surface density in the range of 0.87-2.24 nmol/cm2 elicited the maximum ALP activity/cell at day 6 of culture. The peptide bound ceramic generated an ALP activity/cell that was roughly 3-fold higher than the non-modified ceramic and 2-fold higher than the APTES-grafted ceramic.

  16. Polymeric nanoparticles for co-delivery of synthetic long peptide antigen and poly IC as therapeutic cancer vaccine formulation

    NARCIS (Netherlands)

    Rahimian, Sima; Fransen, Marieke F.; Kleinovink, Jan Willem; Christensen, Jonatan Riis; Amidi, Maryam|info:eu-repo/dai/nl/304834912; Hennink, Wim E.|info:eu-repo/dai/nl/070880409; Ossendorp, Ferry

    2015-01-01

    The aim of the current study was to develop a cancer vaccine formulation for treatment of human papillomavirus (HPV)-induced malignancies. Synthetic long peptides (SLPs) derived from HPV16 E6 and E7 oncoproteins have been used for therapeutic vaccination in clinical trials with promising results. In

  17. Synthetic Peptides Analogue to Enamel Proteins Promote Osteogenic Differentiation of MC3T3-E1 and Mesenchymal Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Rubert, M.; Ramis, J. M.; Vondrášek, Jiří; Gaya, A.; Lyngstadaas, S. P.; Monjo, M.

    2011-01-01

    Roč. 1, č. 2 (2011), s. 198-209 ISSN 2157-9083 Grant - others:GA ČR(CZ) GAP302/10/0427 Institutional research plan: CEZ:AV0Z40550506 Keywords : proline-rich regions * synthetic peptides * bone formation * mineralization * In Vitro Subject RIV: EI - Biotechnology ; Bionics

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    The human peptide transporter, hPepT1, situated in the small intestine, may be exploited to increase absorption of drugs or model drugs by attaching them to a dipeptide, which is recognised by hPepT1. A synthetic protocol for this kind of model prodrugs was developed, in which model drugs...

  19. Characterization of murine B-cell epitopes on the Mycobacterium leprae proline-rich antigen by use of synthetic peptides

    NARCIS (Netherlands)

    Klatser, P. R.; de Wit, M. Y.; Kolk, A. H.; Hartskeerl, R. A.

    1991-01-01

    Using synthetic peptides representing overlapping sequences of the 100-amino-acid-long N-terminal region of the proline-rich antigen of Mycobacterium leprae (PRA), we have mapped the epitopes in the primary structure of PRA recognized by four monoclonal antibodies. The M. leprae-specific monoclonal

  20. Model prodrugs for the intestinal peptide transporter. a synthetic approach for coupling of hydroxy-containing compounds to dieptides

    DEFF Research Database (Denmark)

    Friedrichsen, G; Nielsen, Carsten Uhd; Steffansen, Bente

    2001-01-01

    The human peptide transporter, hPepT1, situated in the small intestine, may be exploited to increase absorption of drugs or model drugs by attaching them to a dipeptide, which is recognised by hPepT1. A synthetic protocol for this kind of model prodrugs was developed, in which model drugs...

  1. Physicochemical Characterization and Skin Permeation of Cationic Transfersomes Containing the Synthetic Peptide PnPP-19.

    Science.gov (United States)

    Almeida, Flavia De Marco; Silva, Carolina Nunes; de Araujo Lopes, Savia Caldeira; Santos, Daniel Moreira; Torres, Fernanda Silva; Cardoso, Felipe Lima; Martinelli, Patricia Massara; da Silva, Elizabeth Ribeiro; de Lima, Maria Elena; Miranda, Lucas Antonio Ferreira; Oliveira, Monica Cristina

    2018-01-08

    PnPP-19 is a 19-amino-acid synthetic peptide previously described as a novel drug for the treatment of erectile dysfunction. The aim of this work was to evaluate the physicochemical properties of cationic transfersomes containing PnPP-19 and the skin permeation of free PnPP-19 and PnPP-19-loaded transfersomes. Three different liposomal preparation methods were evaluated. Cationic transfersomes contained egg phosphatidyl choline: stearylamine (9:1 w/w) and Tween 20 (84.6:15.4 lipid:Tween, w/w). Lipid concentration varied from 20 to 40 mM. We evaluated the entrapment percentage, mean diameter, zeta potential and stability at 4 oC of the formulations. The skin permeation assays were performed with abdominal human skin using Franz diffusion cell with 3 cm2 diffusion area at 32 oC and a fluorescent derivative of the peptide, containing 5-TAMRA, bound to PnPP-19 C-terminal region, where an extra lysine was inserted. Our results showed variable entrapment efficiencies, from 6% to 30%, depending on the preparation method and the lipid concentration used. The reverse phase evaporation method using a total lipid concentration equal to 40 mM led to the best entrapment percentage (30.2 + 4.5%). Free PnPP-19 was able to permeate skin at a rate of 10.8 ng/cm2/h. However, PnPP-19 was specifically hydrolyzed by skin proteases, generating a fragment of 15 amino acid residues. Encapsulated PnPP-19 permeated the skin at a rate of 19.8 ng/cm2/h. The encapsulation of PnPP-19 in cationic transfersomes protected the peptide from degradation, favoring its topical administration. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. A synthetic peptide blocking TRPV1 activation inhibits UV-induced skin responses.

    Science.gov (United States)

    Kang, So Min; Han, Sangbum; Oh, Jang-Hee; Lee, Young Mee; Park, Chi-Hyun; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-10-01

    Transient receptor potential type 1 (TRPV1) can be activated by ultraviolet (UV) irradiation, and mediates UV-induced matrix metalloproteinase (MMP)-1 and proinflammatory cytokines in keratinocytes. Various chemicals and compounds targeting TRPV1 activation have been developed, but are not in clinical use mostly due to their safety issues. We aimed to develop a novel TRPV1-targeting peptide to inhibit UV-induced responses in human skin. We designed and generated a novel TRPV1 inhibitory peptide (TIP) which mimics the specific site in TRPV1 (aa 701-709: Gln-Arg-Ala-Ile-Thr-Ile-Leu-Asp-Thr, QRAITILDT), Thr 705 , and tested its efficacy of blocking UV-induced responses in HaCaT, mouse, and human skin. TIP effectively inhibited capsaicin-induced calcium influx and TRPV1 activation. Treatment of HaCaT with TIP prevented UV-induced increases of MMP-1 and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α. In mouse skin in vivo, TIP inhibited UV-induced skin thickening and prevented UV-induced expression of MMP-13 and MMP-9. Moreover, TIP attenuated UV-induced erythema and the expression of MMP-1, MMP-2, IL-6, and IL-8 in human skin in vivo. The novel synthetic peptide targeting TRPV1 can ameliorate UV-induced skin responses in vitro and in vivo, providing a promising therapeutic approach against UV-induced inflammation and photoaging. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  3. Synthetic anti-endotoxin peptides inhibit cytoplasmic LPS-mediated responses.

    Science.gov (United States)

    Pfalzgraff, Anja; Heinbockel, Lena; Su, Qi; Brandenburg, Klaus; Weindl, Günther

    2017-09-15

    Toll-like receptor (TLR) 4-independent recognition of lipopolysaccharide (LPS) in the cytosol by inflammatory caspases leads to non-canonical inflammasome activation and induction of IL-1 secretion and pyroptosis. The discovery of this novel mechanism has potential implications for the development of effective drugs to treat sepsis since LPS-mediated hyperactivation of caspases is critically involved in endotoxic shock. Previously, we demonstrated that Pep19-2.5, a synthetic anti-endotoxin peptide, efficiently neutralises pathogenicity factors of Gram-negative and Gram-positive bacteria and protects against sepsis in vivo. Here, we report that Pep19-2.5 inhibits the effects of cytoplasmic LPS in human myeloid cells and keratinocytes. In THP-1 monocytes and macrophages, the peptide strongly reduced secretion of IL-1β and LDH induced by intracellular LPS. In contrast, the TLR4 signaling inhibitor TAK-242 abrogates LPS-induced TNF and IL-1β secretion, but not pyroptotic cell death. Furthermore, Pep19-2.5 suppressed LPS-induced HMGB-1 production and caspase-1 activation in THP-1 monocytes. Consistent with this observation, we found impaired IL-1β and IL-1α release in LPS-stimulated primary monocytes in the presence of Pep19-2.5 and reduced LDH release and IL-1B and IL-1A expression in LPS-transfected HaCaT keratinocytes. Additionally, Pep19-2.5 completely abolished IL-1β release induced by LPS/ATP in macrophages via canonical inflammasome activation. In conclusion, we provide evidence that anti-endotoxin peptides inhibit the inflammasome/IL-1 axis induced by cytoplasmic LPS sensing in myeloid cells and keratinocytes and activation of the classical inflammasome by LPS/ATP which may contribute to the protection against bacterial sepsis and skin infections with intracellular Gram-negative bacteria. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Synthetic peptides for efficient discrimination of anti-enterovirus antibodies at the serotype level.

    Science.gov (United States)

    Routsias, John G; Mavrouli, Maria D; Antonaki, Georgia; Spanakis, Nikolaos; Tsakris, Athanassios

    2014-08-01

    Enteroviruses are important human pathogens, causing a broad spectrum of diseases from minor common colds to fatal myocarditis. However, certain disease syndromes are caused by one or few serotypes. Serotype identification is difficult due to the laborious neutralization tests that lack of sensitivity, while in commercial ELISAs homotypic antibodies' activities are largely masked by the recognition of genera-specific epitopes by heterotypic antibodies. In the present study homotypic assays were developed with the ability to discriminate different enterovirus serotypes. Seventy-three children sera, positive for IgM antibodies against enterovirus genus and 49 healthy children were examined for the presence of antibodies against 14 synthetic peptides derived from a non-conserved region of the VP1 protein of coxsackieviruses B2, B3, B4, B5, A9, A16, A24, echoviruses 6, 7, 9, 11, 30, enterovirus 71 and parechovirus 1. 50% of the anti-enterovirus IgM positive sera (>150 BU) reacted with the peptides with the majority of them to preferentially recognize one of them, supporting the homotypic nature of our assay. Inhibition studies yielded homologous inhibition rates 67-95% suggesting that specific peptide recognition actually occurred. The diagnostic value of our assay was tested in blood samples drawn over a 1.5-year period from a 5-year old patient. The anti-enterovirus reactivity was clearly attributed to echovirus serotype 11. The IgM/IgG antibody ratio was reversed 4 months later and subsequently IgM antibodies dropped below the cutoff point. In this paper we demonstrate that our assay can be used to discriminate between antibodies targeting different enterovirus serotypes. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Leishmanicidal activity of synthetic antimicrobial peptides in an infection model with human dendritic cells.

    Science.gov (United States)

    Pérez-Cordero, José Julián; Lozano, José Manuel; Cortés, Jimena; Delgado, Gabriela

    2011-04-01

    Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world [14]. The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania[7,39]. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Per Westermark

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

  7. Inhibition of transmitter release and attenuation of anti-retroviral-associated and tibial nerve injury-related painful peripheral neuropathy by novel synthetic Ca2+ channel peptides.

    Science.gov (United States)

    Wilson, Sarah M; Schmutzler, Brian S; Brittain, Joel M; Dustrude, Erik T; Ripsch, Matthew S; Pellman, Jessica J; Yeum, Tae-Sung; Hurley, Joyce H; Hingtgen, Cynthia M; White, Fletcher A; Khanna, Rajesh

    2012-10-12

    N-type Ca(2+) channels (CaV2.2) are a nidus for neurotransmitter release and nociceptive transmission. However, the use of CaV2.2 blockers in pain therapeutics is limited by side effects resulting from inhibition of the physiological functions of CaV2.2 within the CNS. We identified an anti-nociceptive peptide (Brittain, J. M., Duarte, D. B., Wilson, S. M., Zhu, W., Ballard, C., Johnson, P. L., Liu, N., Xiong, W., Ripsch, M. S., Wang, Y., Fehrenbacher, J. C., Fitz, S. D., Khanna, M., Park, C. K., Schmutzler, B. S., Cheon, B. M., Due, M. R., Brustovetsky, T., Ashpole, N. M., Hudmon, A., Meroueh, S. O., Hingtgen, C. M., Brustovetsky, N., Ji, R. R., Hurley, J. H., Jin, X., Shekhar, A., Xu, X. M., Oxford, G. S., Vasko, M. R., White, F. A., and Khanna, R. (2011) Suppression of inflammatory and neuropathic pain by uncoupling CRMP2 from the presynaptic Ca(2+) channel complex. Nat. Med. 17, 822-829) derived from the axonal collapsin response mediator protein 2 (CRMP2), a protein known to bind and enhance CaV2.2 activity. Using a peptide tiling array, we identified novel peptides within the first intracellular loop (CaV2.2(388-402), "L1") and the distal C terminus (CaV1.2(2014-2028) "Ct-dis") that bound CRMP2. Microscale thermophoresis demonstrated micromolar and nanomolar binding affinities between recombinant CRMP2 and synthetic L1 and Ct-dis peptides, respectively. Co-immunoprecipitation experiments showed that CRMP2 association with CaV2.2 was inhibited by L1 and Ct-dis peptides. L1 and Ct-dis, rendered cell-penetrant by fusion with the protein transduction domain of the human immunodeficiency virus TAT protein, were tested in in vitro and in vivo experiments. Depolarization-induced calcium influx in dorsal root ganglion (DRG) neurons was inhibited by both peptides. Ct-dis, but not L1, peptide inhibited depolarization-stimulated release of the neuropeptide transmitter calcitonin gene-related peptide in mouse DRG neurons. Similar results were obtained in DRGs from mice

  8. Anchor residue motifs of HLA class-I-binding peptides analyzed by the direct binding of synthetic peptides to HLA class I alpha chains.

    Science.gov (United States)

    Fruci, D; Rovero, P; Falasca, G; Chersi, A; Sorrentino, R; Butler, R; Tanigaki, N; Tosi, R

    1993-11-01

    The binding characteristics of the primary anchor residue motifs reported for HLA-A2 (A*0201, A*0205) and HLA-B27 (B*2705) alleles were investigated by a direct binding assay of the pertinent synthetic peptides to HLA class I alpha chains derived from a panel of HLA homozygous B-cell lines of various HLA phenotypes, including four A2 subtypes. The assay is based on a serologic detection of the conformational change of HLA class I alpha chains induced by binding to specific peptides in the presence of beta 2m. It is applicable to test a large number of HLA allelic products and synthetic peptides. Assay data confirmed the high allele specificity of the anchor residue motifs tested, but also revealed the intra- and interlocus cross-reactivity of these motifs. In the case of A2 anchor motifs, not only a broad cross-reactivity within the A2 subgroup, but also cross-reactivities with A24, A26, A28, and A29 were observed. With B27 anchor motifs, an interlocus cross-reactivity with A3 and A31 was seen. Several peptides, even though they carried A2 or B27 major anchor residue motifs, failed to bind to the relevant alpha chains, suggesting that the presence of a primary anchor residue motif is necessary for HLA class-I-peptide binding but is not by itself sufficient to guarantee binding.

  9. Mutant PrPSc Conformers Induced by a Synthetic Peptide and Several Prion Strains

    Science.gov (United States)

    Tremblay, Patrick; Ball, Haydn L.; Kaneko, Kiyotoshi; Groth, Darlene; Hegde, Ramanujan S.; Cohen, Fred E.; DeArmond, Stephen J.; Prusiner, Stanley B.; Safar, Jiri G.

    2004-01-01

    Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrPSc(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4°C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrPSc(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a β-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrPSc(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrPSc in the brains of Tg196 mice. Our results argue that MoPrPSc(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrPC(P101L) as well as in Tg196 mice expressing low levels of MoPrPC(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a β-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy. PMID:14747574

  10. Performance evaluation of phage-displayed synthetic human single-domain antibody libraries: A retrospective analysis.

    Science.gov (United States)

    Henry, Kevin A; Tanha, Jamshid

    2018-05-01

    Fully human synthetic single-domain antibodies (sdAbs) are desirable therapeutic molecules but their development is a considerable challenge. Here, using a retrospective analysis of in-house historical data, we examined the parameters that impact the outcome of screening phage-displayed synthetic human sdAb libraries to discover antigen-specific binders. We found no evidence for a differential effect of domain type (V H or V L ), library randomization strategy, incorporation of a stabilizing disulfide linkage or sdAb display format (monovalent vs. multivalent) on the probability of obtaining any antigen-binding human sdAbs, instead finding that the success of library screens was primarily related to properties of target antigens, especially molecular mass. The solubility and binding affinity of sdAbs isolated from successful screens depended both on properties of the sdAb libraries (primarily domain type) and the target antigens. Taking attrition of sdAbs with major manufacturability concerns (aggregation; low expression) and sdAbs that do not recognize native cell-surface antigens as independent probabilities, we calculate the overall likelihood of obtaining ≥1 antigen-binding human sdAb from a single library-target screen as ~24%. Successful library-target screens should be expected to yield ~1.3 human sdAbs on average, each with average binding affinity of ~2 μM. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat...... antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination against P. falciparum malaria....... were confined to the nonrepeat region. When used as immunogens, the LR67 and LR68 peptides elicited strong IgG responses in outbred mice and LR67 also induced antibodies in mice of different H-2 haplotypes, confirming the presence of T-helper-cell epitopes in these constructs. Mouse antipeptide...

  12. Peripherally applied synthetic peptide isoAsp7-Aβ(1-42) triggers cerebral β-amyloidosis.

    Science.gov (United States)

    Kozin, S A; Cheglakov, I B; Ovsepyan, A A; Telegin, G B; Tsvetkov, P O; Lisitsa, A V; Makarov, A A

    2013-10-01

    Intracerebral and intraperitoneal inoculation with β-amyloid-rich brain extracts originating from patients with Alzheimer's disease as well as intracerebral injection of aggregates composed of synthetic Aβ can induce cerebral β-amyloidosis, and associated cognitive dysfunctions in susceptible animal hosts. We have found that repetitive intravenous administration of 100 μg of synthetic peptide corresponding to isoAsp7-containing Aβ(1-42), an abundant age-dependent Aβ isoform present both in the pathological brain and in synthetic Aβ preparations, robustly accelerates formation of classic dense-core congophilic amyloid plaques in the brain of β-amyloid precursor protein transgenic mice. Our findings indicate this peptide as an inductive agent of cerebral β-amyloidosis in vivo.

  13. Effectiveness of intranasal vaccination against Angiostrongylus costaricensis using a serine/threonine phosphatase 2 A synthetic peptide and recombinant antigens.

    Science.gov (United States)

    Solano-Parada, J; Gonzalez-Gonzalez, G; Torró, L M de Pablos; dos Santos, M F Brazil; Espino, A M; Burgos, M; Osuna, A

    2010-07-19

    Intranasal immunization was assayed in C57BL/6 mice against Angiostrongylus costaricensis using a synthetic and a recombinant peptide belonging to the catalytic region of the serine/threonine phosphatase 2 A (PP2A) of the parasite. Immunization was carried out with the synthetic peptide (SP) polymerized either with itself or with the beta fraction of the cholera toxin (CTB) and then enclosed in nanocapsules of phosphatidyl choline, cholesterol and Quil A (ISCOM). Another group of mice was immunized with recombinant peptide. Immunization consisted of two intranasal inoculations at two-week intervals, and the challenge with L3 larvae was made one month after the last vaccination. The effectiveness of immunization was evaluated 30 days after infection by analysis of the number of parasites in the arteries of the immunized mice, as well as by measuring spleen sizes in the experimental groups. The response induced was determined by identifying the isotypes of IgG as well as the IgE and IgA specific antigen response. The interleukins produced by the splenocyte culture of the different groups were assessed after exposing them to the peptide used in the immunization. From our results, 60%, 80%, and 100% protection against the A. costaricensis challenge was achieved in mice immunized with polymerized synthetic peptide in ISCOM, synthetic peptide polymerized with the CTB in ISCOM and inclusion bodies respectively. Splenomegaly was found to be less evident in the immunized mice than in the controls. A significant increase in IFN gamma and IL-17 levels was observed in the group with 100% protection. The results showed that vaccination through the nasal mucosa may constitute a useful method of immunization and result in a protective immune response against A. costaricensis. (c) 2010 Elsevier Ltd. All rights reserved.

  14. Structural and antigenic features of the synthetic SF23 peptide corresponding to the receptor binding fragment of diphtheria toxin.

    Science.gov (United States)

    Khrustaleva, Tatyana Aleksandrovna; Khrustalev, Vladislav Victorovich; Barkovsky, Eugene Victorovich; Kolodkina, Valentina Leonidovna; Astapov, Anatoly Archipovich

    2015-02-01

    The SF23 peptide corresponding to the receptor binding fragment of diphtheria toxin (residues 508-530) has been synthesized. This fragment forming a protruding beta hairpin has been chosen because it is the less mutable B-cell epitope. Affine chromatography and ELISA show that antibodies from the sera of persons infected by toxigenic Corynebacterium diphtheriae and those immunized by diphtheria toxoid are able to bind the synthetic SF23 peptide. There are antibodies recognizing the SF23 peptide in the serum of horses hyperimmunized with diphtheria toxoid. Analysis of circular dichroism spectra show formation of beta hairpin by the peptide. Taken together, the results showed that the structure of the less mutable epitope of C. diphtheriae toxin was reproduced by the short SF23 peptide. Since antibodies against that epitope should block its interactions with cellular receptor (heparin-binding epidermal growth factor), the SF23 peptide can be considered as a promising candidate for synthetic vaccine development. Fluorescence quenching studies showed the existence of chloride and phosphate binding sites on the SF23 molecule. Phosphate containing adjuvants (aluminum hydroxyphosphate or aluminum hydroxyphosphate sulfate) are recommended to increase the SF23 immunogenic properties. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. A New Synthetic Peptide Having Two Target of Antibacterial Action inE. coliML35.

    Science.gov (United States)

    Barreto-Santamaría, Adriana; Curtidor, Hernando; Arévalo-Pinzón, Gabriela; Herrera, Chonny; Suárez, Diana; Pérez, Walter H; Patarroyo, Manuel E

    2016-01-01

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

  16. 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

  17. Contamination risks in work with synthetic peptides: flg22 as an example of a pirate in commercial peptide preparations.

    Science.gov (United States)

    Mueller, Katharina; Chinchilla, Delphine; Albert, Markus; Jehle, Anna K; Kalbacher, Hubert; Boller, Thomas; Felix, Georg

    2012-08-01

    The pattern recognition receptor FLAGELLIN SENSING2 (FLS2) renders plant cells responsive to subnanomolar concentrations of flg22, the active epitope of bacterial flagellin. We recently observed that a preparation of the peptide IDL1, a signal known to regulate abscission processes via the receptor kinases HAESA and HAESA-like2, apparently triggered Arabidopsis thaliana cells in an FLS2-dependent manner. However, closer investigation revealed that this activity was due to contamination by a flg22-type peptide, and newly synthesized IDL1 peptide was completely inactive in FLS2 signaling. This raised alert over contamination events occurring in the process of synthesis or handling of peptides. Two recent reports have suggested that FLS2 has further specificities for structurally unrelated peptides derived from CLV3 and from Ax21. We thus scrutinized these peptides for activity in Arabidopsis cells as well. While responding to peptides do occur and can be detected even in trace amounts by FLS2.

  18. The detection of a synthetic Interleukin-1 receptor antagonist peptide in a seized product from a racing stable.

    Science.gov (United States)

    Levina, Vita; Timms, Mark; Vine, John; Steel, Rohan

    2016-09-01

    A synthetic Interleukin-1 receptor antagonist peptide with the sequence Acetyl-Phe-Glu-Trp-Thr-Pro-Gly-Tyr-Trp-Gln-Pro-Tyr-Ala-Leu-Pro-Leu-OH has been identified in a vial seized during a stable inspection. The use of peptide-based Interleukin-1 receptor antagonists as anti-inflammatory agents has not been previously reported, making this peptide the first in a new class of sports doping peptides. The peptide has been characterized by high-resolution mass spectrometry and a detection method developed based on solid-phase extraction and liquid chromatography - triple quadrupole mass spectrometry. Using in vitro and in vivo models to study the properties of the peptide after administration, the peptide was shown to be highly unstable in plasma and was not detected in urine after administration in a rat. The poor stability of the peptide makes detection challenging but also suggests that it has limited effectiveness as an anti-inflammatory drug. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

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

    2014-11-07

    The rice blast disease caused by Magnaporthe oryzae is one of the most devastating diseases of cultivated rice. One of the most important stages in the infective cycle of M. oryzae is the formation of the dome-shaped structure called appressorium. The purpose of the present study was to identify novel peptides to control the rice blast disease by blocking the appressorium formation through screening of a synthetic peptide combinatorial library. As result of the screening, a set of 29 putative bioactive peptides were identified, synthesized and assayed in comparison with the previously identified peptide PAF104. The peptides MgAPI24, MgAPI40 and MgAPI47 showed improved inhibitory activity on the M. oryzae appressorium formation. Our data show that these peptides have a differential effect on two developmental structures: appressoria and appressorium-like structures. Antimicrobial assays against M. oryzae and other non-target microorganisms showed a weak or no toxicity of these peptides, demonstrating their specific activity blocking the appressorium formation. Therefore, the outcome of this research would be useful in the development of novel target-oriented peptides to use in plant protection. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

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

  1. [The immunological responses induced by Mycobacterium tuberculosis heat shock protein 16.3 and its synthetic peptide in mice].

    Science.gov (United States)

    Shi, Chang-hong; Zhang, Ting-fen; Zhu, De-sheng; Zhang, Hai; Bai, Bing; Zhao, Yong; Yue, Chen-li; Zhao, Lei; Liu, Jian-li

    2009-08-01

    To evaluate the immune responses and resistance against Mycobacterium tuberculosis (MTB) infection in the mice induced by HSP16.3 of MTB and its synthetic peptide. BALB/c mice were immunized subcutaneously 3 times at 2 week interval at the base of tail. The doses of HSP16.3 protein and synthetic peptide were both 50 microg each time. A single dose of BCG (5 x 10(6) CFU/mouse) was used to immunize the mice. The concentrations of specific antibodies in serum obtained at 0, 2, 4, 6, 8 weeks after the first immunization and the titer of serum obtained at 8th week, were analyzed by enzyme linked immunosorbent assay (ELISA). Four weeks after the final immunization, 8 mice from each group were sacrificed and single-cell suspensions of splenocytes were prepared, some of which were used for lymphocyte proliferation by MTT colorimetry with HSP16.3 stimulation, and the remaining cells were used for IFN-gamma level assay by sandwich ELISA. The remaining mice in each group were challenged intravenously with 10(5) colony forming units (CFU) of MTB H(37)Rv and were sacrificed 4 weeks after infection, and the number of bacteria in the spleens and lungs were determined by plating serial dilutions of homogenized tissue on Middlebrook 7H10 agar. The statistical significance of differences among means was assessed by an LSD-t test. The level of specific antibody to HSP16.3 protein and the peptide increased rapidly in the former 4 weeks and moderately in the later weeks. The average antibody-specific titers of 3 experiment groups (HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA) were higher than the BCG group. The indexes of spleen lymphocyte proliferation (SI) of the 3 experiment groups (3.13 +/- 0.18, 3.21 +/- 0.21 and 2.40 +/- 0.15) were significantly higher than the BCG group (1.67 +/- 0.12) and the saline group (1.04 +/- 0.09) respectively. The SI of HSP16.3 protein + DDA + MPL group (3.13 +/- 0.18) and synthetic peptide + DDA + MPL group (3

  2. Micromagnetic analysis of current-induced domain wall motion in a bilayer nanowire with synthetic antiferromagnetic coupling

    Energy Technology Data Exchange (ETDEWEB)

    Komine, Takashi, E-mail: komine@mx.ibaraki.ac.jp; Aono, Tomosuke [Faculty of Engineering, Ibaraki University 4-12-1, Nakanarusawa, Hitachi, Ibaraki, 316-8511 (Japan)

    2016-05-15

    We demonstrate current-induced domain wall motion in bilayer nanowire with synthetic antiferromagnetic (SAF) coupling by modeling two body problems for motion equations of domain wall. The influence of interlayer exchange coupling and magnetostatic interactions on current-induced domain wall motion in SAF nanowires was also investigated. By assuming the rigid wall model for translational motion, the interlayer exchange coupling and the magnetostatic interaction between walls and domains in SAF nanowires enhances domain wall speed without any spin-orbit-torque. The enhancement of domain wall speed was discussed by energy distribution as a function of wall angle configuration in bilayer nanowires.

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

    Directory of Open Access Journals (Sweden)

    Jawahar Babu Peravali

    2013-01-01

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

  4. Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2

    Directory of Open Access Journals (Sweden)

    Lennick Michael

    2003-01-01

    Full Text Available Abstract Background Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. Results Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75 into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFα and TNFβ and induced an unexpected biological response in a TNFR2-specific manner. Conclusions To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.

  5. DBAASP v.2: an enhanced database of structure and antimicrobial/cytotoxic activity of natural and synthetic peptides.

    Science.gov (United States)

    Pirtskhalava, Malak; Gabrielian, Andrei; Cruz, Phillip; Griggs, Hannah L; Squires, R Burke; Hurt, Darrell E; Grigolava, Maia; Chubinidze, Mindia; Gogoladze, George; Vishnepolsky, Boris; Alekseyev, Vsevolod; Rosenthal, Alex; Tartakovsky, Michael

    2016-01-04

    Antimicrobial peptides (AMPs) are anti-infectives that may represent a novel and untapped class of biotherapeutics. Increasing interest in AMPs means that new peptides (natural and synthetic) are discovered faster than ever before. We describe herein a new version of the Database of Antimicrobial Activity and Structure of Peptides (DBAASPv.2, which is freely accessible at http://dbaasp.org). This iteration of the database reports chemical structures and empirically-determined activities (MICs, IC50, etc.) against more than 4200 specific target microbes for more than 2000 ribosomal, 80 non-ribosomal and 5700 synthetic peptides. Of these, the vast majority are monomeric, but nearly 200 of these peptides are found as homo- or heterodimers. More than 6100 of the peptides are linear, but about 515 are cyclic and more than 1300 have other intra-chain covalent bonds. More than half of the entries in the database were added after the resource was initially described, which reflects the recent sharp uptick of interest in AMPs. New features of DBAASPv.2 include: (i) user-friendly utilities and reporting functions, (ii) a 'Ranking Search' function to query the database by target species and return a ranked list of peptides with activity against that target and (iii) structural descriptions of the peptides derived from empirical data or calculated by molecular dynamics (MD) simulations. The three-dimensional structural data are critical components for understanding structure-activity relationships and for design of new antimicrobial drugs. We created more than 300 high-throughput MD simulations specifically for inclusion in DBAASP. The resulting structures are described in the database by novel trajectory analysis plots and movies. Another 200+ DBAASP entries have links to the Protein DataBank. All of the structures are easily visualized directly in the web browser. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay.

    Science.gov (United States)

    Bottino, Carolina G; Gomes, Luciano P; Pereira, José B; Coura, José R; Provance, David William; De-Simone, Salvatore G

    2013-12-03

    The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides. Twenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay. The peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%. The epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the development of diagnostic reagents that could

  7. Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

    Science.gov (United States)

    2013-01-01

    Background The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides. Methods Twenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay. Results The peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%. Conclusions The epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the

  8. Central injection of a synthetic chicken partial leptin peptide does not affect food intake in chicks.

    Science.gov (United States)

    Sims, Wil; Yi, Jiaqing; Cline, Mark A; Gilbert, Elizabeth R

    2017-08-24

    Leptin is an adipose tissue-derived hormone in mammals that plays an important role in whole body energy balance via its inhibitory effects on food intake mediated through the hypothalamus. Chicken leptin has a low sequence homology to mammalian leptin and its role in appetite regulation is not reported; hence the objective of this study was to determine effects of central injection of chicken leptin on food and water intake and associated behaviors in chicks. Chicks were intracerebroventricularly injected with 0 (vehicle), 0.3, 1.0, or 3.0 nmol of a synthetic chicken leptin partial peptide and food and water intake were monitored. There were no effects observed and a second experiment was conducted to evaluate food and water intake at higher doses; after injection of 0, 2.5, 5.0, or 10.0 nmol leptin. Again, there were no effects on food or water intake. In the third experiment, behaviors were analyzed during the first 30 min post-injection of vehicle or 10 nmol leptin. At 5 min post-injection, vehicle-injected chicks spent more time sitting than leptin-injected chicks. A wide dose range was evaluated however, the absence of an effect on food intake or behavior suggests that the chicken leptin peptide that was tested does not mediate effects on appetite in the brain and that chicken leptin likely has a different physiological role in birds than in mammals. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Synthetic peptide TEKKRRETVEREKE derived from ezrin induces differentiation of NIH/3T3 fibroblasts.

    Science.gov (United States)

    Chulkina, Marina; Negmadjanov, Ulugbek; Lebedeva, Ekaterina; Pichugin, Aleksey; Mazurov, Dmitriy; Ataullakhanov, Ravshan; Holmuhamedov, Ekhson

    2017-09-15

    Synthetic 14 AA peptide (Gepon) derived from the hinge region of ezrin, a protein that links cell surface molecules to intracellular actin filaments, accelerates and facilitates wound and ulcer healing in clinical applications. However, the molecular mechanisms underlying this phenomenon and involved in enhanced healing of wounds with Gepon are not yet understood. The purpose of current study was to investigate intracellular signaling pathways involved in the effect of this peptide on wild type and genetically modified (CD44 KO) NIH/3T3 embryonic mouse fibroblasts. Gepon treatment of NIH/3T3 cells resulted in morphological and biochemical changes, characteristic of differentiated fibroblasts. While treatment of NIH/3T3 cells with TGF-β1 triggered the activation of both canonical and non-canonical signaling pathways, exposure of fibroblasts to Gepon activated only the ERK1/2 dependent pathway without modulating SMAD dependent signaling pathway. Knocking out hyaluronic acid CD44 receptor did not change Gepon or TGF-β1 dependent activation of intracellular signaling pathways and assembling of α-SMA-positive filaments. Gepon dependent differentiation of NIH/3T3 fibroblasts is based on activation of ERK1/2 kinase, non-canonical intracellular signaling pathway. Our data suggest that the treatment of fibroblasts with Gepon triggers activation of the non-canonical (SMAD independent) intracellular signaling pathway that involves ERK1/2kinase phosphorylation. Activation of the MAPK signaling pathway and the increase in formation of α-SMA containing stress filaments induced by Gepon were independent on presence of CD44 receptor in NIH/3T3 fibroblasts. Thus, our observation designates the significance and sufficiency of MAPK pathway mediated activation of fibroblasts with Gepon for healing of erosion, ulcers and wounds. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    Science.gov (United States)

    Volkmann, Gerrit; Liu, Xiang-Qin

    2009-01-01

    Background Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. Methodology A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. Conclusions We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups. PMID:20027230

  11. The Synthetic Antimicrobial Peptide 19-2.5 Interacts with Heparanase and Heparan Sulfate in Murine and Human Sepsis.

    Science.gov (United States)

    Martin, Lukas; De Santis, Rebecca; Koczera, Patrick; Simons, Nadine; Haase, Hajo; Heinbockel, Lena; Brandenburg, Klaus; Marx, Gernot; Schuerholz, Tobias

    2015-01-01

    Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains from their proteoglycans. Thereby, heparanase liberates highly potent circulating heparan sulfate-fragments (HS-fragments) and triggers the fatal and excessive inflammatory response in sepsis. As a potential anti-inflammatory agent for sepsis therapy, peptide 19-2.5 belongs to the class of synthetic anti-lipopolysaccharide peptides; however, its activity is not restricted to Gram-negative bacterial infection. We hypothesized that peptide 19-2.5 interacts with heparanase and/or HS, thereby reducing the levels of circulating HS-fragments in murine and human sepsis. Our data indicate that the treatment of septic mice with peptide 19-2.5 compared to untreated control animals lowers levels of plasma heparanase and circulating HS-fragments and reduces heparanase activity. Additionally, mRNA levels of heparanase in heart, liver, lung, kidney and spleen are downregulated in septic mice treated with peptide 19-2.5 compared to untreated control animals. In humans, plasma heparanase level and activity are elevated in septic shock. The ex vivo addition of peptide 19-2.5 to plasma of septic shock patients decreases heparanase activity but not heparanase level. Isothermal titration calorimetry revealed a strong exothermic reaction between peptide 19-2.5 and heparanase and HS-fragments. However, a saturation character has been identified only in the peptide 19-2.5 and HS interaction. In conclusion, the findings of our current study indicate that peptide 19-2.5 interacts with heparanase, which is elevated in murine and human sepsis and consecutively attenuates the generation of circulating HS-fragments in systemic inflammation. Thus, peptide 19-2.5 seems to be a potential anti-inflammatory agent in sepsis.

  12. The Synthetic Antimicrobial Peptide 19-2.5 Interacts with Heparanase and Heparan Sulfate in Murine and Human Sepsis.

    Directory of Open Access Journals (Sweden)

    Lukas Martin

    Full Text Available Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains from their proteoglycans. Thereby, heparanase liberates highly potent circulating heparan sulfate-fragments (HS-fragments and triggers the fatal and excessive inflammatory response in sepsis. As a potential anti-inflammatory agent for sepsis therapy, peptide 19-2.5 belongs to the class of synthetic anti-lipopolysaccharide peptides; however, its activity is not restricted to Gram-negative bacterial infection. We hypothesized that peptide 19-2.5 interacts with heparanase and/or HS, thereby reducing the levels of circulating HS-fragments in murine and human sepsis. Our data indicate that the treatment of septic mice with peptide 19-2.5 compared to untreated control animals lowers levels of plasma heparanase and circulating HS-fragments and reduces heparanase activity. Additionally, mRNA levels of heparanase in heart, liver, lung, kidney and spleen are downregulated in septic mice treated with peptide 19-2.5 compared to untreated control animals. In humans, plasma heparanase level and activity are elevated in septic shock. The ex vivo addition of peptide 19-2.5 to plasma of septic shock patients decreases heparanase activity but not heparanase level. Isothermal titration calorimetry revealed a strong exothermic reaction between peptide 19-2.5 and heparanase and HS-fragments. However, a saturation character has been identified only in the peptide 19-2.5 and HS interaction. In conclusion, the findings of our current study indicate that peptide 19-2.5 interacts with heparanase, which is elevated in murine and human sepsis and consecutively attenuates the generation of circulating HS-fragments in systemic inflammation. Thus, peptide 19-2.5 seems to be a potential anti-inflammatory agent in sepsis.

  13. Antibacterial activity of synthetic peptides derived from lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212.

    Science.gov (United States)

    León-Calvijo, María A; Leal-Castro, Aura L; Almanzar-Reina, Giovanni A; Rosas-Pérez, Jaiver E; García-Castañeda, Javier E; Rivera-Monroy, Zuly J

    2015-01-01

    Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i) the incorporation of unnatural amino acids in the sequence, the (ii) reduction or (iii) elongation of the peptide chain length, and (iv) synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR) and I.4 ((RRWQWR)4K2Ahx2C2) exhibit bigger or similar activity against E. coli (MIC 4-33 μM) and E. faecalis (MIC 10-33 μM) when they were compared with lactoferricin protein (LF) and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA) and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE). It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield.

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

    Directory of Open Access Journals (Sweden)

    Hannah Priyadarshini Gideon

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

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

    Directory of Open Access Journals (Sweden)

    Robert H. Notter

    2016-10-01

    Full Text Available Background This study examines the biophysical and preclinical pulmonary activity of synthetic lung surfactants containing novel phospholipase-resistant phosphonolipids or synthetic glycerophospholipids combined with Super Mini-B (S-MB DATK and/or SP-Css ion-lock 1 peptides that replicate the functional biophysics of surfactant proteins (SP-B and SP-C. Phospholipase-resistant phosphonolipids used in synthetic surfactants are DEPN-8 and PG-1, molecular analogs of dipalmitoyl phosphatidylcholine (DPPC and palmitoyl-oleoyl phosphatidylglycerol (POPG, while glycerophospholipids used are active lipid components of native surfactant (DPPC:POPC:POPG 5:3:2 by weight. The objective of the work is to test whether these novel lipid/peptide synthetic surfactants have favorable preclinical activity (biophysical, pulmonary for therapeutic use in reversing surfactant deficiency or dysfunction in lung disease or injury. Methods Surface activity of synthetic lipid/peptide surfactants was assessed in vitro at 37 °C by measuring adsorption in a stirred subphase apparatus and dynamic surface tension lowering in pulsating and captive bubble surfactometers. Shear viscosity was measured as a function of shear rate on a Wells-Brookfield micro-viscometer. In vivo pulmonary activity was determined by measuring lung function (arterial oxygenation, dynamic lung compliance in ventilated rats and rabbits with surfactant deficiency/dysfunction induced by saline lavage to lower arterial PO2 to <100 mmHg, consistent with clinical acute respiratory distress syndrome (ARDS. Results Synthetic surfactants containing 5:3:2 DPPC:POPC:POPG or 9:1 DEPN-8:PG-1 combined with 3% (by wt of S-MB DATK, 3% SP-Css ion-lock 1, or 1.5% each of both peptides all adsorbed rapidly to low equilibrium surface tensions and also reduced surface tension to ≤1 mN/m under dynamic compression at 37 °C. However, dual-peptide surfactants containing 1.5% S-MB DATK + 1.5% SP-Css ion-lock 1 combined with

  16. Early in-situ cellularization of a supramolecular vascular graft is modified by synthetic stromal cell-derived factor-1α derived peptides.

    Science.gov (United States)

    Muylaert, Dimitri E P; van Almen, Geert C; Talacua, Hanna; Fledderus, Joost O; Kluin, Jolanda; Hendrikse, Simone I S; van Dongen, Joost L J; Sijbesma, Eline; Bosman, Anton W; Mes, Tristan; Thakkar, Shraddha H; Smits, Anthal I P M; Bouten, Carlijn V C; Dankers, Patricia Y W; Verhaar, Marianne C

    2016-01-01

    In an in-situ approach towards tissue engineered cardiovascular replacement grafts, cell-free scaffolds are implanted that engage in endogenous tissue formation. Bioactive molecules can be incorporated into such grafts to facilitate cellular recruitment. Stromal cell derived factor 1α (SDF1α) is a powerful chemoattractant of lymphocytes, monocytes and progenitor cells and plays an important role in cellular signaling and tissue repair. Short SDF1α-peptides derived from its receptor-activating domain are capable of activating the SDF1α-specific receptor CXCR4. Here, we show that SDF1α-derived peptides can be chemically modified with a supramolecular four-fold hydrogen bonding ureido-pyrimidinone (UPy) moiety, that allows for the convenient incorporation of the UPy-SDF1α-derived peptides into a UPy-modified polymer scaffold. We hypothesized that a UPy-modified material bioactivated with these UPy-SDF1α-derived peptides can retain and stimulate circulating cells in an anti-inflammatory, pro-tissue formation signaling environment. First, the early recruitment of human peripheral blood mononuclear cells to the scaffolds was analyzed in vitro in a custom-made mesofluidic device applying physiological pulsatile fluid flow. Preferential adhesion of lymphocytes with reduced expression of inflammatory factors TNFα, MCP1 and lymphocyte activation marker CD25 was found in the bioactivated scaffolds, indicating a reduction in inflammatory signaling. As a proof of concept, in-vivo implantation of the bioactivated scaffolds as rat abdominal aorta interposition grafts showed increased cellularity by CD68+ cells after 7 days. These results indicate that a completely synthetic, cell-free biomaterial can attract and stimulate specific leukocyte populations through supramolecular incorporation of short bioactive SDF1α derived peptides. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Designed low amphipathic peptides with alpha-helical propensity exhibiting antimicrobial activity via a lipid domain formation mechanism.

    Science.gov (United States)

    Yamamoto, Naoki; Tamura, Atsuo

    2010-05-01

    Although several low amphipathic peptides have been known to exhibit antimicrobial activity, their mode of action has not been completely elucidated. In this study, using designed low amphipathic peptides that retain different alpha-helical content and hydrophobicity, we attempted to investigate the mechanism of these properties. Calorimetric and thermodynamic analyses demonstrated that the peptides induce formation of two lipid domains in an anionic liposome at a high peptide-to-lipid ratio. On the other hand, even at a low peptide-to-lipid ratio, they caused minimal membrane damage, such as flip-flop of membrane lipids or leakage of calcein molecules from liposomes, and never translocated across membranes. Interaction energies between the peptides and anionic liposomes showed good correlation with antimicrobial activity for both Escherichia coli and Bacillus subtilis. We thus propose that the domain formation mechanism in which antimicrobial peptides exhibit activity solely by forming lipid domains without membrane damage is a major determinant of the antimicrobial activity of low amphipathic peptides. These peptides appear to stiffen the membrane such that it is deprived of the fluidity necessary for biological functions. We also showed that to construct the lipid domains, peptides need not form stable and cooperative structures. Rather, it is essential for peptides to only interact tightly with the membrane interface via strong electrostatic interactions, and slight differences in binding strength are invoked by differences in hydrophobicity. The peptides thus designed might pave the way for "clean" antimicrobial reagents that never cause release of membrane elements and efflux of their inner components. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  18. Biofunctionalization of the implant surface with different concentrations of a synthetic peptide (P-15).

    Science.gov (United States)

    Lutz, R; Prechtl, C; Nonhoff, J; Weisel, T; Damien, C J; Schlegel, K A

    2013-07-01

    This study aimed at identifying the ideal concentration of a biofunctional surface coating of dental implants with a synthetic peptide (P-15). In a previous study, P-15 was shown to enhance osseointegration parameters. Implants (modified ANKYLOS(®) A8; FRIADENT Plus(®) surface) with five different concentrations (0-400 μg/ml) of a P-15 coating as well as uncoated controls were inserted in the frontal bone of 45 adult domestic pigs. The histomorphometric and microradiographic findings for the coated implants were compared to those for the uncoated ones after 7, 14, and 30 days. No significant differences were observed comparing the peri-implant bone density between the coated and uncoated implants The bone-to-implant contact, as the primary histological parameter for osseointegration, showed high rates for all surfaces investigated (between 73.3 ± 17.9% for the control and 81.9 ± 15.2% for P15 20 μg/ml after 30 days). No significant benefit on osseointegration of a biofunctional P-15 coating of dental implants could be displayed in the present study. © 2012 John Wiley & Sons A/S.

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

    Directory of Open Access Journals (Sweden)

    Stephanie Speck

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

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

    Science.gov (United States)

    Nadal, Anna; Montero, Maria; Company, Nuri; Badosa, Esther; Messeguer, Joaquima; Montesinos, Laura; Montesinos, Emilio; Pla, Maria

    2012-09-04

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

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

    Directory of Open Access Journals (Sweden)

    Nadal Anna

    2012-09-01

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

  2. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Berbís, M. Álvaro [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); André, Sabine [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Cañada, F. Javier [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); Pipkorn, Rüdiger [Central Peptide Synthesis Unit, German Cancer Research Center, 69120 Heidelberg (Germany); Ippel, Hans [Department of Biochemistry, CARIM, University of Maastricht, Maastricht (Netherlands); Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Mayo, Kevin H. [Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Kübler, Dieter [Biomolecular Interactions, German Cancer Research Center, 69120 Heidelberg (Germany); Gabius, Hans-Joachim [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Jiménez-Barbero, Jesús, E-mail: jjbarbero@cib.csic.es [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain)

    2014-01-03

    Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

  3. Different domains of the glucagon and glucagon-like peptide-1 receptors provide the critical determinants of ligand selectivity

    DEFF Research Database (Denmark)

    Runge, S; Wulff, B S; Madsen, K

    2003-01-01

    (1) Glucagon and glucagon-like peptide-1 (GLP-1) are homologous peptide hormones with important functions in glucose metabolism. The receptors for glucagon and GLP-1 are homologous family B G-protein coupled receptors. The GLP-1 receptor amino-terminal extracellular domain is a major determinant...

  4. Generalized frequency-domain synthetic aperture focusing technique for ultrasonic imaging of irregularly layered objects.

    Science.gov (United States)

    Qin, Kaihuai; Yang, Chun; Sun, Feng

    2014-01-01

    In ultrasonic nondestructive testing (NDT), the phase shift migration (PSM) technique, as a frequency-domain implementation of the synthetic aperture focusing technique (SAFT), can be adopted for imaging of regularly layered objects that are inhomogeneous only in depth but isotropic and homogeneous in the lateral direction. To deal with irregularly layered objects that are anisotropic and inhomogeneous in both the depth and lateral directions, a generalized frequency- domain SAFT, called generalized phase shift migration (GPSM), is proposed in this paper. Compared with PSM, the most significant innovation of GPSM is that the phase shift factor is generalized to handle anisotropic media with lateral velocity variations. The generalization is accomplished by computer programming techniques without modifying the PSM model. In addition, SRFFT (split-radix fast Fourier transform) input/output pruning algorithms are developed and employed in the GPSM algorithm to speed up the image reconstructions. The experiments show that the proposed imaging techniques are capable of reconstructing accurate shapes and interfaces of irregularly layered objects. The computing time of the GPSM algorithm is much less than the time-domain SAFT combined with the ray-tracing technique, which is, at present, the common method used in ultrasonic NDT industry for imaging layered objects. Furthermore, imaging regularly layered objects can be regarded as a special case of the presented technique.

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

    Science.gov (United States)

    Notter, Robert H; Gupta, Rohun; Schwan, Adrian L; Wang, Zhengdong; Shkoor, Mohanad Gh; Walther, Frans J

    2016-01-01

    This study examines the biophysical and preclinical pulmonary activity of synthetic lung surfactants containing novel phospholipase-resistant phosphonolipids or synthetic glycerophospholipids combined with Super Mini-B (S-MB) DATK and/or SP-Css ion-lock 1 peptides that replicate the functional biophysics of surfactant proteins (SP)-B and SP-C. Phospholipase-resistant phosphonolipids used in synthetic surfactants are DEPN-8 and PG-1, molecular analogs of dipalmitoyl phosphatidylcholine (DPPC) and palmitoyl-oleoyl phosphatidylglycerol (POPG), while glycerophospholipids used are active lipid components of native surfactant (DPPC:POPC:POPG 5:3:2 by weight). The objective of the work is to test whether these novel lipid/peptide synthetic surfactants have favorable preclinical activity (biophysical, pulmonary) for therapeutic use in reversing surfactant deficiency or dysfunction in lung disease or injury. Surface activity of synthetic lipid/peptide surfactants was assessed in vitro at 37 °C by measuring adsorption in a stirred subphase apparatus and dynamic surface tension lowering in pulsating and captive bubble surfactometers. Shear viscosity was measured as a function of shear rate on a Wells-Brookfield micro-viscometer. In vivo pulmonary activity was determined by measuring lung function (arterial oxygenation, dynamic lung compliance) in ventilated rats and rabbits with surfactant deficiency/dysfunction induced by saline lavage to lower arterial PO 2 to surfactants containing 5:3:2 DPPC:POPC:POPG or 9:1 DEPN-8:PG-1 combined with 3% (by wt) of S-MB DATK, 3% SP-Css ion-lock 1, or 1.5% each of both peptides all adsorbed rapidly to low equilibrium surface tensions and also reduced surface tension to ≤1 mN/m under dynamic compression at 37 °C. However, dual-peptide surfactants containing 1.5% S-MB DATK + 1.5% SP-Css ion-lock 1 combined with 9:1 DEPN-8:PG-1 or 5:3:2 DPPC:POPC:POPG had the greatest in vivo activity in improving arterial oxygenation and dynamic lung

  6. YY-39, a tick anti-thrombosis peptide containing RGD domain.

    Science.gov (United States)

    Tang, Jing; Fang, Yaqun; Han, Yajun; Bai, Xuewei; Yan, Xiuwen; Zhang, Yun; Lai, Ren; Zhang, Zhiye

    2015-06-01

    Ticks are obligatory blood feeding ectoparasites, which continuously attach to their hosts for 1-2 weeks. There are many biologically active compounds in tick salivary glands interfering host haemostatic system and to successfully obtain blood meal. Several platelet aggregation inhibitors have been identified from ticks. A family of conserved peptides, which were identified from transcriptome analysis of many tick salivary glands, were found to contain unique primary structure including predicted mature peptides of 39-47 amino acid residues in length and a Pro/Glu(P/E)-Pro/His(P/H)-Lys-Gly-Asp(RGD) domain. Given their unique structure and RGD domain, they are considered a novel family of disintegrins that inhibit platelet aggregation. One of them (YY-39) was tested for its effects on platelets and thrombosis in vivo. YY-39 was found effectively to inhibit platelet aggregation induced by adenosine diphosphate (ADP), thrombin and thromboxane A2 (TXA2). Furthermore, YY-39 blocked platelet adhesion to soluble collagen and bound to purified GPIIb/IIIa in a dose-dependent manner. In in vivo experiments, YY-39 reduced thrombus weight effectively in a rat arteriovenous shunt model and inhibited thrombosis in a carrageenan-induced mouse tail thrombosis model. Combined with their prevalence in ticks and platelet inhibitory functions, this family of peptides might be conserved tick anti-haemostatic molecules. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Peripheral blood mononuclear cell responses to heat shock proteins and their derived synthetic peptides in juvenile idiopathic arthritis patients

    Czech Academy of Sciences Publication Activity Database

    Sedláčková, L.; Velek, Jiří; Vavřincová, P.; Hromadníková, I.

    2006-01-01

    Roč. 55, č. 4 (2006), s. 153-159 ISSN 1023-3830 Grant - others:Transeurope(XE) QLK3-2002-01936; Transnet(XE) MRTN-CT-2004-512253 Institutional research plan: CEZ:AV0Z40550506 Keywords : heat shock proteins * proliferative response * juvenile idiopathic arthritis * Hsp-derived synthetic peptides Subject RIV: CC - Organic Chemistry Impact factor: 1.485, year: 2006

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

    Directory of Open Access Journals (Sweden)

    Vordemvenne Thomas

    2011-11-01

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

  9. Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2.

    Science.gov (United States)

    Niu, Xufeng; Feng, Qingling; Wang, Mingbo; Guo, Xiaodong; Zheng, Qixin

    2009-03-04

    It is advantageous to incorporate controlled growth factor delivery into tissue engineering strategies. The purpose of the present study was to develop a novel tissue engineering scaffold with the capability of controlled releasing BMP-2-derived synthetic peptide. Porous nano-hydroxyapatite/collagen/poly(L-lactic acid)/chitosan microspheres (nHAC/PLLA/CMs) composite scaffolds containing different quantities of chitosan microspheres (CMs) were prepared by a thermally induced phase separation method. Dioxane was used as the solvent for PLLA. Introduction of less than 30% of CMs (on PLLA weight basis) did not remarkably affect the morphology and porosity of the nHAC/PLLA/CMs scaffolds. However, as the microspheres contents increased to 50%, the porosity of the composite decreased rapidly. The compressive modulus of the composite scaffolds increased from 15.4 to 25.5 MPa, while the compressive strength increased from 1.42 to 1.63 MPa as the microspheres contents increased from 0% to 50%. The hydrolytic degradation and synthetic peptide release kinetics in vitro were investigated by incubation in phosphate buffered saline solution (pH 7.4). The results indicated that the degradation rate of the scaffolds was increased with the enhancement of CMs dosage. The synthetic peptide was released in a temporally controlled manner, depending on the degradation of both incorporated chitosan microspheres and PLLA matrix. In vitro bioactivity assay revealed that the encapsulated synthetic peptide was biologically active as evidenced by stimulation of rabbit marrow mesenchymal stem cells (MSCs) alkaline phosphatase (ALP) activity. The successful microspheres-scaffold system offers a new delivery method of growth factors and a novel scaffold design for bone regeneration.

  10. Reversal of H-bonding direction by N-sulfonation in a synthetic reverse-turn peptide motif.

    Science.gov (United States)

    Vijayadas, Kuruppanthara N; Kotmale, Amol S; Thorat, Shridhar H; Gonnade, Rajesh G; Nair, Roshna V; Rajamohanan, Pattuparambil R; Sanjayan, Gangadhar J

    2015-03-14

    This communication depicts an intriguing example of hydrogen-bonding reversal upon introduction of a sulfonamide linkage at the N-terminus of a synthetic reverse-turn peptide motif. The ready availability of two sulfonyl oxygen atoms, as hydrogen-bonding acceptors, combined with the inherent twisted conformation of sulfonamides are seen to act as switches that engage/disengage the hydrogen-bond at the sticky ends/termini.

  11. Antitumor potential of a synthetic interferon-alpha/PLGF-2 positive charge peptide hybrid molecule in pancreatic cancer cells.

    Science.gov (United States)

    Yin, Hongmei; Chen, Naifei; Guo, Rui; Wang, Hong; Li, Wei; Wang, Guanjun; Cui, Jiuwei; Jin, Haofan; Hu, Ji-Fan

    2015-11-20

    Pancreatic cancer is the most aggressive malignant disease, ranking as the fourth leading cause of cancer-related death among men and women in the United States. Interferon alpha (IFNα) has been used to treat pancreatic cancer, but its clinical application has been significantly hindered due to the low antitumor activity. We used a "cDNA in-frame fragment library" screening approach to identify short peptides that potentiate the antitumor activity of interferons. A short positively charged peptide derived from the C-terminus of placental growth factor-2 (PLGF-2) was selected to enhance the activity of IFNα. For this, we constructed a synthetic interferon hybrid molecule (SIFα) by fusing the positively charged PLGF-2 peptide to the C-terminus of the human IFNα. Using human pancreatic cell lines (ASPC and CFPAC1) as a model system, we found that SIFα exhibited a significantly higher activity than did the wild-type IFNα in inhibiting the tumor cell growth. The enhanced activity of the synthetic SIFα was associated with the activation of interferon pathway target genes and the increased binding of cell membrane receptor. This study demonstrates the potential of a synthetic SIFα as a novel antitumor agent.

  12. NMR structure of a biologically active peptide containing the RNA-binding domain of human immunodeficiency virus type 1 Tat.

    Science.gov (United States)

    Mujeeb, A; Bishop, K; Peterlin, B M; Turck, C; Parslow, T G; James, T L

    1994-01-01

    The Tat protein of human immunodeficiency virus type 1 enhances transcription by binding to a specific RNA element on nascent viral transcripts. Binding is mediated by a 10-amino acid basic domain that is rich in arginines and lysines. Here we report the three-dimensional peptide backbone structure of a biologically active 25-mer peptide that contains the human immunodeficiency virus type 1 Tat basic domain linked to the core regulatory domain of another lentiviral Tat--i.e., that from equine infectious anemia virus. Circular dichroism and two-dimensional proton NMR studies of this hybrid peptide indicate that the Tat basic domain forms a stable alpha-helix, whereas the adjacent regulatory sequence is mostly in extended form. These findings suggest that the tendency to form stable alpha-helices may be a common property of arginine- and lysine-rich RNA-binding domains. Images PMID:8058789

  13. Modeling induced polarization effects in helicopter time domain electromagnetic data: Synthetic case studies

    DEFF Research Database (Denmark)

    Viezzoli, Andrea; Kaminskiy, Vladislav; Fiandaca, Gianluca

    2017-01-01

    We have developed a synthetic multiparametric modeling and inversion exercise undertaken to study the robustness of inverting airborne time-domain electromagnetic (TDEM) data to extract Cole-Cole parameters. The following issues were addressed: nonuniqueness, ill posedness, dependency on manual...... constrained multiparametric inversion was evaluated, including recovery of chargeability distributions from shallow and deep targets based on analysis of induced polarization (IP) effects, simulated in airborne TDEM data. Different scenarios were studied, including chargeable targets associated...... by a shallower chargeable target, became possible only when full Cole-Cole modeling was used in the inversion. Lateral constraints improved the recoverability of model parameters. Finally, modeling IP effects increased the accuracy of recovered electrical resistivity models....

  14. Evaluation of the Immunogenicity of a Potyvirus-Like Particle as an Adjuvant of a Synthetic Peptide.

    Science.gov (United States)

    Cárdenas-Vargas, Albertina; Elizondo-Quiroga, Darwin; Gutierrez-Ortega, Abel; Charles-Niño, Claudia; Pedroza-Roldán, César

    2016-12-01

    Improvement of current vaccines is highly necessary to increase immunogenicity levels and protection against several pathogens. Virus-like particles (VLPs) are promising approaches for vaccines because they emulate infectious virus structure, but lack any genetic material needed for replication. Plant viruses have emerged as a potential framework for VLP design, mainly because there is no preexisting immunity in mammals. In this study, we evaluated the scaffold of the papaya ringspot virus (PRSV) as a VLP adjuvant for a short synthetic peptide derived from the Hemagglutinin protein of AH1 N1 influenza virus-hemagglutinin (VLP-HA). Our results demonstrated that the adjuvant property of this VLP is highly similar to the trivalent influenza vaccine, showing comparable levels of IgG- and IgA-specific antibodies to HA-derived peptide in serum and feces of vaccinated mice, respectively. Furthermore, VLP-HA-immunized mice showed Th1-biased immune response as suggested by measuring IgG subclasses in comparison with the predominance of Th2-biased immune response in trivalent influenza vaccine dose-vaccinated mice. VLP-HA administration in mice induced comparable levels of activated CD4 + - and CD8 + -specific T lymphocytes for the HA-derived peptide. These results suggest the potential adjuvant capacity of the PRSV-VLP as a carrier for short synthetic peptides.

  15. ARSENITE BINDING TO SYNTHETIC PEPTIDES: THE EFFECT OF INCREASING LENGTH BETWEEN TWO CYSTEINES

    Science.gov (United States)

    Binding of trivalent arsenicals to peptides and proteins can alter peptide/protein structure and enzyme function and thereby contribute to arsenic toxicity and carcinogenicity. We utilized radioactive 73As- labeled arsenite and vacuum filtration methodology to determine the bindi...

  16. From the Cover: Twisting macromolecular chains: Self-assembly of a chiral supermolecule from nonchiral polythiophene polyanions and random-coil synthetic peptides

    Science.gov (United States)

    Nilsson, K. Peter R.; Rydberg, Johan; Baltzer, Lars; Inganäs, Olle

    2004-08-01

    The self-assembly of a negatively charged conjugated polythiophene derivative and a positively charged synthetic peptide will create a chiral, well ordered supermolecule. This supermolecule has the three-dimensional ordered structure of a biomolecule and the electronic properties of a conjugated polymer. The molecular complex being formed clearly affects the conformation of the polymer backbone. A main-chain chirality, such as a predominantly one-handed helical structure induced by the acid-base complexation between the conjugated polymer and the synthetic peptide, is seen. The alteration of the polymer backbone influences the optical properties of the polymer, seen as changes in the absorption, emission, and Raman spectra of the polymer. The complexation of the polythiophene and the synthetic peptide also induce a change from random-coil to helical structure of the synthetic peptide. The supermolecule described in this article may be used in a wide range of applications such as biomolecular devices, artificial enzymes, and biosensors.

  17. Polystyrene beads coated with antibodies directed to HLA class I intracytoplasmic domain: the use in quantitative measurement of peptide-HLA class I binding by flow cytometry.

    Science.gov (United States)

    Chersi, A; Rosano, L; Tanigaki, N

    2000-12-01

    Protein-reactive, conformation-independent anti-peptide antibodies were raised in rabbits against a C-terminal sequence SDSAQGSDVSLA, common to most HLA-A and -B locus products. Antibodies were coupled to 4.5-microm polystyrene beads through the Fc portion by the use of protein A. The antibody-coupled beads showed a high capacity to bind HLA-A and -B proteins as well as their alpha chains by the intracytoplasmic domain, keeping the extracellular domains solvent exposed. The density of HLA class I proteins bound on the beads was approximately the same as that on cultured B cells. The antibody beads made it possible to quantitate peptide-HLA class I binding, i.e., in vitro HLA class I assembly by flow cytometry. The assembly rate determined by the provisionally called flow cytometric HLA class I assay was 15%-19% for the reassembly of dissociated HLA class I proteins with the released selfpeptides. With single synthetic peptides, the highest rate so far obtained was 6.5%. The assay specificity and reproducibility were satisfactory.

  18. Modulation of HIV peptide antigen specific cellular immune response by synthetic α- and β-defensin peptides.

    Science.gov (United States)

    Mohan, Teena; Sharma, Chandresh; Bhat, Ajaz A; Rao, D N

    2013-03-25

    Defensin peptides have their direct role in host defense against microbial infection as innate molecules and also thought to contribute to adaptive immunity by recruiting naïve T-cells and immature dendritic cells at the site of infection through CCR6 receptor. The main aim of the present study is to investigate the efficacy of defensins for the induction of cell mediated immune response against the peptide antigen of HIV-1 encapsulated in PLG microparticles through intranasal (IN) route in mice model. To characterized, we have analyzed T-cell proliferation, Th1/Th2 cytokines, β-chemokines production and IFN-γ/perforin secretion from CD4(+)/CD8(+) T-cells in response to HIV immunogen alone and with defensins at different mucosal site i.e. lamina propria (LP), spleen (SP) and peyer's patches (PP). The cellular immunogenicity of HIV peptide with defensin formulations showed a significantly higher (ppeptide. The enhanced cytokines measurement profile showed mixed Th1 and Th2 type of peptide specific immune response by the incorporation of defensins. In the continuation, enhancement in MIP-1α and RANTES level was also observed in HIV peptide-defensin formulations. The FACS data had revealed that CD4(+)/CD8(+) T-cells showed significantly (ppeptide formulations than HIV antigen alone group. Thus, the study emphasized here that defensin peptides have a potential role as mucosal adjuvant, might be responsible for the induction of cell mediated immunity when administered in mice through IN route with HIV peptide antigen. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases.

    Science.gov (United States)

    Quadri, L E; Weinreb, P H; Lei, M; Nakano, M M; Zuber, P; Walsh, C T

    1998-02-10

    The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carrier protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16-17-kDa peptidyl carrier protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56-104 min-1 and K(m) values of 1.3-1.8 microM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl carrier protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl carrier protein (ACP) subunit, suggesting Sfp as a good candidate for heterologous coexpression with peptide and polyketide synthase genes to overproduce holo-synthase enzymes. Cosubstrate coenzyme A (CoA), the phosphopantetheinyl group donor, has a K(m) of 0.7 microM. Desulfo-CoA and homocysteamine-CoA are also substrates of Sfp, and benzoyl-CoA and phenylacetyl-CoA are also utilized by Sfp, resulting in direct transfer of acyl phosphopantetheinyl moieties into the carrier protein substrate. Mutagenesis in Sfp of five residues conserved across the PPTase family was assessed for in vivo effects on surfactin production and in vitro effects on PPTase activity.

  20. Membrane anchorage brings about fusogenic properties in a short synthetic peptide

    NARCIS (Netherlands)

    Pecheur, EI; Hoekstra, D; SainteMarie, J; Maurin, L; Bienvenue, A; Philippot, [No Value

    1997-01-01

    The fusogenic properties of an amphipathic net-negative peptide (wae 11), consisting of 11 amino acid residues, were studied. We demonstrate that, whereas the free peptide displays no significant fusion activity, membrane fusion is strongly promoted when the peptide is anchored to a liposomal

  1. Natural and synthetic cathelicidin peptides with anti-microbial and anti-biofilm activity against Staphylococcus aureus

    Science.gov (United States)

    2011-01-01

    Background Chronic, infected wounds typically contain multiple genera of bacteria, including Staphylococcus aureus, many of which are strong biofilm formers. Bacterial biofilms are thought to be a direct impediment to wound healing. New therapies that focus on a biofilm approach may improve the recovery and healing rate for infected wounds. In this study, cathelicidins and related short, synthetic peptides were tested for their anti-microbial effectiveness as well as their ability to inhibit the ability of S. aureus to form biofilms. Results The helical human cathelicidin LL-37 was tested against S. aureus, and was found to exhibit effective anti-microbial, anti-attachment as well as anti-biofilm activity at concentrations in the low μg/ml range. The effect of peptide chirality and associated protease-resistance was explored through the use of an all-D amino acid peptide, D-LL-37, and in turn compared to scrambled LL-37. Helical cathelicidins have been identified in other animals such as the Chinese cobra, Naja atra (NA-CATH). We previously identified an 11-residue imperfectly repeated pattern (ATRA motif) within the sequence of NA-CATH. A series of short peptides (ATRA-1, -2, -1A), as well as a synthetic peptide, NA-CATH:ATRA1-ATRA1, were designed to explore the significance of the conserved residues within the ATRA motif for anti-microbial activity. The CD spectrum of NA-CATH and NA-CATH:ATRA1-ATRA1 revealed the structural properties of these peptides and suggested that helicity may factor into their anti-microbial and anti-biofilm activities. Conclusions The NA-CATH:ATRA1-ATRA1 peptide inhibits the production of biofilm by S. aureus in the presence of salt, exhibiting anti-biofilm activity at lower peptide concentrations than NA-CATH, LL-37 and D-LL-37; and demonstrates low cytoxicity against host cells but does not affect bacterial attachment. The peptides utilized in this anti-biofilm approach may provide templates for a new group of anti-microbials and

  2. Synthetic peptide monolayers: Probing structure, attachment, and hydration conduction with STM

    Science.gov (United States)

    Bergeron, David Joseph

    2000-10-01

    Although the scanning tunneling microscope (STM) has been used in many studies of proteins and other biological molecules, the interpretation of the data has been hampered by a limited understanding of image contrast and tip-sample interactions. It has been recognized that adsorbed water can carry the currents necessary for STM imaging, and it has been speculated that the mechanism of charge transport is based on the exchange of protons along hydrogen-bonded networks. While a water film facilitates the imaging of proteins, it hampers efforts to use STM to probe the electronic structure of redox proteins. The physics of charge transfer in proteins and the intricacies of imaging large molecules with STM can be studied using synthetic proteins. A protein designed with engineerable electron transfer properties, a hydrophilic exterior and mechanisms for attachment to surfaces has been studied using both STM and fixed-electrode conductivity measurements. The effects of immobilization strategy on image quality were investigated by preparing monolayers using self-assembly, Langmuir-Blodgett and monolayer insertion techniques and imaging these films in both humid air and vacuum. The molecules were easily imaged in humid air due to the adsorbed water film, but dehydration in vacuum introduced a conductance gap which prevented the proteins from being imaged within a wide voltage range. The molecules were subjected to large forces when the tip scanned over them without retracting from the surface and peptides were displaced in ways which reflected the strength of their attachment to the surface. In the course of these studies, new STM imaging phenomena have been observed including meniscus-limited lateral resolution, and intermittent imaging of covalently attached molecules. The conductivity of protein films deposited between fixed electrodes depended strongly on hydration, and the experimental arrangement provides a platform for further study of the mechanism of conduction

  3. Cytological Profile of Antibacterial FtsZ Inhibitors and Synthetic Peptide MciZ.

    Science.gov (United States)

    Araújo-Bazán, Lidia; Ruiz-Avila, Laura B; Andreu, David; Huecas, Sonia; Andreu, José M

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Lidia Araujo-Bazan

    2016-10-01

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

  5. Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: Effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes

    International Nuclear Information System (INIS)

    Nair, M.P.N.; Pottathil, R.; Heimer, E.P.; Schwartz, S.A.

    1988-01-01

    Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by [ 3 H]thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3 + lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3 - lymphocytes. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection

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

    Directory of Open Access Journals (Sweden)

    Frederick Verbeke

    2015-06-01

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

  7. Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration.

    Science.gov (United States)

    Pfalzgraff, Anja; Heinbockel, Lena; Su, Qi; Gutsmann, Thomas; Brandenburg, Klaus; Weindl, Günther

    2016-08-11

    The stagnation in the development of new antibiotics and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. Antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that Pep19-2.5, a synthetic anti-lipopolysaccharide (LPS) peptide (SALP), efficiently neutralises pathogenicity factors of Gram-negative (LPS) and Gram-positive (lipoprotein/-peptide, LP) bacteria and protects against sepsis. Here, we investigated the potential of Pep19-2.5 and the structurally related compound Pep19-4LF for their therapeutic application in bacterial skin infections. SALPs inhibited LP-induced phosphorylation of NF-κB p65 and p38 MAPK and reduced cytokine release and gene expression in primary human keratinocytes and dermal fibroblasts. In LPS-stimulated human monocyte-derived dendritic cells and Langerhans-like cells, the peptides blocked IL-6 secretion, downregulated expression of maturation markers and inhibited dendritic cell migration. Both SALPs showed a low cytotoxicity in all investigated cell types. Furthermore, SALPs markedly promoted cell migration via EGFR transactivation and ERK1/2 phosphorylation and accelerated artificial wound closure in keratinocytes. Peptide-induced keratinocyte migration was mediated by purinergic receptors and metalloproteases. In contrast, SALPs did not affect proliferation of keratinocytes. Conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections.

  8. A synthetic cyclic peptide derived from Limulus anti-lipopolysaccharide factor neutralizes endotoxin in vitro and in vivo.

    Science.gov (United States)

    Ren, Jian-Dong; Gu, Jin-Song; Gao, Hong-Fu; Xia, Pei-Yuan; Xiao, Guang-Xia

    2008-06-01

    Endotoxin, also known as lipopolysaccharide (LPS), is the major mediator of septic shock due to Gram-negative bacterial infections. Recently, much attention has been focused on cationic peptides which possess the potential to detoxify LPS. Limulus anti-LPS factor (LALF), a protein found in the horseshoe crab (Limulus polyphemus), has been proved with striking anti-LPS effects. We synthesized a cyclic peptide (CLP-19), and then investigated its bioactivity both in vitro and in vivo. The ability of CLP-19 to neutralize LPS in vitro was tested using a Limulus amebocyte lysate (LAL) assay and the LPS-binding affinity was measured with an affinity biosensor method. The synthetic peptide LALF31-52 (residues 31 to 52 of LALF) was used as the positive control peptide in this study. It was found that CLP-19 exhibited the significant activity to antagonize LPS without observable cytotoxicity effect on mouse macrophages. CLP-19 directly bound to LPS, and neutralized it in a dose-dependent manner in the LAL assay. Moreover, CLP-19 also showed the remarkable ability to protect mice from lethal LPS attack and to inhibit the LPS-induced tumor necrosis factor alpha (TNF-alpha) release by decreasing serum LPS in vivo. Our work suggests that this peptide is worthy of further investigation as a potential anti-LPS agent in the treatment of septic shock.

  9. Anti-infective activity of apolipoprotein domain derived peptides in vitro: identification of novel antimicrobial peptides related to apolipoprotein B with anti-HIV activity

    Directory of Open Access Journals (Sweden)

    McKnight Áine

    2010-03-01

    Full Text Available Abstract Background Previous reports have shown that peptides derived from the apolipoprotein E receptor binding region and the amphipathic α-helical domains of apolipoprotein AI have broad anti-infective activity and antiviral activity respectively. Lipoproteins and viruses share a similar cell biological niche, being of overlapping size and displaying similar interactions with mammalian cells and receptors, which may have led to other antiviral sequences arising within apolipoproteins, in addition to those previously reported. We therefore designed a series of peptides based around either apolipoprotein receptor binding regions, or amphipathic α-helical domains, and tested these for antiviral and antibacterial activity. Results Of the nineteen new peptides tested, seven showed some anti-infective activity, with two of these being derived from two apolipoproteins not previously used to derive anti-infective sequences. Apolipoprotein J (151-170 - based on a predicted amphipathic alpha-helical domain from apolipoprotein J - had measurable anti-HSV1 activity, as did apolipoprotein B (3359-3367 dp (apoBdp, the latter being derived from the LDL receptor binding domain B of apolipoprotein B. The more active peptide - apoBdp - showed similarity to the previously reported apoE derived anti-infective peptide, and further modification of the apoBdp sequence to align the charge distribution more closely to that of apoEdp or to introduce aromatic residues resulted in increased breadth and potency of activity. The most active peptide of this type showed similar potent anti-HIV activity, comparable to that we previously reported for the apoE derived peptide apoEdpL-W. Conclusions These data suggest that further antimicrobial peptides may be obtained using human apolipoprotein sequences, selecting regions with either amphipathic α-helical structure, or those linked to receptor-binding regions. The finding that an amphipathic α-helical region of

  10. Molecular Diversity of the Antimicrobial Domain of Beta-Defensin 3 and Homologous Peptides

    Science.gov (United States)

    Nava, Gerardo M.; Escorcia, Magdalena; Castañeda, M. Pilar

    2009-01-01

    Human β-defensin 3 has received great interest for possible pharmaceutical applications. To characterize the biology of this antimicrobial peptide, the mouse β-defensin 14 has been selected as a prototypical model. This report provides definite evidence of true orthology between these defensins and reveals molecular diversity of a mammalian specific domain responsible for their antimicrobial activity. Specifically, this analysis demonstrates that eleven amino acid residues of the antimicrobial domain have been mutated by positive selection to confer protein niche specialization. These data support the notion that natural selection acts as evolutionary force driving the proliferation and diversification of defensins and introduce a novel strategy for the design of more effective antibiotics. PMID:19888439

  11. High throughput screening methods for assessing antibiofilm and immunomodulatory activities of synthetic peptides.

    Science.gov (United States)

    Haney, Evan F; Mansour, Sarah C; Hilchie, Ashley L; de la Fuente-Núñez, César; Hancock, Robert E W

    2015-09-01

    The recent observation that certain cationic peptides possess potent antibiofilm activity demonstrated that small peptides could be used to treat biofilm-associated infections. Other so-called innate defense regulator peptides possess potent immunomodulatory properties such as leukocyte recruitment and suppression of harmful inflammation. A peptide that directly targets biofilm cells while favorably modulating the immune response would be particularly advantageous for treating serious skin infections caused by Staphylococcus aureus. In the present work, using SPOT-synthesized peptide arrays on cellulose membranes, we outline a strategy for systematically assessing the antibiofilm activity of hundreds of IDR-1002 (VQRWLIVWRIRK-NH2) and IDR-HH2 (VQLRIRVAVIRA-NH2) peptide variants against MRSA biofilms. In addition, the ability of these peptides to stimulate production of a monocyte chemoattractant protein (MCP-1) and suppress LPS-induced interleukin (IL)-1β production in human peripheral blood mononuclear cells (PBMCs) was evaluated. These results informed the synthesis of second-generation peptides resulting in a new peptide, IDR-2009 (KWRLLIRWRIQK-NH2), with enhanced MCP-1 stimulatory activity, favorable IL-1β suppression characteristics and strong antibiofilm activity against MRSA and Pseudomonas aeruginosa biofilms. This work provides a proof-of-concept that multiple peptide activities can be optimized simultaneously to generate novel sequences that possess a variety of biological properties. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Antimicrobial Activity of Novel Synthetic Peptides Derived from Indolicidin and Ranalexin against Streptococcus pneumoniae.

    Science.gov (United States)

    Jindal, Hassan Mahmood; Le, Cheng Foh; Mohd Yusof, Mohd Yasim; Velayuthan, Rukumani Devi; Lee, Vannajan Sanghiran; Zain, Sharifuddin Md; Isa, Diyana Mohd; Sekaran, Shamala Devi

    2015-01-01

    Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics in order to defeat multidrug-resistant bacteria such as Streptococcus pneumoniae. In this study, thirteen antimicrobial peptides were designed based on two natural peptides indolicidin and ranalexin. Our results revealed that four hybrid peptides RN7-IN10, RN7-IN9, RN7-IN8, and RN7-IN6 possess potent antibacterial activity against 30 pneumococcal clinical isolates (MIC 7.81-15.62µg/ml). These four hybrid peptides also showed broad spectrum antibacterial activity (7.81µg/ml) against S. aureus, methicillin resistant S. aureus (MRSA), and E. coli. Furthermore, the time killing assay results showed that the hybrid peptides were able to eliminate S. pneumoniae within less than one hour which is faster than the standard drugs erythromycin and ceftriaxone. The cytotoxic effects of peptides were tested against human erythrocytes, WRL-68 normal liver cell line, and NL-20 normal lung cell line. The results revealed that none of the thirteen peptides have cytotoxic or hemolytic effects at their MIC values. The in silico molecular docking study was carried out to investigate the binding properties of peptides with three pneumococcal virulent targets by Autodock Vina. RN7IN6 showed a strong affinity to target proteins; autolysin, pneumolysin, and pneumococcal surface protein A (PspA) based on rigid docking studies. Our results suggest that the hybrid peptides could be suitable candidates for antibacterial drug development.

  13. Antibodies against a Synthetic Peptide of SagA Neutralize the Cytolytic Activity of Streptolysin S from Group A Streptococci†

    Science.gov (United States)

    Dale, James B.; Chiang, Edna Y.; Hasty, David L.; Courtney, Harry S.

    2002-01-01

    Virtually all group A streptococci (GAS) produce streptolysin S (SLS), a cytolytic toxin that is responsible for the beta-hemolysis surrounding colonies of the organisms grown on blood agar. SLS is an important virulence determinant of GAS, and recent studies have identified a nine-gene locus that is responsible for synthesis and transport of the toxin. SLS is not immunogenic; thus, no neutralizing antibodies are evoked during the course of natural infection. In the present study, we show that a synthetic peptide containing amino acid residues 10 to 30 of the putative SLS (SagA) propeptide [SLS(10-30)] coupled to keyhole limpet hemocyanin evoked antibodies in rabbits that completely neutralized the hemolytic activity of the toxin in vitro. Inhibition of hemolysis was reversed by preincubation of the immune serum with soluble, unconjugated peptide, indicating the specificity of the antibodies. In addition, antibodies that were affinity purified over an SLS(10-30) peptide column completely inhibited SLS-mediated hemolysis. The SLS(10-30) antisera did not opsonize group A streptococci; however, when combined with type-specific M protein antisera, the SLS antibodies significantly enhanced phagocytosis mediated by M protein antibodies. Thus, we have shown for the first time that it is possible to raise neutralizing antibodies against one of the most potent bacterial cytolytic toxins known. Our data also provide convincing evidence that the sagA gene actually encodes the SLS peptide of GAS. The synthetic peptide may prove to be an important component of vaccines designed to prevent GAS infections. PMID:11895983

  14. Use of synthetic peptides to represent surface-exposed epitopes defined by neutralizing dengue complex- and flavivirus group-reactive monoclonal antibodies on the native dengue type-2 virus envelope glycoprotein.

    Science.gov (United States)

    Falconar, Andrew K I

    2008-07-01

    The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393-401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101-109 sequence containing 105-C by G substitution and the 393-401 sequence are good candidates for diagnostic assays and cross-protection experiments.

  15. The use of synthetic peptides for detection of anti-citrullinated protein antibodies in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Holm, Bettina Eide; Heiden, Julie

    2018-01-01

    and prognostic. As a result, several assays for detection of ACPAs exist, which vary in sensitivity and specificity. In this study, we analyzed the reactivity of RA sera to selected peptides by solid-phase immunoassays in order to develop an ACPA assay with improved sensitivity and specificity. ACPA levels were...... determined with respect to sensitivity and specificity in 332 serum samples using the newly developed peptide panel, which was compared to the commercial assays CCPlus (Eurodiagnostica) and CCP3.1 (Inova Diagnostics). A primary panel (peptides 814, 33062 and 33156) was identified, which obtained...... a sensitivity of 71%, while the complete peptide panel reacted with 79% of RA sera screened. Total specificities of 89% and 80% were obtained for the primary peptide panel and the complete peptide panel. Sensitivities for the commercial assays ranged between 71% and 76% and specificities between 88% and 90...

  16. Mapping the interactions between the Alzheimer's Aβ-peptide and human serum albumin beyond domain resolution.

    Science.gov (United States)

    Algamal, Moustafa; Milojevic, Julijana; Jafari, Naeimeh; Zhang, William; Melacini, Giuseppe

    2013-10-01

    Human serum albumin (HSA) is a potent inhibitor of Aβ self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer's disease. It is known that HSA interacts with Aβ oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aβ interactions beyond domain resolution. Here, we map the HSA-Aβ interactions at subdomain and peptide resolution. We show that each separate subdomain of HSA domain 3 inhibits Aβ self-association. We also show that fatty acids (FAs) compete with Aβ oligomers for binding to domain 3, but the determinant of the HSA/Aβ oligomer interactions are markedly distinct from those of FAs. Although salt bridges with the FA carboxylate determine the FA binding affinities, hydrophobic contacts are pivotal for Aβ oligomer recognition. Specifically, we identified a site of Aβ oligomer recognition that spans the HSA (494-515) region and aligns with the central hydrophobic core of Aβ. The HSA (495-515) segment includes residues affected by FA binding and this segment is prone to self-associate into β-amyloids, suggesting that sites involved in fibrilization may provide a lead to develop inhibitors of Aβ self-association. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Modular design of synthetic protein mimics. Characterization of the helical conformation of a 13-residue peptide in crystals

    International Nuclear Information System (INIS)

    Karle, I.L.; Flippen-Anderson, J.L.; Uma, K.; Balaram, P.

    1989-01-01

    The incorporation of α-aminoisobutyryl (Aib) residues into peptide sequences facilitates helical folding. Aib-containing sequences have been chosen for the design of rigid helical segments in a modular approach to the construction of a synthetic protein mimic. The helical conformation of the synthetic peptide Boc-Aib-(Val-Ala-Leu-Aib) 3 -OMe in crystals is established by X-ray diffraction. The 13-residue apolar peptide adopts a helical form in the crystal with seven α-type hydrogen bonds in the middle and 3 10 -type hydrogen bonds at either end. The helices stack in columns, zigzag rather than linear, by means of direct NH hor-ellipsis OC head to tail hydrogen bonds. Leucyl side chains are extended on one side of the helix and valyl side chains on the other side. Water molecules form hydrogen bonds with several backbone carbonyl oxygens that also participate in α-helix hydrogen bonds. There is no apparent distortion of the helix caused by hydration

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

    Science.gov (United States)

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

    2015-01-01

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

  19. Biosensors based on modularly designed synthetic peptides for recognition, detection and live/dead differentiation of pathogenic bacteria.

    Science.gov (United States)

    Liu, Xiaobo; Marrakchi, Mouna; Xu, Dawei; Dong, He; Andreescu, Silvana

    2016-06-15

    Rapid and sensitive detection of bacterial pathogens is critical for assessing public health, food and environmental safety. We report the use of modularly designed and site-specifically oriented synthetic antimicrobial peptides (sAMPs) as novel recognition agents enabling detection and quantification of bacterial pathogens. The oriented assembly of the synthetic peptides on electrode surfaces through an engineered cysteine residue coupled with impedimetric detection facilitated rapid and sensitive detection of bacterial pathogens with a detection limit of 10(2)CFU/mL for four bacterial strains including Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). The approach enabled differentiation between live and dead bacteria. The fabrication of the sAMPs functionalized surface and the importance of the sAMPs orientation for providing optimum recognition and detection ability against pathogens are discussed. The proposed methodology provides a universal platform for the detection of bacterial pathogens based on engineered peptides, as alternative to the most commonly used immunological and gene based assays. The method can also be used to fabricate antimicrobial coatings and surfaces for inactivation and screening of viable bacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Lipidated peptides via post-synthetic thioalkylation promoted by molecular sieves.

    Science.gov (United States)

    Calce, Enrica; Leone, Marilisa; Monfregola, Luca; De Luca, Stefania

    2014-08-01

    A thioalkylation procedure, which uses molecular sieves to promote the reaction, was exploited to provide peptides with useful functional groups (lipidic moieties), naturally occurring on proteins as post-translational modifications. The procedure was further implemented to synthesize tailor-made lipidated peptides, interesting tools to investigate biological processes involving their Ras parent proteins. Moreover, the one-pot preparation of multi-alkylated peptides confirms the versatility and flexibility of the employed methodology.

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

    Science.gov (United States)

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

    2014-01-01

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

  2. Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture.

    Science.gov (United States)

    Miller, Bradley R; Drake, Eric J; Shi, Ce; Aldrich, Courtney C; Gulick, Andrew M

    2016-10-21

    Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-like Proteins Support a Highly Dynamic Domain Architecture

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Bradley R.; Drake, Eric J.; Shi, Ce; Aldrich, Courtney C.; Gulick, Andrew M. (UMM); (HWMRI)

    2016-09-05

    Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein, an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might be derived from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full-length NRPS EntF bound to the MLPs from Escherichia coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatics support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally, the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.

  4. Characterization of a peptide domain within the GB virus C NS5A phosphoprotein that inhibits HIV replication.

    Directory of Open Access Journals (Sweden)

    Jinhua Xiang

    2008-07-01

    Full Text Available GBV-C infection is associated with prolonged survival in HIV-infected people and GBV-C inhibits HIV replication in co-infection models. Expression of the GBV-C nonstructural phosphoprotein 5A (NS5A decreases surface levels of the HIV co-receptor CXCR4, induces the release of SDF-1 and inhibits HIV replication in Jurkat CD4+ T cell lines.Jurkat cell lines stably expressing NS5A protein and peptides were generated and HIV replication in these cell lines assessed. HIV replication was significantly inhibited in all cell lines expressing NS5A amino acids 152-165. Substitution of an either alanine or glycine for the serine at position 158 (S158A or S158G resulted in a significant decrease in the HIV inhibitory effect. In contrast, substituting a phosphomimetic amino acid (glutamic acid; S158E inhibited HIV as well as the parent peptide. HIV inhibition was associated with lower levels of surface expression of the HIV co-receptor CXCR4 and increased release of the CXCR4 ligand, SDF-1 compared to control cells. Incubation of CD4+ T cell lines with synthetic peptides containing amino acids 152-167 or the S158E mutant peptide prior to HIV infection resulted in HIV replication inhibition compared to control peptides.Expression of GBV-C NS5A amino acids 152-165 are sufficient to inhibit HIV replication in vitro, and the serine at position 158 appears important for this effect through either phosphorylation or structural changes in this peptide. The addition of synthetic peptides containing 152-167 or the S158E substitution to Jurkat cells resulted in HIV replication inhibition in vitro. These data suggest that GBV-C peptides or a peptide mimetic may offer a novel, cellular-based approach to antiretroviral therapy.

  5. Anti-peptide antibodies that recognize conformational differences of HLA class I intracytoplasmic domains.

    Science.gov (United States)

    Chersi, Alberto; Galati, Rossella; Ogino, Takeshi; Butler, Richard H; Tanigaki, Nobuyuki

    2002-09-01

    Rabbit antibodies were raised against both long and short peptides derived from exon 7 sequences of human leukocyte antigen (HLA) class I alpha chains; anti-A/B against a 13-mer shared by most HLA-A alpha and HLA-B alpha chains, anti-C against a 15-mer characteristic of HLA-C alpha chains, anti-ACT against a 6-mer specific to HLA-A alpha chains, and anti-CCT against a 5-mer specific to HLA-C alpha chains. Binding activity of the antibodies was determined with peptides by enzyme-linked immunoabsorbent assay (ELISA) and with HLA class I transfectants and the parental cells by FACS analysis. Anti-A/B and anti-C were to a greater or lesser extent crossreactive with the long and short peptides, whereas anti-ACT and anti-CCT were specific to the corresponding short peptides. No binding was seen for anti-ACT and anti-CCT with HLA class I transfectants, C1R-A2, C1R-B7, and 221-Cw1 and the parental cells, C1R (Cw4, E) and 721.221 (E, F). Anti-A/B and anti-C were substantially protein-reactive and the binding order was C1R-B7 > C1R-A2, 721.221 > C1R, 221-Cw1 for anti-A/B, and C1R-B7 > 721.221 > C1R, 221-Cw1, C1R-A2 for anti-C. Thus, anti-A/B and anti-C bound better to HLA-B and HLA-E rather than to HLA-A and HLA-C. Computer modeling of the three-dimensional structure of the intracytoplasmic domains demonstrated that this may be due to structural differences despite the sequence similarities.

  6. Parasiticidal effect of synthetic bovine lactoferrin peptides on the enteric parasite Giardia intestinalis.

    Science.gov (United States)

    Aguilar-Diaz, Hugo; Canizalez-Roman, Adrian; Nepomuceno-Mejia, Tomas; Gallardo-Vera, Francisco; Hornelas-Orozco, Yolanda; Nazmi, Kamran; Bolscher, Jan G M; Carrero, Julio Cesar; Leon-Sicairos, Claudia; Leon-Sicairos, Nidia

    2017-02-01

    Giardia intestinalis is the most common infectious protozoan parasite in children. Despite the effectiveness of some drugs, the disease remains a major worldwide problem. Consequently, the search for new treatments is important for disease eradication. Biological molecules with antimicrobial properties represent a promising alternative to combat pathogens. Bovine lactoferrin (bLF) is a key component of the innate host defense system, and its peptides have exhibited strong antimicrobial activity. Based on these properties, we evaluated the parasiticidal activity of these peptides on G. intestinalis. Trophozoites were incubated with different peptide concentrations for different periods of time, and the growth or viability was determined by carboxyfluorescein-succinimidyl-diacetate-ester (CFDA) and propidium iodide (PI) staining. Endocytosis of peptides was investigated by confocal microscopy, damage was analyzed by transmission and scanning electron microscopy, and the type of programmed cell death was analyzed by flow cytometry. Our results showed that the LF peptides had giardicidal activity. The LF peptides interacted with G. intestinalis and exposure to LF peptides correlated with an increase in the granularity and vacuolization of the cytoplasm. Additionally, the formation of pores, extensive membrane disruption, and programmed cell death was observed in trophozoites treated with LF peptides. Our results demonstrate that LF peptides exhibit potent in vitro antigiardial activity.

  7. Enhancing Vaccine Efficacy by Engineering a Complex Synthetic Peptide To Become a Super Immunogen.

    Science.gov (United States)

    Nordström, Therése; Pandey, Manisha; Calcutt, Ainslie; Powell, Jessica; Phillips, Zachary N; Yeung, Grace; Giddam, Ashwini K; Shi, Yun; Haselhorst, Thomas; von Itzstein, Mark; Batzloff, Michael R; Good, Michael F

    2017-10-15

    Peptides offer enormous promise as vaccines to prevent and protect against many infectious and noninfectious diseases. However, to date, limited vaccine efficacy has been reported and none have been licensed for human use. Innovative ways to enhance their immunogenicity are being tested, but rational sequence modification as a means to improve immune responsiveness has been neglected. Our objective was to establish a two-step generic protocol to modify defined amino acids of a helical peptide epitope to create a superior immunogen. Peptide variants of p145, a conserved helical peptide epitope from the M protein of Streptococcus pyogenes , were designed by exchanging one amino acid at a time, without altering their α-helical structure, which is required for correct antigenicity. The immunogenicities of new peptides were assessed in outbred mice. Vaccine efficacy was assessed in a skin challenge and invasive disease model. Out of 86 variants of p145, seven amino acid substitutions were selected and made the basis of the design for 18 new peptides. Of these, 13 were more immunogenic than p145; 7 induced Abs with significantly higher affinity for p145 than Abs induced by p145 itself; and 1 peptide induced more than 10,000-fold greater protection following challenge than the parent peptide. This peptide also only required a single immunization (compared with three immunizations with the parent peptide) to induce complete protection against invasive streptococcal disease. This study defines a strategy to rationally improve the immunogenicity of peptides and will have broad applicability to the development of vaccines for infectious and noninfectious diseases. Copyright © 2017 by The American Association of Immunologists, Inc.

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

    Directory of Open Access Journals (Sweden)

    Protti Maria

    2005-12-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Generation of rabbit antipeptide antibodies to HLA-class II antigens by the use of synthetic peptides.

    Science.gov (United States)

    Chersi, A; Morganti, M C; Chillemi, F; Houghten, R; Cenciarelli, C

    1988-07-01

    A group of eight synthetic peptides, corresponding in sequence to selected regions of HLA-DQ histocompatibility antigens, was used for rabbit immunization to examine their antigenicity and for localizing exposed regions in the native glycoproteins. Those antibodies were then tested in their ability to recognize the HLA-DQ alloantigens. Seven peptides elicited rabbit antibodies, four of which reacted with human glycoproteins prepared from chronic lymphocytic leukaemia cells. The results indicate that sequence stretches 63 to 79 and probably 82 to 93 of the beta chain correspond to exposed regions in DQw1, DQw2 and DQw3 molecules. However, the specificity of those antipeptide antibodies was low, due to extensive crossreactions with amino acid sequencies of high homology occurring in DQ alloantigens.

  11. Improved Canine and Human Visceral Leishmaniasis Immunodiagnosis Using Combinations of Synthetic Peptides in Enzyme-Linked Immunosorbent Assay

    Science.gov (United States)

    Costa, Míriam Maria; Penido, Marcos; dos Santos, Mariana Silva; Doro, Daniel; de Freitas, Eloísa; Michalick, Marilene Susan Marques; Grimaldi, Gabriel; Gazzinelli, Ricardo Tostes; Fernandes, Ana Paula

    2012-01-01

    Background Zoonotic visceral leishmaniasis (VL) is a severe infectious disease caused by protozoan parasites of the genus Leishmania and the domestic dogs are the main urban parasite reservoir hosts. In Brazil, indirect fluorescence antibody tests (IFAT) and indirect enzyme linked immunosorbent assay (ELISA) using promastigote extracts are widely used in epidemiological surveys. However, their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens, which reduces their accuracy allowing the maintenance of infected animals that favors transmission to humans. In this context, the use of combinations of defined peptides appears favorable. Therefore, they were tested by combinations of five peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39. Methodology/Principal Findings Combinations of peptides derived A2, NH, LACK and K39 antigens were used in ELISA with sera from 44 human patients and 106 dogs. Improved sensitivities and specificities, close to 100%, were obtained for both sera of patients and dogs. Moreover, high sensitivity and specificity were observed even for canine sera presenting low IFAT anti-Leishmania antibody titers or from asymptomatic animals. Conclusions/Significance The use of combinations of B cell predicted synthetic peptides derived from antigens A2, NH, LACK and K39 may provide an alternative for improved sensitivities and specificities for immunodiagnostic assays of VL. PMID:22629475

  12. Short Synthetic α-Helical-Forming Peptide Amphiphiles for Fungal Keratitis Treatment In Vivo.

    Science.gov (United States)

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

    2017-03-01

    The emergence of fungal keratitis is on the rise globally. However, current antifungal therapeutics are ineffective in severe keratomycosis. Previously reported α-helical peptides comprising 8-14 amino acids demonstrate broad-spectrum antimicrobial activity both in vitro and in vivo. Here, α-helical peptides of the optimized sequences are investigated for antifungal biofilm in vitro and in vivo using a fungal biofilm-caused mouse keratitis model. The peptides with the optimal composition demonstrate higher α-helical propensity and improve antifungal activity in dispersing Candida albicans biofilm in vitro. Moreover, the optimized α-helical peptides are not only effective in treating C. albicans biofilm-induced keratitis in mice, they are also nontoxic to the mice eyes. These peptides have the potential to be developed as antifungal agents for the treatment of C. albicans biofilm-caused keratitis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. A synthetic prestin reveals protein domains and molecular operation of outer hair cell piezoelectricity.

    Science.gov (United States)

    Schaechinger, Thorsten J; Gorbunov, Dmitry; Halaszovich, Christian R; Moser, Tobias; Kügler, Sebastian; Fakler, Bernd; Oliver, Dominik

    2011-06-24

    Prestin, a transporter-like protein of the SLC26A family, acts as a piezoelectric transducer that mediates the fast electromotility of outer hair cells required for cochlear amplification and auditory acuity in mammals. Non-mammalian prestin orthologues are anion transporters without piezoelectric activity. Here, we generated synthetic prestin (SynPres), a chimera of mammalian and non-mammalian prestin exhibiting both, piezoelectric properties and anion transport. SynPres delineates two distinct domains in the protein's transmembrane core that are necessary and sufficient for generating electromotility and associated non-linear charge movement (NLC). Functional analysis of SynPres showed that the amplitude of NLC and hence electromotility are determined by the transport of monovalent anions. Thus, prestin-mediated electromotility is a dual-step process: transport of anions by an alternate access cycle, followed by an anion-dependent transition generating electromotility. The findings define structural and functional determinants of prestin's piezoelectric activity and indicate that the electromechanical process evolved from the ancestral transport mechanism.

  14. Frequency domain synthetic aperture focusing technique for variable-diameter cylindrical components.

    Science.gov (United States)

    Jin, Haoran; Wu, Eryong; Han, Ye; Yang, Keji; Chen, Jian

    2017-09-01

    Ultrasonic non-destructive testing (UNDT) plays an important role in ensuring the quality of cylindrical components of equipment such as pipes and axles. As the acoustic beam width widens along propagation depths, the diffraction of acoustic wave becomes serious and the images of defects will be interfered with. To precisely evaluate the dimensions of defects and flaws concealed in components, the synthetic aperture focusing technique (SAFT) is introduced to enhance the image resolutions. Conventional SAFTs have been successfully implemented for the ultrasonic imaging of normal cylinders, while solutions for complex ones, such as variable-diameter cylinders, are still lacking. To overcome this problem, a frequency-domain SAFT for variable-diameter cylindrical components is proposed. This algorithm is mainly based on acoustic field extrapolation, which is modified from cylindrical phase shift migration with the aid of split-step Fourier. After a series of extrapolations, a high-resolution ultrasound image can be reconstructed using a particular imaging condition. According to the experimental results, the proposed method yields low side lobes and high resolutions for flat transducers. Its attainable angular resolution relies on the transducer diameter D and scanning radius R and approximates D/(2R).

  15. Identification of functional peptides from natural and synthetic products on their anticancer activities by tumor targeting.

    Science.gov (United States)

    Ko, Joshua K; Auyeung, Kathy K

    2014-01-01

    Cancer cells can express specific membrane proteins, which act as biomarkers for chemotherapeutic targeting. Functional peptides possess unique properties that will ensure efficacy, selectivity, specificity and low toxicity when used as therapeutic agents. Therapeutic peptides have been derived in treatment of cancers through improvement of cellular uptake, drug targeting and vaccine development. Peptides from natural source have been used for chemoprevention and therapy of various cancers. These include peptides derived from food, marine products, venom components and other animal constituents. Besides, chemically- and recombinantly-synthesized peptides have also been produced and extensively studied in contemporary applications. Improvement of tumor targeting is essential for chemotherapeutic development. This can be achieved through enhancement of intracellular delivery and/or increased specific binding affinity to cancer cells by pore-forming and cytotoxic peptides. Cytotoxic peptides such as the Bcl-2 family members can induce receptor-specific binding to tumor cells and promote apoptosis by targeting lipid membranes. This approach has some limitations in targeting, penetration and localization within tumors. Cell-penetrating peptides (CPPs) belong to a new class of tumor-targeting peptides that can facilitate internalization of tumor markers and/or chemotherapeutic drugs. In order to overcome the problem of serum instability in classical CPPs (e.g. Tat), newer classes of CPPs has been recently introduced. Nevertheless, some cyclized CPPs can further enhance cellular uptake and binding selectivity when compared to activities of their linear counterpart, especially when treating chemoresistant tumors. This review compiles the use of effective tumor-targeting peptides including novel CPPs that represents new therapeutic strategies for the treatment of cancers.

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

    Science.gov (United States)

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

    2015-03-01

    Ambient exposure to a short synthetic peptide has enhanced fecundity (number of offspring) in invertebrates and vertebrates, ostensibly by disinhibiting reproduction. In separate experiments, nematodes (Caenorhabditis elegans) and guppy fish (Poecilia reticulata) were exposed via their aqueous environment to a dissolved synthetic hexamer (6mer) peptide, IEPVFT (EPL036), at a concentration of 1 μmol l(-1). In the case of the worms, peptide was added to their aqueous buffer daily throughout the experiment (14 days); for the guppies, peptide administration was on the first 15 alternate days in a 50 week experiment. Fecundity rose by 79% among the worms. The number of descendants of the treated guppies was more than four times that of controls by week 26 (103 versus 25, including 72 juveniles versus 6), with 15.4% more estimated biomass in the test tank in total (i.e. including founders). It was deduced that treated females bred earlier, at a smaller size, and had larger brood sizes. The total number of fish in the control tank had caught up by termination, but biomass continued to lag the test tank. There were no overt signs of toxicity among either the worms or the fish. Bioinformatics has been unilluminating in explaining these results in terms, for example, of mimicry of an endogenous regulator. A mass spectrometric campaign to identify a receptor, using murine brain for expediency, proved inconclusive. Molecular modelling in silico indicated unexpectedly that the hexamer EPL036 might be acting as an antagonist, to pro-fecundity effect; that is, as a blocker of an inhibitor. This suggests that there awaits discovery an evolutionarily conserved reproductive inhibitor and its (anti-fecundity) receptor. © 2015. Published by The Company of Biologists Ltd.

  17. Synthetic antibodies and peptides recognizing progressive multifocal leukoencephalopathy-specific point mutations in polyomavirus JC capsid viral protein 1.

    Science.gov (United States)

    Chen, Gang; Gorelik, Leonid; Simon, Kenneth J; Pavlenco, Alevtina; Cheung, Anne; Brickelmaier, Margot; Chen, Ling Ling; Jin, Ping; Weinreb, Paul H; Sidhu, Sachdev S

    2015-01-01

    Polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a rare and frequently fatal brain disease that afflicts a small fraction of the immune-compromised population, including those affected by AIDS and transplantation recipients on immunosuppressive drug therapy. Currently there is no specific therapy for PML. The major capsid viral protein 1 (VP1) involved in binding to sialic acid cell receptors is believed to be a key player in pathogenesis. PML-specific mutations in JCV VP1 sequences present at the binding pocket of sialic acid cell receptors, such as L55F and S269F, abolish sialic acid recognition and might favor PML onset. Early diagnosis of these PML-specific mutations may help identify patients at high risk of PML, thus reducing the risks associated with immunosuppressive therapy. As a first step in the development of such early diagnostic tools, we report identification and characterization of affinity reagents that specifically recognize PML-specific mutations in VP1 variants using phage display technology. We first identified 2 peptides targeting wild type VP1 with moderate specificity. Fine-tuning via selection of biased libraries designed based on 2 parental peptides yielded peptides with different, yet still moderate, bindinspecificities. In contrast, we had great success in identifying synthetic antibodies that recognize one of the PML-specific mutations (L55F) with high specificity from the phage-displayed libraries. These peptides and synthetic antibodies represent potential candidates for developing tailored immune-based assays for PML risk stratification in addition to complementing affinity reagents currently available for the study of PML and JCV.

  18. Impaired Hippocampal Neuroligin-2 Function by Chronic Stress or Synthetic Peptide Treatment is Linked to Social Deficits and Increased Aggression

    DEFF Research Database (Denmark)

    van der Kooij, Michael A; Fantin, Martina; Kraev, Igor

    2014-01-01

    Neuroligins (NLGNs) are cell adhesion molecules that are important for proper synaptic formation and functioning and are critical regulators of the balance between neural excitation/inhibition (E/I). Mutations in NLGNs have been linked to psychiatric disorders in humans involving social dysfunction....... Furthermore, using synthetic peptides that comprise sequences in either NLGN-1 (neurolide-1) or NLGN-2 (neurolide-2) involved in the interaction with their presynaptic partner NRXN1, intra-hippocampal administration of neurolide-2 led also to reduced sociability and increased aggression. These results...

  19. The Safety Evaluation of a Potent Angiogenic Activator, Synthetic Peptide (SFKLRY-NH2) for the Skin Application

    Science.gov (United States)

    Kim, Dong Ha; Lim, Yun Young; Kim, Hyeong Mi; Kim, So Young; Park, Sung-Gil; Lee, Taehoon

    2012-01-01

    A novel synthetic hexapeptide (SFKLRY-NH2) that displays angiogenic activity has been identified by positional scanning of a synthetic peptide combinatorial library (PS-SPCL). This study was carried out to investigate the irritation of the SFKLRY-NH2 on the skin. The tests were performed on the basis of Korea Food and Drug Administration (KFDA) guidelines. In results, cell toxicity is not appeared for SFKLRY-NH2 in HaCaT cells and B16F10 cells. SFKLRY-NH2 induced no skin irritation at low concentration (10 μM), mild irritation at high concentration (10mM). We consider that this result is helpful for saying about the safety of SFKLRY-NH2 in clinical use. PMID:24278589

  20. Cell penetrating synthetic antimicrobial peptides (SAMPs) exhibiting potent and selective killing of mycobacterium by targeting its DNA.

    Science.gov (United States)

    Sharma, Aashish; Pohane, Amol Arunrao; Bansal, Sandhya; Bajaj, Avinash; Jain, Vikas; Srivastava, Aasheesh

    2015-02-23

    Naturally occurring antimicrobial peptides (AMPs) are powerful defence tools to tackle pathogenic microbes. However, limited natural production and high synthetic costs in addition to poor selectivity limit large-scale use of AMPs in clinical settings. Here, we present a series of synthetic AMPs (SAMPs) that exhibit highly selective and potent killing of Mycobacterium (minimum inhibitory concentration <20 μg mL(-1)) over E. coli or mammalian cells. These SAMPs are active against rapidly multiplying as well as growth saturated Mycobacterium cultures. These SAMPs are not membrane-lytic in nature, and are readily internalized by Mycobacterium and mammalian cells; whereas in E. coli, the lipopolysaccharide layer inhibits their cellular uptake, and hence, their antibacterial action. Upon internalization, these SAMPs interact with the unprotected genomic DNA of mycobacteria, and impede DNA-dependent processes, leading to bacterial cell death. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Characterization of desmoglein-3 epitope region peptides as synthetic antigens: analysis of their in vitro T cell stimulating efficacy, cytotoxicity, stability, and their conformational features.

    Science.gov (United States)

    Szabados, Hajnalka; Uray, Katalin; Majer, Zsuzsa; Silló, Pálma; Kárpáti, Sarolta; Hudecz, Ferenc; Bősze, Szilvia

    2015-09-01

    Desmoglein-3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full-length peptide (Dsg3/189-205, Dsg3/206-222, Dsg3/342-358, and Dsg3/761-777) and its N-terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure-activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20 h of stimulation, the interferon (IFN)-γ content of the supernatants was measured by enzyme-linked immunosorbent assay. In the in vitro conditions, peptides were stable and non-cytotoxic. The in vitro IFN-γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342-358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192-205, Dsg3/763-777, and Dsg3/764-777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were the most capable to distinguish between healthy and PV donors. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  2. Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, A. Patricia [University of Washington, Department of Medicinal Chemistry, School of Pharmacy (United States); Spyracopoulos, Leo [Department of Biochemistry (Canada); Irvin, Randall T. [University of Alberta, Department of Medical Microbiology and Immunology (Canada); Sykes, Brian D. [Department of Biochemistry (Canada)

    2000-07-15

    The backbone dynamics of a {sup 15}N-labeled recombinant PAK pilin peptide spanning residues 128-144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys{sup 128}-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Ser-Lys{sup 144}) were probed by measurements of {sup 15}N NMR relaxation. This PAK(128-144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The {sup 15}N longitudinal (T{sub 1}) and transverse (T{sub 2}) relaxation rates and the steady-state heteronuclear {l_brace}{sup 1}H{r_brace}-{sup 15}N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25 deg. C ) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the 'model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559 and 4559-4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153-162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II {beta}-turns spanning residues Asp{sup 134}-Glu-Gln-Phe{sup 137} and Pro{sup 139}-Lys-Gly-Cys{sup 142}, respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.

  3. Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily

    DEFF Research Database (Denmark)

    Bikker, Floris J; Ligtenberg, Antoon J M; Nazmi, Kamran

    2002-01-01

    SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment...

  4. Identification of a Tsal152-75 salivary synthetic peptide to monitor cattle exposure to tsetse flies.

    Science.gov (United States)

    Somda, Martin Bienvenu; Cornelie, Sylvie; Bengaly, Zakaria; Mathieu-Daudé, Françoise; Poinsignon, Anne; Dama, Emilie; Bouyer, Jeremy; Sidibé, Issa; Demettre, Edith; Seveno, Martial; Remoué, Franck; Sanon, Antoine; Bucheton, Bruno

    2016-03-15

    The saliva of tsetse flies contains a cocktail of bioactive molecules inducing specific antibody responses in hosts exposed to bites. We have previously shown that an indirect-ELISA test using whole salivary extracts from Glossina morsitans submorsitans was able to discriminate between (i) cattle from tsetse infested and tsetse free areas and (ii) animals experimentally exposed to low or high numbers of tsetse flies. In the present study, our aim was to identify specific salivary synthetic peptides that could be used to develop simple immunoassays to measure cattle exposure to tsetse flies. In a first step, 2D-electrophoresis immunoblotting, using sera from animals exposed to a variety of bloodsucking arthropods, was performed to identify specific salivary proteins recognised in cattle exposed to tsetse bites. Linear epitope prediction software and Blast analysis were then used to design synthetic peptides within the identified salivary proteins. Finally, candidate peptides were tested by indirect-ELISA on serum samples from tsetse infested and tsetse free areas, and from exposure experiments. The combined immunoblotting and bioinformatics analyses led to the identification of five peptides carrying putative linear epitopes within two salivary proteins: the tsetse salivary gland protein 1 (Tsal1) and the Salivary Secreted Adenosine (SSA). Of these, two were synthesised and tested further based on the absence of sequence homology with other arthropods or pathogen species. IgG responses to the Tsal152-75 synthetic peptide were shown to be specific of tsetse exposure in both naturally and experimentally exposed hosts. Nevertheless, anti-Tsal152-75 IgG responses were absent in animals exposed to high tsetse biting rates. These results suggest that Tsal152-75 specific antibodies represent a biomarker of low cattle exposure to tsetse fly. These results are discussed in the light of the other available tsetse saliva based-immunoassays and in the perspective of developing

  5. D-SAL and NAP: Two Peptides Sharing a SIP Domain.

    Science.gov (United States)

    Gozes, Illana; Sragovich, Shlomo; Schirer, Yulie; Idan-Feldman, Anat

    2016-06-01

    NAPVSIPQ (NAP) and all D-amino acid SALLRSIPA (D-SAL) are neuroprotective peptides derived from activity-dependent neuroprotective protein (ADNP) and activity-dependent neurotrophic factor (ADNF), respectively. Both proteins were shown to protect against cognitive impairment, using different animal models and to increase neuronal survival following exposure to neurotoxins. NAP was extensively tested and found to increase microtubule stability, protect axonal transport, and inhibit apoptosis. Here, we aimed to further evaluate and correlate effects at the behavioral level, in a rat model of diabetes. Diabetes is primarily a metabolic disorder which presents secondary neurological manifestations. Diabetes induces peripheral nervous system damage which is translated into impaired sensory perception and is termed diabetic neuropathy. Diabetes-related central nervous system damage causes cognitive decline. The behavioral study aimed to evaluate the effect of NAP and D-SAL on peripheral neuropathy and cognitive decline. Peripheral neuropathy was tested by measuring the response to a thermal stimulus, and cognitive ability was measured by a social memory test and a spatial memory test using long- and short-term dependent tasks and a reference memory task. Results indicated an immediate sensory neuropathy in the diabetic model, which was prevented by both peptides and a later neuropathic phase, prevented only by NAP treatment. Cognitive tests revealed impaired performance in both social and spatial memory tests in the diabetes model. Each of the peptides improved different aspects of cognitive behavior, with NAP being more potent than D-SAL. Mechanistically, both NAP and SAL contain a SIP (SxIP) domain that has been shown to interact with microtubule end-binding proteins (EBs). Specifically, we have previously shown a direct interaction of NAP with EB1 and EB3; we have further shown an interaction of the NAP-derived 4 amino acid SKIP peptide with EB3, stimulating axonal

  6. Synthetic peptides from heat-shock protein 65 inhibit proinflammatory cytokine secretion by peripheral blood mononuclear cells from rheumatoid arthritis patients.

    Science.gov (United States)

    Zhou, Jun; Wang, Li-Ping; Feng, Xuan; Fan, Dan-Dan; Zang, Wei-Jin; Wang, Bing

    2014-01-01

    1. Rheumatoid arthritis (RA) is a systemic autoimmune disease mediated by T cells. Proinflammatory cytokines plays a critical role in the pathogenesis of RA. The aim of the present study was to investigate the effects of synthetic peptides (HP-R1, HP-R2 and HP-R3), derived from the sequence of 65 kDa mycobacterial heat shock protein (HSP), on the proliferation of and cytokine secretion by peripheral blood mononuclear cells (PBMC) from RA patients. 2. The PBMC were obtained from RA patients and collected by Ficoll-Hypaque density centrifugation. Peripheral blood mononuclear cells were treated with one of the three synthetic peptides for 4 h, after which time proliferation and cytokine production were determined. The effects of the three peptides on the proliferation of PBMC were analysed by the colorimetric cell proliferation (CCK-8) assay. Cytokine production was measured in culture supernatants using specific ELISAs. 3. None of the three peptides had any significant effect on the proliferation of PBMC from healthy controls. However, the proliferation of PBMC from RA patients was inhibited by all three peptides. The production of tumour necrosis factor-α from RA patients was significantly inhibited by all three peptides. The secretion of interferon-γ was significantly suppressed by HP-R1 and HP-R2. Unlike the other two peptides, HP-R2 increased the secretion of interleukin (IL)-4. None of the peptides had any significant effect on the production of IL-10. 4. The results of the present study suggest that the synthetic peptides derived from HSP65 exhibit antiproliferative and anti-inflammatory activity, and support the potential use of synthetic peptides as therapeutic drugs in RA patients. © 2013 Wiley Publishing Asia Pty Ltd.

  7. A Protocol for the Design of Protein and Peptide Nanostructure Self-Assemblies Exploiting Synthetic Amino Acids.

    Science.gov (United States)

    Haspel, Nurit; Zheng, Jie; Aleman, Carlos; Zanuy, David; Nussinov, Ruth

    2017-01-01

    In recent years there has been increasing interest in nanostructure design based on the self-assembly properties of proteins and polymers. Nanodesign requires the ability to predictably manipulate the properties of the self-assembly of autonomous building blocks, which can fold or aggregate into preferred conformational states. The design includes functional synthetic materials and biological macromolecules. Autonomous biological building blocks with available 3D structures provide an extremely rich and useful resource. Structural databases contain large libraries of protein molecules and their building blocks with a range of sizes, shapes, surfaces, and chemical properties. The introduction of engineered synthetic residues or short peptides into these building blocks can greatly expand the available chemical space and enhance the desired properties. Herein, we summarize a protocol for designing nanostructures consisting of self-assembling building blocks, based on our recent works. We focus on the principles of nanostructure design with naturally occurring proteins and synthetic amino acids, as well as hybrid materials made of amyloids and synthetic polymers.

  8. Reversion of prion protein conformational changes by synthetic beta-sheet breaker peptides.

    Science.gov (United States)

    Soto, C; Kascsak, R J; Saborío, G P; Aucouturier, P; Wisniewski, T; Prelli, F; Kascsak, R; Mendez, E; Harris, D A; Ironside, J; Tagliavini, F; Carp, R I; Frangione, B

    2000-01-15

    Transmissible spongiform encephalopathies are associated with a structural transition in the prion protein that results in the conversion of the physiological PrPc to pathological PrP(Sc). We investigated whether this conformational transition can be inhibited and reversed by peptides homologous to the PrP fragments implicated in the abnormal folding, which contain specific residues acting as beta-sheet blockers (beta-sheet breaker peptides). We studied the effect of a 13-residue beta-sheet breaker peptide (iPrP13) on the reversion of the abnormal structure and properties of PrP(Sc) purified from the brains of mice with experimental scrapie and from human beings affected by sporadic and variant Creutzfeldt-Jakob disease. In a cellular model of familial prion disease, we studied the effect of the peptide in the production of the abnormal form of PrP in intact cells. The influence of the peptide on prion infectivity was studied in vivo by incubation time assays in mice with experimental scrapie. The beta-sheet breaker peptide partly reversed in-vitro PrP(Sc) to a biochemical and structural state similar to that of PrPc. The effect of the peptide was also detected in intact cells. Treatment of prion infectious material with iPrP13 delayed the appearance of clinical symptoms and decreased infectivity by 90-95% in mice with experimental scrapie. Beta-sheet breaker peptides reverse PrP conformational changes implicated in the pathogenesis of spongiform encephalopathies. These peptides or their derivatives provide a useful tool to study the role of PrP conformation and might represent a novel therapeutic approach for prion-related disorders.

  9. Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2.

    Science.gov (United States)

    Kafurke, Uwe; Erijman, Ariel; Aizner, Yonatan; Shifman, Julia M; Eichler, Jutta

    2015-09-01

    Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein-ligand interactions. Such mimics of protein binding sites can currently be generated through structure-based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin-2 (Fas) because the structure and function of this complex is well understood. Structure-based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE-specific peptide-Fas interaction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

  10. Extracellular Matrix Glycoprotein-Derived Synthetic Peptides Differentially Modulate Glioma and Sarcoma Cell Migration.

    Science.gov (United States)

    Brösicke, Nicole; Sallouh, Muhammad; Prior, Lisa-Marie; Job, Albert; Weberskirch, Ralf; Faissner, Andreas

    2015-07-01

    Glycoproteins of the extracellular matrix (ECM) regulate proliferation, migration, and differentiation in numerous cell lineages. ECM functions are initiated by small peptide sequences embedded in large constituents that are recognized by specific cellular receptors. In this study, we have investigated the biological effects of peptides derived from collagen type IV and tenascin-C compared to the well-known RGD peptide originally discovered in fibronectin. The influence of glycoproteins and corresponding peptides on the migration of the glioma cell lines U-251-MG and U-373-MG and the sarcoma line S-117 was studied. When the cell lines were tested in a modified Boyden chamber assay on filters coated with the ECM glycoproteins, glioma cells showed a strong migration response on tenascin-C and the basal lamina constituent collagen IV, in contrast to S-117 cells. In order to identify relevant stimulatory motifs, peptides derived from fibronectin (6NHX-GRGDSF), tenascin-C (TN-C, VSWRAPTA), and collagen type IV (MNYYSNS) were compared, either applied in solution in combination with ECM glycoprotein substrates, in solution in the presence of untreated membranes, or coated on the filters of the Boyden chambers. Using this strategy, we could identify the novel tenascin-C-derived peptide motif VSWRAPTA as a migration stimulus for glioma cells. Furthermore, while kin peptides generally blocked the effects of the respective homologous ECM proteins, unexpected effects were observed in heterologous situations. There, in several cases, addition of soluble peptides strongly boosted the response to the coated ECM proteins. We propose that peptides may synergize or antagonize each other by stimulating different signaling pathways.

  11. Different domains of the glucagon and glucagon-like peptide-1 receptors provide the critical determinants of ligand selectivity

    OpenAIRE

    Runge, S; Wulff, B S; Madsen, K; Bräuner-Osborne, H; Knudsen, L B

    2003-01-01

    Glucagon and glucagon-like peptide-1 (GLP-1) are homologous peptide hormones with important functions in glucose metabolism. The receptors for glucagon and GLP-1 are homologous family B G-protein coupled receptors. The GLP-1 receptor amino-terminal extracellular domain is a major determinant of glucagon/GLP-1 selectivity of the GLP-1 receptor. However, the divergent residues in glucagon and GLP-1 that determine specificity for the GLP-1 receptor amino-terminal extracellular domain are not kno...

  12. 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.

  13. Protection of Cattle against Foot-and-Mouth Disease by a Synthetic Peptide

    Science.gov (United States)

    Dimarchi, Richard; Brooke, Gerald; Gale, Charles; Cracknell, Victor; Doel, Timothy; Mowat, Noel

    1986-05-01

    A chemically synthesized peptide consisting essentially of two separate regions (residues 141 to 158 and 200 to 213) of a virus coat protein (VP1) from the 01 Kaufbeuren strain of foot-and-mouth disease virus was prepared free of any carrier protein. It elicited high levels of neutralizing antibody and protected cattle against intradermolingual challenge by inoculation with infectious virus. Comparative evaluation of this peptide with a single-site peptide (residues 141 to 158) in guinea pigs suggests the importance of the VP1 carboxyl terminal residues in enhancing the protective response.

  14. Synthetic Peptides to Target Stringent Response-Controlled Virulence in a Pseudomonas aeruginosa Murine Cutaneous Infection Model

    Directory of Open Access Journals (Sweden)

    Daniel Pletzer

    2017-09-01

    Full Text Available Microorganisms continuously monitor their surroundings and adaptively respond to environmental cues. One way to cope with various stress-related situations is through the activation of the stringent stress response pathway. In Pseudomonas aeruginosa this pathway is controlled and coordinated by the activity of the RelA and SpoT enzymes that metabolize the small nucleotide secondary messenger molecule (pppGpp. Intracellular ppGpp concentrations are crucial in mediating adaptive responses and virulence. Targeting this cellular stress response has recently been the focus of an alternative approach to fight antibiotic resistant bacteria. Here, we examined the role of the stringent response in the virulence of P. aeruginosa PAO1 and the Liverpool epidemic strain LESB58. A ΔrelA/ΔspoT double mutant showed decreased cytotoxicity toward human epithelial cells, exhibited reduced hemolytic activity, and caused down-regulation of the expression of the alkaline protease aprA gene in stringent response mutants grown on blood agar plates. Promoter fusions of relA or spoT to a bioluminescence reporter gene revealed that both genes were expressed during the formation of cutaneous abscesses in mice. Intriguingly, virulence was attenuated in vivo by the ΔrelA/ΔspoT double mutant, but not the relA mutant nor the ΔrelA/ΔspoT complemented with either gene. Treatment of a cutaneous P. aeruginosa PAO1 infection with anti-biofilm peptides increased animal welfare, decreased dermonecrotic lesion sizes, and reduced bacterial numbers recovered from abscesses, resembling the phenotype of the ΔrelA/ΔspoT infection. It was previously demonstrated by our lab that ppGpp could be targeted by synthetic peptides; here we demonstrated that spoT promoter activity was suppressed during cutaneous abscess formation by treatment with peptides DJK-5 and 1018, and that a peptide-treated relA complemented stringent response double mutant strain exhibited reduced peptide

  15. Magnetic characteristics of synthetic pseudo-single-domain and multi-domain greigite (Fe3S4)

    Czech Academy of Sciences Publication Activity Database

    Chang, L.; Roberts, A. P.; Muxworthy, A. R.; Tang, Y.; Chen, Q.; Rowan, Ch. J.; Liu, Q.; Pruner, Petr

    2007-01-01

    Roč. 34, č. 24 (2007), L24304-L24304 ISSN 0094-8276 R&D Projects: GA AV ČR IAA3013406 Institutional research plan: CEZ:AV0Z30130516 Keywords : greigite * magnetic properties * grain size * pseudo-single-domain * multi-domain Subject RIV: DE - Earth Magnetism, Geodesy, Geography Impact factor: 2.744, year: 2007

  16. Synthetic antimicrobial peptides of the halictines family disturb the membrane integrity of Candida cells.

    Science.gov (United States)

    Kodedová, Marie; Sychrová, Hana

    2017-10-01

    We compared the potency of four derivatives of the antimicrobial peptide halictine-2 against six Candida species. Observed activity was peptide and species specific. Halictines rapidly permeabilized cell membranes and caused the leakage of cytosolic components. Their killing potential was enhanced by the commercial antimicrobial agent octenidine dihydrochloride. The effect on C. glabrata cells did not depend on the activity of Cdr pumps, but was influenced by their lipid composition. The pre-treatment of cells with myriocin, an inhibitor of sphingolipid synthesis, enhanced the peptides' activity, whereas pre-treatment with terbinafine and fluconazole, inhibitors of sterol synthesis, significantly weakened their efficacy. The killing efficacy of peptides increased in combination with amphotericin B. Thus the mode of action of halictines is likely to depend on the plasma-membrane sterols, which might explain the observed differences among the tested Candida species. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Post-synthetic modification of tryptophan containing peptides via NIS mediation.

    Science.gov (United States)

    Gu, Chen-Xue; Bi, Qing-Wei; Gao, Chu-Kun; Wen, Jian; Zhao, Zhi-Gang; Chen, Zili

    2017-04-18

    A new efficient method was developed to provide modified tryptophan peptides through NIS (N-iodosuccinimide) mediated N 2 -selective coupling of a Trp unit with 1,2,3-triazoles, of which, the preliminary spectral properties were also studied.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  19. Synthetic peptides for in vitro evaluation of the neutralizing potency of Loxosceles antivenoms.

    Science.gov (United States)

    Ramada, Juliani Salvini; Becker-Finco, Alessandra; Minozzo, João Carlos; Felicori, Liza Figueiredo; Machado de Avila, Ricardo Andrez; Molina, Franck; Nguyen, Christophe; de Moura, Juliana; Chávez-Olórtegui, Carlos; Alvarenga, Larissa Magalhães

    2013-10-01

    An important step in the development of therapeutic antivenoms is the pre-clinical testing using in vivo methods to assess their neutralizing potency. For spider antivenoms (Loxosceles species), horse serum potency against the necrotizing activities of Loxosceles intermedia crude venom is currently tested in rabbits. These procedures are time consuming and involve a large number of animals. The aim of this study was to develop an in vitro method to assess the neutralizing potency of anti-Loxosceles sera. We first demonstrated that it was not possible to establish a correlation between the ELISA antibody reactivity of horse anti-Loxosceles serum and their neutralizing potency. We then showed that the antivenoms recognized several peptide epitopes from different regions of SMase-D proteins, which are toxic antigens from Loxosceles venoms. The recognition of some peptides was observed only when high neutralizing potency sera was used. Based on these results, three peptides (peptide 1, DNRRPIWNLAHMVNA and peptide 3, DFSGPYLPSLPTLDA corresponding to residues 2-16 and 164-178, respectively, of SMase-1 protein from Loxosceles laeta, and peptide 2, EFVNLGANSIETDVS corresponding to residues 22-36 of A1H - LoxGa protein from Loxosceles gaucho and LiD1 protein from L. intermedia) were selected. The peptides were synthesized, coupled to bovine serum albumin (BSA), and used as antigens in indirect ELISA to test their reactivity with horse anti-Loxosceles serum of varying neutralizing potencies. We found certain assay conditions that discriminated between the high and low neutralizing potency sera. This study introduced an in vitro and peptide-based neutralization assay for anti-Loxosceles antivenoms. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Jorge F. Correia-Pinto

    2015-09-01

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

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  2. Immunogenicity of synthetic peptides representing neutralizing epitopes on the glycoprotein of infectious hematopoietic necrosis virus

    Science.gov (United States)

    Emmenegger, E.; Landolt, M.; LaPatra, S.; Winton, J.

    1997-01-01

    Three peptides, P76, P226, and P268 representing 3 putative antigen~c determinants on the glycoprotein of infectious hematopoietic necrosis virus (IHNV), were synthesized and injected into rainbow trout Oncorhynchus mykiss to assess their immunogen~city. Antisera extracted from the immunized trout were analyzed uslng an enzyme linked imrnunosorbent assay (ELISA) for the presence of antibodies that could bind to the peptides or to intact virions of IHNV. The antisera were also tested for neutralizing activity against IHNV by a complement-mediated neutralization assay. In general, recognition of the peptides and IHNV was low and only a few antibody binding patterns were demonstrated. Antisera from fish injected with P76 constructs recognized the homologous peptide more than the heterologous peptides, whereas antisera from fish inoculated with either P226 or P268 constructs recognized P76 equally, or better, than the homologous peptide; however, there was a high degree of individual variation within each treatment group. Neutralization actlvlty was demonstrated by serum from a single flsh lnlected with one of the pept~des (P268) and from 7 of 10 positive control f~sh Infected with an attenuated strain of IHNV Possible explanations for the dichotomous immune responses are discussed. These results indicate we need to improve our overall understanding of the

  3. Cooperative phosphoinositide and peptide binding by PSD-95/discs large/ZO-1 (PDZ) domain of polychaetoid, Drosophila zonulin.

    Science.gov (United States)

    Ivarsson, Ylva; Wawrzyniak, Anna Maria; Wuytens, Gunther; Kosloff, Mickey; Vermeiren, Elke; Raport, Marie; Zimmermann, Pascale

    2011-12-30

    PDZ domains are well known protein-protein interaction modules that, as part of multidomain proteins, assemble molecular complexes. Some PDZ domains have been reported to interact with membrane lipids, in particular phosphatidylinositol phosphates, but few studies have been aimed at elucidating the prevalence or the molecular details of such interactions. We screened 46 Drosophila PDZ domains for phosphoinositide-dependent cellular localization and discovered that the second PDZ domain of polychaetoid (Pyd PDZ2) interacts with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) at the plasma membrane. Surface plasmon resonance binding experiments with recombinant protein established that Pyd PDZ2 interacts with phosphatidylinositol phosphates with apparent affinities in the micromolar range. Electrostatic interactions involving an extended positively charged surface of Pyd PDZ2 are crucial for the PtdIns(4,5)P(2)-dependent membrane interactions as shown by a combination of three-dimensional modeling, mutagenesis, binding, and localization studies. In vivo localization studies further suggested that both lipid and peptide binding contribute to membrane localization. We identified the transmembrane protein Crumbs as a Pyd PDZ2 ligand and probed the relation between peptide and PtdIns(4,5)P(2) binding. Contrary to the prevalent view on PDZ/peptide/lipid binding, we did not find competition between peptide and lipid ligands. Instead, preloading the protein with the 10-mer Crb3 peptide increased the apparent affinity of Pyd PDZ2 for PtdIns(4,5)P(2) 6-fold. Our results suggest that membrane localization of Pyd PDZ2 may be driven by a combination of peptide and PtdIns(4,5)P(2) binding, which raises the intriguing possibility that the domain may coordinate protein- and phospholipid-mediated signals.

  4. Synthetic

    Directory of Open Access Journals (Sweden)

    Anna Maria Manferdini

    2010-06-01

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

  5. Disease resistance conferred by the expression of a gene encoding a synthetic peptide in transgenic cotton (Gossypium hirsutum L.) plants.

    Science.gov (United States)

    Rajasekaran, Kanniah; Cary, Jeffrey W; Jaynes, Jesse M; Cleveland, Thomas E

    2005-11-01

    Fertile, transgenic cotton plants expressing the synthetic antimicrobial peptide, D4E1, were produced through Agrobacterium-mediated transformation. PCR products and Southern blots confirmed integration of the D4E1 gene, while RT-PCR of cotton RNA confirmed the presence of D4E1 transcripts. In vitro assays with crude leaf protein extracts from T0 and T1 plants confirmed that D4E1 was expressed at sufficient levels to inhibit the growth of Fusarium verticillioides and Verticillium dahliae compared to extracts from negative control plants transformed with pBI-d35S(Omega)-uidA-nos (CGUS). Although in vitro assays did not show control of pre-germinated spores of Aspergillus flavus, bioassays with cotton seeds in situ or in planta, inoculated with a GFP-expressing A. flavus, indicated that the transgenic cotton seeds inhibited extensive colonization and spread by the fungus in cotyledons and seed coats. In planta assays with the fungal pathogen, Thielaviopsis basicola, which causes black root rot in cotton, showed typical symptoms such as black discoloration and constriction on hypocotyls, reduced branching of roots in CGUS negative control T1 seedlings, while transgenic T1 seedlings showed a significant reduction in disease symptoms and increased seedling fresh weight, demonstrating tolerance to the fungal pathogen. Significant advantages of synthetic peptides in developing transgenic crop plants that are resistant to diseases and mycotoxin-causing fungal pathogens are highlighted in this report.

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

    Science.gov (United States)

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

    2014-06-01

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

  7. Determining the Mode of Action Involved in the Antimicrobial Activity of Synthetic Peptides: A Solid-State NMR and FTIR Study

    Science.gov (United States)

    Lorin, Aurélien; Noël, Mathieu; Provencher, Marie-Ève; Turcotte, Vanessa; Cardinal, Sébastien; Lagüe, Patrick; Voyer, Normand; Auger, Michèle

    2012-01-01

    We have previously shown that leucine to lysine substitution(s) in neutral synthetic crown ether containing 14-mer peptide affect the peptide structure and its ability to permeabilize bilayers. Depending on the substitution position, the peptides adopt mainly either a α-helical structure able to permeabilize dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) vesicles (nonselective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). In this study, we have used a combination of solid-state NMR and Fourier transform infrared spectroscopy to investigate the effects of nonselective α-helical and selective intermolecular β-sheet peptides on both types of bilayers. 31P NMR results indicate that both types of peptides interact with the headgroups of DMPC and DMPG bilayers. 2H NMR and Fourier transform infrared results reveal an ordering of the hydrophobic core of bilayers when leakage is noted, i.e., for DMPG vesicles in the presence of both types of peptides and DMPC vesicles in the presence of nonselective peptides. However, selective peptides have no significant effect on the ordering of DMPC acyl chains. The ability of these 14-mer peptides to permeabilize lipid vesicles therefore appears to be related to their ability to increase the order of the bilayer hydrophobic core. PMID:23062339

  8. A graph kernel approach for alignment-free domain-peptide interaction prediction with an application to human SH3 domains.

    Science.gov (United States)

    Kundu, Kousik; Costa, Fabrizio; Backofen, Rolf

    2013-07-01

    State-of-the-art experimental data for determining binding specificities of peptide recognition modules (PRMs) is obtained by high-throughput approaches like peptide arrays. Most prediction tools applicable to this kind of data are based on an initial multiple alignment of the peptide ligands. Building an initial alignment can be error-prone, especially in the case of the proline-rich peptides bound by the SH3 domains. Here, we present a machine-learning approach based on an efficient graph-kernel technique to predict the specificity of a large set of 70 human SH3 domains, which are an important class of PRMs. The graph-kernel strategy allows us to (i) integrate several types of physico-chemical information for each amino acid, (ii) consider high-order correlations between these features and (iii) eliminate the need for an initial peptide alignment. We build specialized models for each human SH3 domain and achieve competitive predictive performance of 0.73 area under precision-recall curve, compared with 0.27 area under precision-recall curve for state-of-the-art methods based on position weight matrices. We show that better models can be obtained when we use information on the noninteracting peptides (negative examples), which is currently not used by the state-of-the art approaches based on position weight matrices. To this end, we analyze two strategies to identify subsets of high confidence negative data. The techniques introduced here are more general and hence can also be used for any other protein domains, which interact with short peptides (i.e. other PRMs). The program with the predictive models can be found at http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/SH3PepInt.tar.gz. We also provide a genome-wide prediction for all 70 human SH3 domains, which can be found under http://www.bioinf.uni-freiburg.de/Software/SH3PepInt/Genome-Wide-Predictions.tar.gz. Supplementary data are available at Bioinformatics online.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    Merozoite surface protein 2 (MSP2) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the peripheral blood mononuclear cell (PBMC) response to synthetic peptides corresponding to conserved and variant regions of the FCQ-27 allelic form of MSP2 in Ghanaian individuals...... from an area of hyperendemic malaria transmission and in Danes without exposure to malaria. PBMC from 20-39% of Ghanaians responded to each of the peptides by proliferation and 29-36% had PBMC which produced interferon-gamma (IFN-gamma) in response to peptide stimulation. In Danes......, there was no proliferation to two of the peptides and only PBMC from 5% of the individuals proliferated to the other three peptides. IFN-gamma production was not detected to any peptide. In both Danes and Ghanaians in only a few instances was IL-4 detected in the PBMC cultures. Overall PBMC from 79% of the Ghanaians...

  10. Synthetic Peptide dendrimers block the development and expression of experimental allergic encephalomyelitis.

    Science.gov (United States)

    Wegmann, Keith W; Wagner, Cynthia R; Whitham, Ruth H; Hinrichs, David J

    2008-09-01

    Multiple Ag peptides (MAPs) containing eight proteolipid protein (PLP)(139-151) peptides arranged around a dendrimeric branched lysine core were used to influence the expression and development of relapsing experimental allergic encephalomyelitis (EAE) in SJL mice. The PLP(139-151) MAPs were very efficient agents in preventing the development of clinical disease when administered after immunization with the PLP(139-151) monomeric encephalitogenic peptide in CFA. The treatment effect with these MAPs was peptide specific; irrelevant multimeric peptides such as guinea pig myelin basic protein GPBP(72-84) MAP (a dendrimeric octamer composed of the 72-84 peptide) and PLP(178-191) MAP (a dendrimeric octamer composed of the PLP(178-191) peptide) had no treatment effect on PLP(139-151)-induced EAE. PLP(139-151) MAP treatment initiated after clinical signs of paralysis also altered the subsequent course of EAE; it limited developing signs of paralysis and effectively limited the severity and number of disease relapses in MAP-treated mice over a 60-day observation period. PLP(139-151) MAP therapy initiated before disease onset acts to limit the numbers of Th17 and IFN-gamma-producing cells that enter into the CNS. However, Foxp3(+) cells entered the CNS in numbers equivalent for nontreated and PLP(139-151) MAP-treated animals. The net effect of PLP(139-151) MAP treatment dramatically increases the ratio of Foxp3(+) cells to Th17 and IFN-gamma-producing cells in the CNS of PLP(139-151) MAP-treated animals.

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

    Science.gov (United States)

    Razeghifard, Reza

    2015-11-01

    A synthetic peptide was used as a scaffold to bind Zn-Chlorophyll (ZnChl) analogues through histidine ligation to study their photochemistry in the presence of different type of quinones. The Chl analogues were chlorin e6 (Ce6), chlorin e6 trimethyl ester, pyropheophorbide a, and pheophorbide a while the quinones were PPBQ, DMBQ, NPHQ, DBTQ, DCBQ and PBQ. The binding of each ZnChl analogue to the peptide was verified by native gel electrophoresis. First the photo-stability of the ZnChl analogues were tested under continuous light. The ZnCe6 and ZnCe6TM analogues showed the least stability judged by the loss of optical signal intensity at their Qy band. The photoactivity of each ZnChl analogue was measured in the presence of each of the six quinones using time-resolved EPR spectroscopy. DMBQ was found to be the most efficient electron acceptor when all four ZnChl analogues were compared. The light-induced electron transfer between the ZnChl analogues complexed with the peptide and DMBQ were also measured using time-resolved EPR spectroscopy. The ZnCe6-peptide complex exhibited the highest photoactivity. The electron transfer in the complex was faster and the photoactivity yield was higher than those values obtained for free ZnCe6 and DMBQ. The fast phase of kinetics can be attributed to intra-protein electron transfer in the complex since it was not observed in the presence of DMBQ-glutathione adduct. Unlike free ZnCe6, the ZnCe6-peptide complex was robust and demonstrated very similar photoactivity efficiency in pH values 10, 8.0 and 5.0. The electron transfer kinetics were pH dependent and appeared to be modulated by the peptide charge and possibly fold. The charge recombination rate was slowed by an order of magnitude when the pH value was changed from 10.0 to 5.0. The implications of constructing the photoactive peptide complexes in terms of artificial photosynthesis are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR

    International Nuclear Information System (INIS)

    Lindfors, Hanna E.; Koning, Peter E. de; Wouter Drijfhout, Jan; Venezia, Brigida; Ubbink, Marcellus

    2008-01-01

    Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints

  13. Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR

    Energy Technology Data Exchange (ETDEWEB)

    Lindfors, Hanna E. [Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories (Netherlands); Koning, Peter E. de; Wouter Drijfhout, Jan [Leiden University Medical Centre, Department of Immunohematology and Blood Transfusion (Netherlands); Venezia, Brigida; Ubbink, Marcellus [Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories (Netherlands)], E-mail: m.ubbink@chem.leidenuniv.nl

    2008-07-15

    Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

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

    Directory of Open Access Journals (Sweden)

    Ruodan Xu

    2015-04-01

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

  15. Effects of synthetic neural adhesion molecule mimetic peptides and related proteins on the cardiomyogenic differentiation of mouse embryonic stem cells.

    Science.gov (United States)

    Xu, Ruodan; Srinivasan, Sureshkumar Perumal; Sureshkumar, Poornima; Nembo, Erastus Nembu; Schäfer, Christoph; Semmler, Judith; Matzkies, Matthias; Albrechtsen, Morten; Hescheler, Jürgen; Nguemo, Filomain

    2015-01-01

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

  16. A novel synthetic peptide microarray assay detects Chlamydia species-specific antibodies in animal and human sera.

    Science.gov (United States)

    Sachse, Konrad; Rahman, Kh Shamsur; Schnee, Christiane; Müller, Elke; Peisker, Madlen; Schumacher, Thomas; Schubert, Evelyn; Ruettger, Anke; Kaltenboeck, Bernhard; Ehricht, Ralf

    2018-03-16

    Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

  17. Short Synthetic β-Sheet Antimicrobial Peptides for the Treatment of Multidrug-Resistant Pseudomonas aeruginosa Burn Wound Infections.

    Science.gov (United States)

    Zhong, Guansheng; Cheng, Junchi; Liang, Zhen Chang; Xu, Liang; Lou, Weiyang; Bao, Chang; Ong, Zhan Yuin; Dong, Huihui; Yang, Yi Yan; Fan, Weimin

    2017-04-01

    Pseudomonas aeruginosa is often implicated in burn wound infections; its inherent drug resistance often renders these infections extremely challenging to treat. This is further compounded by the problem of emerging drug resistance and the dearth of novel antimicrobial drug discovery in recent years. In the perennial search for effective antimicrobial compounds, the authors identify short synthetic β-sheet folding peptides, IRIKIRIK (IK8L), IRIkIrIK (IK8-2D), and irikirik (IK8D) as prime candidates owing to their high potency against Gram-negative bacteria. In this study, the peptides are first assayed against 20 clinically isolated multidrug-resistant P. aeruginosa strains in comparison with the conventional antibiotics imipenem and ceftazidime, and IK8L is demonstrated to be the most effective. IK8L also exhibits superior antibacterial killing kinetics compared to imipenem and ceftazidime. From transmission electron microscopy, confocal microscopy, and protein release analyses, IK8L shows membrane-lytic antimicrobial mechanism. Repeated use of IK8L does not induce drug resistance, while the bacteria develop resistance against the antibiotics after several times of treatment at sublethal doses. Analysis of mouse blood serum chemistry reveals that peptide does not induce systemic toxicity. The potential utility of IK8L in the in vivo treatment of P. aeruginosa-infected burn wounds is further demonstrated in a mouse model. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2014-08-01

    Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. A new class of synthetic anti-lipopolysaccharide peptides inhibits influenza A virus replication by blocking cellular attachment.

    Science.gov (United States)

    Hoffmann, Julia; Schneider, Carola; Heinbockel, Lena; Brandenburg, Klaus; Reimer, Rudolph; Gabriel, Gülsah

    2014-04-01

    Influenza A viruses are a continuous threat to human health as illustrated by the 2009 H1N1 pandemic. Since circulating influenza virus strains become increasingly resistant against currently available drugs, the development of novel antivirals is urgently needed. Here, we have evaluated a recently described new class of broad-spectrum antiviral peptides (synthetic anti-lipopolysaccharide peptides; SALPs) for their potential to inhibit influenza virus replication in vitro and in vivo. We found that particularly SALP PEP 19-2.5 shows high binding affinities for the influenza virus receptor molecule, N-Acetylneuraminic acid, leading to impaired viral attachment and cellular entry. As a result, replication of several influenza virus subtypes (H7N7, H3N2 and 2009 pandemic H1N1) was strongly reduced. Furthermore, mice co-treated with PEP 19-2.5 were protected against an otherwise 100% lethal H7N7 influenza virus infection. These findings show that SALPs exhibit antiviral activity against influenza viruses by blocking virus attachment and entry into host cells. Thus, SALPs present a new class of broad-spectrum antiviral peptides for further development for influenza virus therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. First characterization of Plasmodium vivax liver stage antigen (PvLSA) using synthetic peptides.

    Science.gov (United States)

    Goo, Youn-Kyoung; Seo, Eun-Jeong; Choi, Yeon-Kyung; Shin, Hyun-Il; Sattabongkot, Jetsumon; Ji, So-Young; Chong, Chom-Kyu; Cho, Shin-Hyung; Lee, Won-Ja; Kim, Jung-Yeon

    2014-02-12

    Plasmodium vivax is the most widespread human malaria in tropical and subtropical countries, including the Republic of Korea. Vivax malaria is characterized by hypnozoite relapse and long latency infection by the retained liver stage of P. vivax, and somewhat surprisingly, little is known of the liver stage antigens of this parasite. Here, we report for the first time the characterization of a liver stage antigen of P. vivax (PvLSA). Five peptides located inside PvLSA were synthesized, and specific anti-sera to the respective peptides were used to localize PvLSA on P. vivax parasites in human liver cells by immunofluorescence. Western blotting and enzyme-linked immunosorbent assay were performed using the five peptides and sera collected from vivax malaria patients and from normal healthy controls. PvLSA was localized on P. vivax parasites in human liver cells. Vivax malaria-infected patients were detected using the five peptides by western blotting. Furthermore, the peptides reacted with the sera of vivax malaria patients. These results suggest that PvLSA may function during the liver stage of P. vivax.

  1. Silicon-Containing Amino Acids: Synthetic Aspects, Conformational Studies, and Applications to Bioactive Peptides.

    Science.gov (United States)

    Rémond, Emmanuelle; Martin, Charlotte; Martinez, Jean; Cavelier, Florine

    2016-10-12

    Unnatural α-amino acids form a family of essential molecules used for, among other applications, the synthesis of modified peptides, to improve resistance to proteolytic enzyme degradation, and to modulate physico- and biochemical properties of bioactive peptides as well as chiral inducers in asymmetric synthesis. Among them, silicon-containing unnatural amino acids are becoming an interesting new class of building blocks. The replacement of carbon atoms in bioactive substances with silicon is becoming increasingly popular. Peptides containing silyl amino acids hold great promise for maintaining or reinforcing the biological activity of active compounds, while they simultaneously enhance their resistance to enzyme degradation. In addition, the lipophilicity of the silicon atom facilitates their membrane crossing and their bioavailability. Nowadays, the interest of the pharmaceutical industry in peptide- and protein-based therapies is increasing. In this respect, silicon-containing amino acids and peptides are likely to be a significant part of future innovations in this area, and more generally in the area of biomolecules. In this process, commercial availability of silicon-containing amino acids is necessary: new syntheses have been developed, and work in this area is ongoing. This review aims to be a comprehensive and general summary of the different methods used to prepare silicon-containing amino acids and their implications on conformational structures and biological applications when they are incorporated into bioactive molecules.

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

    Directory of Open Access Journals (Sweden)

    Robert H. Notter

    2016-01-01

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

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

    DEFF Research Database (Denmark)

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

    1998-01-01

    Native kinetoplastid membrane protein-11 (KMP-11), purified from crude extracts of Leishmania donovani parasites, activates T cells from individuals who have recovered from visceral leishmaniasis. In this work we used three 38-mer peptides spanning the amino acid sequence of the L. donovani KMP-11...... as solid-phase ligands in enzyme-linked immunosorbent assays (ELISAs) and as stimulating antigens in lymphoproliferative assays in order to evaluate humoral and cellular immune responses to well-defined sequences of the protein. Antibody reactivity against the three peptides was measured in plasma from 63......-11 peptides was detected in plasma from Sudanese patients suffering from Leishmania major infections and in plasma from Sudanese and Danish patients infected with Plasmodium falciparum. In lymphoproliferative assays, 10 of 17 PBMC isolates from donors previously infected with L. donovani showed...

  4. High specific selectivity and Membrane-Active Mechanism of the synthetic centrosymmetric α-helical peptides with Gly-Gly pairs.

    Science.gov (United States)

    Wang, Jiajun; Chou, Shuli; Xu, Lin; Zhu, Xin; Dong, Na; Shan, Anshan; Chen, Zhihui

    2015-11-04

    We used a template-assisted approach to develop synthetic antimicrobial peptides, which differ from naturally occurring antimicrobial peptides that can compromise host natural defenses. Previous researches have demonstrated that symmetrical distribution patterns of amino acids contribute to the antimicrobial activity of natural peptides. However, there is little research describing such design ideas for synthetic α-helical peptides. Therefore, here, we established a centrosymmetric α-helical sequence template (y + hhh + y)n (h, hydrophobic amino acid; +, cationic amino acid; y, Gly or hydrophobic amino acid), which contributed to amphipathicity, and a series of centrosymmetric peptides was designed with pairs of small amino acids (Ala and Gly), which were utilized to modulate the biological activity. The centrosymmetric peptides with 3 repeat units exhibited strong antimicrobial activity; in particular, the Gly-rich centrosymmetric peptide GG3 showed stronger selectivity for gram-negative bacteria without hemolysis. Furthermore, beyond our expectation, fluorescence spectroscopy and electron microscopy analyses indicated that the GG3, which possessed poor α-helix conformation, dramatically exhibited marked membrane destruction via inducing bacterial membrane permeabilization, pore formation and disruption, even bound DNA to further exert antimicrobial activity. Collectively, the Gly-rich centrosymmetric peptide GG3 was an ideal candidate for commercialization as a clinical therapeutic to treat gram-negative bacterial infections.

  5. Short synthetic peptides derived from viral proteins compete with HIV gp120 for the binding to CD4 receptors.

    Science.gov (United States)

    Chersi, A; Pugliese, O; Federico, A; Viora, M

    2000-01-01

    In the complex mechanism of adhesion, internalization, and infection of cells by human immunodeficiency virus (HIV) viral particles, a determinant role is played by the viral envelope glycoprotein gp120, which binds to CD4 receptors of T cells and monocytes. We tested the ability of a panel of 7- to 12-residue synthetic peptides, selected from the region 414-434 of the HIV-1 gp120, to inhibit the binding of the viral protein to CD4 receptors of cultured human lymphoid cells. The assay was based on the observation that the binding of gp120 to the receptors interferes with the binding of a specific anti-CD4 monoclonal antibody, as a result of the masking of the antibody epitope; thus, we tested whether preincubation of cells with the peptides before gp120 addition might restore the recognition of the CD4 molecule by the antibody. High expression of CD4 receptors was thus assumed as indication that the binding of the viral protein had been inhibited. Maximum activity was displayed by a 9-residue peptide located near the amino terminal end of the 414-434 fragment. In addition, several fragments deduced from other viral proteins, possessing partial amino acid sequence homology with the HIV gp120 fragment, exhibited a similar type of interaction with the CD4 receptor. All active peptides contain the Cys residue (position 423 of gp120). This residue is essential, although not sufficient, for inhibiting gp120 binding, as few other amino acid residues within the fragment play a complementary role in increasing or decreasing the inhibitory ability.

  6. Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

    Directory of Open Access Journals (Sweden)

    Graham M West

    Full Text Available Activation of the glucagon-like peptide-1 receptor (GLP-1R in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM. Like other class B G protein-coupled receptors (GPCRs, the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

  7. Combinatorial synthetic peptide vaccine strategy protects against hypervirulent CovR/S mutant streptococci

    DEFF Research Database (Denmark)

    Pandey, Manisha; Mortensen, Rasmus; Calcutt, Ainslie

    2016-01-01

    ), and it would be to the organism's advantage if the host did not induce a strong Ab response against it. However, S2 conjugated to diphtheria toxoid is highly immunogenic and induces Abs that recognize and neutralize SpyCEP. Hence, we describe a two-component peptide vaccine that induces Abs (anti-S2...

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

    Science.gov (United States)

    Pontrello, Jason K.

    2015-01-01

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

  9. CXCR4-derived synthetic peptides inducing anti-HIV-1 antibodies.

    Science.gov (United States)

    Hashimoto, Chie; Nomura, Wataru; Narumi, Tetsuo; Fujino, Masayuki; Nakahara, Toru; Yamamoto, Naoki; Murakami, Tsutomu; Tamamura, Hirokazu

    2013-11-15

    Despite almost 30 years since the identification of the human immunodeficiency virus type I (HIV-1), development of effective AIDS vaccines has been hindered by the high mutability of HIV-1. The HIV-1 co-receptors CCR5 and CXCR4 are genetically stable, but viral proteins may mutate rapidly during the course of infection. CXCR4 is a seven transmembrane G protein-coupled receptor, possessing an N-terminal region (NT) and three extracellular loops (ECL1-3). Previous studies have shown that the CXCR4-ED-derived peptides inhibit the entry of HIV-1 by interacting with gp120, an HIV-1 envelope glycoprotein. In the present study, antigenicity of CXCR4-derived peptides has been investigated and the anti-HIV-1 effects of induced antisera have been assessed. It was found that CXCR4-ED-derived antigen molecules immunize mice, showing that the linear peptides have higher antigenicity than the cyclic peptides. The L1- and L2-induced antisera inhibited the HIV-1 entry significantly, while anti-N1 antibodies have no inhibitory activity. This study produced promising examples for the design of AIDS vaccines which target the human protein and can overcome mutability of HIV-1. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Solution structure of the first SH3 domain of human vinexin and its interaction with vinculin peptides

    International Nuclear Information System (INIS)

    Zhang, Jiahai; Li, Xiang; Yao, Bo; Shen, Weiqun; Sun, Hongbin; Xu, Chao; Wu, Jihui; Shi, Yunyu

    2007-01-01

    Solution structure of the first Src homology (SH) 3 domain of human vinexin (V S H3 1 ) was determined using nuclear magnetic resonance (NMR) method and revealed that it was a canonical SH3 domain, which has a typical β-β-β-β-α-β fold. Using chemical shift perturbation and surface plasmon resonance experiments, we studied the binding properties of the SH3 domain with two different peptides from vinculin hinge regions: P856 and P868. The observations illustrated slightly different affinities of the two peptides binding to V S H3 1 . The interaction between P868 and V S H3 1 belonged to intermediate exchange with a modest binding affinity, while the interaction between P856 and V S H3 1 had a low binding affinity. The structure and ligand-binding interface of V S H3 1 provide a structural basis for the further functional study of this important molecule

  11. Oil-based formulation as a sustained-released injection for a novel synthetic peptide.

    Science.gov (United States)

    Zhang, Guiying; Li, Jinglai; Wang, Tao; Gao, Lijun; Quan, Dongqin

    2015-01-01

    In this study, sustained-release of GnRH antagonist peptide LXT-101 was realized through oil formulation, and their releasing characteristics in vitro and in vivo were investigated. In this formulation, the static interaction between cationic charged peptide LXT-101 and the negative charged phospholipid led to the formation of the phospholipid-peptide complex, by which LXT-101 was completely dissolved in oils. This formulation was prepared by mixing an aqueous solution of LXT-101 and empty SUV (small unilamellar liposomes) containing EPC (phosphatidylcholine) and DPPG (1, 2-dipalmitog-sn-glycero-3- phosphoglycerol) at an appropriate ratio, the mixture was subsequently lyophilized, and the resultant was dissolved in the oil to form a clear oily solution containing solubilized peptide LXT-101. With atomic force microscopy combined with Langmuir-Blodgett technology, the morphology of the particles in the oily solution were examined to be oval-shaped and the mean particle size was 150 nm in diameter. In pure water at 37°C, about 70~90 % of LXT-101 was released slowly from the oily formulation over 7 days. An effective sustained suppression of testosterone in beagle dogs could be achieved over a period of seven days with this LXT-101 oily formulation, by i.m. at a dose of 0.2 mg/kg (2 mg/ml). This formulation dramatically improved the bioactivity of LXT-101 compared to its aqueous solution. It was also found that when the concentration of peptide LXT-101 was up to or over 10 mg/ml in aqueous solution, there was no significant difference between the oily formulation and aqueous solution. This fact meant that LXT-101 itself could conduct sustained release in vivo by self-assembly of nanofibers.

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

    Directory of Open Access Journals (Sweden)

    Rina Ogawa

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

  13. Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control

    Directory of Open Access Journals (Sweden)

    Mareike Kessenbrock

    2017-09-01

    Full Text Available Ethylene signaling is decisive for many plant developmental processes. Among these, control of senescence, abscission and fruit ripening are of fundamental relevance for global agriculture. Consequently, detailed knowledge of the signaling network along with the molecular processes of signal perception and transfer are expected to have high impact on future food production and agriculture. Recent advances in ethylene research have demonstrated that signaling of the plant hormone critically depends on the interaction of the ethylene receptor family with the NRAMP-like membrane protein ETHYLENE INSENSITIVE 2 (EIN2 at the ER membrane, phosphorylation-dependent proteolytic processing of ER-localized EIN2 and subsequent translocation of the cleaved EIN2 C-terminal polypeptide (EIN2-CEND to the nucleus. EIN2 nuclear transport, but also interaction with the receptors sensing the ethylene signal, both, depend on a nuclear localization signal (NLS located at the EIN2 C-terminus. Loss of the tight interaction between receptors and EIN2 affects ethylene signaling and impairs plant ethylene responses. Synthetic peptides derived from the NLS sequence interfere with the EIN2–receptor interaction and have utility in controlling plant ethylene responses such as ripening. Here, we report that a synthetic peptide (NOP-1 corresponding to the NLS motif of Arabidopsis EIN2 (aa 1262–1269 efficiently binds to tomato ethylene receptors LeETR4 and NR and delays ripening in the post-harvest phase when applied to the surface of sampled green fruits pre-harvest. In particular, degradation of chlorophylls was delayed by several days, as monitored by optical sensors and confirmed by analytical methods. Similarly, accumulation of β-carotene and lycopene in the fruit pulp after NOP-1 application was delayed, without having impact on the total pigment concentration in the completely ripe fruits. Likewise, the peptide had no negative effects on fruit quality. Our molecular

  14. Solution structure of the human BTK SH3 domain complexed with a proline-rich peptide from p120cbl

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, S.-R.; Lou, Y.-C.; Pai, M.-T.; Jain, Moti L.; Cheng, J.-W. [National Tsing Hua University, Division of Structural Biology and Biomedical Science, Department of Life Science (China)

    2000-04-15

    X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B cell differentiation which incapacitates antibody production in XLA patients leading to, sometimes lethal, bacterial infections. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in one patient family. To understand the role of BTK in B cell development, we have determined the solution structure of BTK SH3 domain complexed with a proline-rich peptide from the protein product of c-cbl protooncogene (p120{sup cbl}). Like other SH3 domains, BTK SH3 domain consists of five {beta}-strands packed in two {beta}-sheets forming a {beta}-barrel-like structure. The rmsd calculated from the averaged coordinates for the BTK SH3 domain residues 218-271 and the p120{sup cbl} peptide residues 6-12 of the complex was 0.87 A ({+-}0.16 A) for the backbone heavy atoms (N, C, and C{sub {alpha}}) and 1.64 A ({+-}0.16 A) for all heavy atoms. Based on chemical shift changes and inter-molecular NOEs, we have found that the residues located in the RT loop, n-Src loop and helix-like loop between {beta}4 and {beta}5 of BTK SH3 domain are involved in ligand binding. We have also determined that the proline-rich peptide from p120{sup cbl} binds to BTK SH3 domain in a class I orientation. These results correlate well with our earlier observation that the truncated BTK SH3 domain (deletion of {beta}4, {beta}5 and the helix-like loop) exhibits weaker affinity for the p120{sup cbl} peptide. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context and hence the weaker binding. These results delineate the importance of the C-terminus in the binding of SH3 domains and also indicate that improper folding and the altered binding behavior of mutant BTK SH3 domain likely lead to XLA.

  15. 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...... peptides known as cell-penetrating peptides (CPPs) is attracting wide attention for a variety of biologically active molecules. CPP-mediated delivery is typically based on the covalent conjugation of the (therapeutic) cargo to CPPs, and is particularly relevant for the delivery of noncharged RNA...

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

    Directory of Open Access Journals (Sweden)

    Nataly De Jesús Huertas Méndez

    2017-03-01

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

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

    Science.gov (United States)

    Huertas Méndez, Nataly De Jesús; Vargas Casanova, Yerly; Gómez Chimbi, Anyelith Katherine; Hernández, Edith; Leal Castro, Aura Lucia; Melo Diaz, Javier Mauricio; Rivera Monroy, Zuly Jenny; García Castañeda, Javier Eduardo

    2017-03-12

    Linear, dimeric, tetrameric, and cyclic peptides derived from lactoferricin B-containing non-natural amino acids and the RWQWR motif were synthesized, purified, and characterized using RP-HPLC, MALDI-TOF mass spectrometry, and circular dichroism. The antibacterial activity of peptides against Escherichia coli ATCC 11775, Stenotrophomonas maltophilia ATCC 13636, and Salmonella enteritidis ATCC 13076 was evaluated. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The synthetic bovine lactoferricin exhibited antibacterial activity against E. coli ATCC 11775 and S. enteritidis ATCC 13076. The dimeric peptide (RRWQWR)₂K-Ahx exhibited the highest antibacterial activity against the tested bacterial strain. The monomeric, cyclic, tetrameric, and palindromic peptides containing the RWQWR motif exhibited high and specific activity against E. coli ATCC 11775. The results suggest that short peptides derived from lactoferricin B could be considered as potential candidates for the development of antibacterial agents against infections caused by E. coli .

  18. Induction of tolerance to one determinant on a synthetic peptide does not affect the response to a second linked determinant. Implications for the mechanism of neonatal tolerance induction

    Science.gov (United States)

    1986-01-01

    To investigate the mechanism underlying neonatal T cell tolerance, we used synthetic peptides to induce tolerance. We found that induction of tolerance to one determinant on a 23-amino acid peptide did not affect the response to an adjacent determinant on the same peptide. There was no evidence of suppression of the response to the second determinant. Furthermore, even small peptides near the minimal size for a determinant, which would be very unlikely to possess a suppressor T cell-inducing determinant as well as a proliferative T cell-inducing determinant, could induce tolerance. These studies provide in vivo experiments supporting clonal inactivation as the mechanism of neonatal tolerance to immunogenic peptides. PMID:2425038

  19. Synthetic peptide antigens derived from long-chain alpha-neurotoxins: Immunogenicity effect against elapid venoms.

    Science.gov (United States)

    de la Rosa, Guillermo; Pastor, Nina; Alagón, Alejandro; Corzo, Gerardo

    2017-02-01

    Three-finger toxins (3FTXs), especially α-neurotoxins, are the most poorly neutralized elapid snake toxins by current antivenoms. In this work, the conserved structural similarity and motif arrangements of long-chain α-neurotoxins led us to design peptides with consensus sequences. Eight long-chain α-neurotoxins (also known as Type II) were used to generate a consensus sequence from which two peptides were chemically synthesized, LCP1 and LCP2. Rabbit sera raised against them were able to generate partially-neutralizing antibodies, which delayed mice mortality in neutralization assays against Naja haje, Dendrospis polylepis and Ophiophagus hannah venoms. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. The role of polar and facial amphipathic character in determining lipopolysaccharide-binding properties in synthetic cationic peptides.

    Science.gov (United States)

    David, S A; Awasthi, S K; Balaram, P

    2000-01-01

    Two series of peptides, designated K and NK were synthesized and tested for lipid A binding and neutralizing properties. K2, which has an 11-residue amphiphilic core, and a branched N-terminus bearing two branched lysinyl residues does not bind lipid A, while NK2, also with an 11-residue amphiphilic core comprised entirely of non-ionizable residues, and a similarly branched, cationic N-terminus, binds lipid A very weakly. Both peptides do not inhibit lipopolysaccharide (LPS) activity in the Limulus assay, nor do they inhibit LPS-induced TNF-alpha and NO production in J774 cells. These results are entirely unlike a homologous peptide with an exclusively hydrophobic core whose LPS-binding and neutralizing properties are very similar to that of polymyxin B [David SA, Awasthi SK, Wiese A et al. Characterization of the interactions of a polycationic, amphiphilic, terminally branched oligopeptide with lipid A and lipopolysaccharide from the deep rough mutant of Salmonella minnesota. J Endotoxin Res 1996; 3: 369-379]. These data suggest that a clear segregation of charged and apolar domains is crucial in molecules designed for purposes of LPS sequestration and that head-tail (polar) orientation of the cationic/hydrophobic regions is preferable to molecules with mixed or facial cationic/amphipathic character.

  1. Efficacy of Synthetic Peptide Corresponding to the ACTH-Like Sequence of Human Immunoglobulin G1 in Experimental Autoimmune Encephalomyelitis.

    Science.gov (United States)

    Turobov, Valery I; Danilkovich, Alexey V; Shevelev, Alexei B; Biryukova, Yulia K; Pozdniakova, Natalia V; Azev, Viatcheslav N; Murashev, Arkady N; Lipkin, Valery M; Udovichenko, Igor P

    2018-01-01

    Peptide immunocortin sequence corresponds to the amino acid residues 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain. Since immunocortin was shown previously to inhibit phagocytosis in peritoneal macrophages and ConA-induced T-lymphocytes proliferation in culture, we suggested that immunocortin administering may be of use for patients with self-immune syndrome. Immunocortin in concentration 10 μM inhibited proliferation of both antigen (myelin)-induced and ConA-induced LN lymphocytes isolated from the lymph nodes of Dark Agouti (DA) rats immunized with chorda shear. The biological trials of the synthetic immunocortin were carried out on the DA rats with induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. These in vivo experiments have shown that intraperitoneal injections of immunocortin in a daily dosage 100 μg per animal reduced symptoms of EAE in DA rats.

  2. Efficacy of Synthetic Peptide Corresponding to the ACTH-Like Sequence of Human Immunoglobulin G1 in Experimental Autoimmune Encephalomyelitis

    Directory of Open Access Journals (Sweden)

    Valery I. Turobov

    2018-02-01

    Full Text Available Peptide immunocortin sequence corresponds to the amino acid residues 11–20 of the variable part of human immunoglobulin G1 (IgG1 heavy chain. Since immunocortin was shown previously to inhibit phagocytosis in peritoneal macrophages and ConA-induced T-lymphocytes proliferation in culture, we suggested that immunocortin administering may be of use for patients with self-immune syndrome. Immunocortin in concentration 10 μM inhibited proliferation of both antigen (myelin-induced and ConA-induced LN lymphocytes isolated from the lymph nodes of Dark Agouti (DA rats immunized with chorda shear. The biological trials of the synthetic immunocortin were carried out on the DA rats with induced experimental autoimmune encephalomyelitis (EAE, an animal model of multiple sclerosis. These in vivo experiments have shown that intraperitoneal injections of immunocortin in a daily dosage 100 μg per animal reduced symptoms of EAE in DA rats.

  3. The Effect of Osteopontin and an Osteopontin-Derived Synthetic Peptide Coating on Osseointegration of Implants in a Canine Model.

    Science.gov (United States)

    Fiorellini, Joseph P; Glindmann, Sven; Salcedo, Jairo; Weber, Hans-Peter; Park, Chang-Joo; Sarmiento, Hector L

    Osteopontin (OPN) and an OPN-derived synthetic peptide, OC-1016, have demonstrated their potential to enhance osseointegration in vitro. The purpose of this study was to evaluate bone-to-implant contact (BIC) and surrounding bone density (BD) of implants coated with either recombinant human OPN (rhOPN) or OC-1016 as compared with noncoated titanium plasma sprayed (TPS) surface in a canine model. Histomorphometric analysis revealed that at 4 weeks, %BIC and %BD of coated implants were significantly higher than those of noncoated TPS implants. At 12 weeks, %BIC of coated implants was also significantly higher than that of noncoated implants; however, there was no statistically significant difference in %BD. The rhOPN and OC-1016 were concluded to be capable of significantly accelerating the early stage of osseointegration and bone healing around implants.

  4. Peptides modeled after the alpha-domain of metallothionein induce neurite outgrowth and promote survival of cerebellar granule neurons

    DEFF Research Database (Denmark)

    Asmussen, Johanne Wirenfeldt; Ambjørn, Malene; Bock, Elisabeth

    2009-01-01

    Metallothionein (MT) is a metal-binding protein capable of preventing oxidative stress and apoptotic cell death in the central nervous system of mammals, and hence is of putative therapeutic value in the treatment of neurodegenerative disorders. Recently, we demonstrated that a peptide modeled...... amino acids, as potent stimulators of neuronal differentiation and survival of primary neurons. In addition, we show that a peptide derived from the N-terminus of the MT beta-domain, EmtinBn, promotes neuronal survival. The neuritogenic and survival promoting effects of EmtinAc, similar to MT and Emtin...

  5. Synthetic study on derivatives of dimeric peptide from human IgG1 hinge region

    Czech Academy of Sciences Publication Activity Database

    Niederhafner, Petr; Gut, Vladimír; Hulačová, Hana; Maloň, Petr; Slaninová, Jiřina; Hlaváček, Jan

    2006-01-01

    Roč. 12, Supplement (2006), s. 110 ISSN 1075-2617. [European Peptide Symposium /29./. 03.09.2006-08.09.2006, Gdansk] R&D Projects: GA ČR(CZ) GA203/03/1362; GA AV ČR(CZ) IAA400550614 Institutional research plan: CEZ:AV0Z40550506 Keywords : hinge region * PEG syntheses * vaccination study Subject RIV: CC - Organic Chemistry

  6. Immunologic and functional characterization of anti-HLA-DR rabbit antibodies induced by synthetic peptides.

    Science.gov (United States)

    Chersi, A; Schulz, G; Houghten, R A

    1984-10-01

    Three peptides selected from the amino acid sequence of the alpha- and beta-chains of DR2 histocompatibility antigens were chemically synthesized and coupled to carrier proteins to be used as immunogens in rabbits. This immunization resulted in the production of specific antibodies that readily recognized the antigen. However, only one of the four antibody preparations, antibody 6148, elicited by a short peptide from the beta-chain (residues 61-73), reacts with native membrane glycoproteins as well as intact human lymphoblastoid cells in enzyme-linked immunosorbant assays. This antibody was found to react also with membrane glycoproteins solubilized by nonionic detergents from cells bearing a different HLA-DR specificity: therefore it is likely that the peptide responsible for eliciting antibody 6148 represents a common framework determinant of DR alloantigens that is accessible on the surface of lymphoblastoid cells. The ability of antibody 6148 to bind to intact cells was confirmed by indirect immunofluorescence and by fluorescein-activated cell sorter analysis. This antibody is also capable of mediating antibody-dependent cellular cytotoxicity as determined by a 51Cr-release assay.

  7. Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

    Directory of Open Access Journals (Sweden)

    Pero Stephanie C

    2007-09-01

    Full Text Available Abstract Background Human growth factor receptor bound protein 7 (Grb7 is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines. Results As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding. Conclusion Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of

  8. A synthetic, xeno-free peptide surface for expansion and directed differentiation of human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Sha Jin

    Full Text Available Human induced pluripotent stem cells have the potential to become an unlimited cell source for cell replacement therapy. The realization of this potential, however, depends on the availability of culture methods that are robust, scalable, and use chemically defined materials. Despite significant advances in hiPSC technologies, the expansion of hiPSCs relies upon the use of animal-derived extracellular matrix extracts, such as Matrigel, which raises safety concerns over the use of these products. In this work, we investigated the feasibility of expanding and differentiating hiPSCs on a chemically defined, xeno-free synthetic peptide substrate, i.e. Corning Synthemax(® Surface. We demonstrated that the Synthemax Surface supports the attachment, spreading, and proliferation of hiPSCs, as well as hiPSCs' lineage-specific differentiation. hiPSCs colonies grown on Synthemax Surfaces exhibit less spread and more compact morphology compared to cells grown on Matrigel™. The cytoskeleton characterization of hiPSCs grown on the Synthemax Surface revealed formation of denser actin filaments in the cell-cell interface. The down-regulation of vinculin and up-regulation of zyxin expression were also observed in hiPSCs grown on the Synthemax Surface. Further examination of cell-ECM interaction revealed that hiPSCs grown on the Synthemax Surface primarily utilize α(vβ(5 integrins to mediate attachment to the substrate, whereas multiple integrins are involved in cell attachment to Matrigel. Finally, hiPSCs can be maintained undifferentiated on the Synthemax Surface for more than ten passages. These studies provide a novel approach for expansion of hiPSCs using synthetic peptide engineered surface as a substrate to avoid a potential risk of contamination and lot-to-lot variability with animal derived materials.

  9. Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides.

    Science.gov (United States)

    Pan, Xichun; Li, Bin; Kuang, Mei; Liu, Xin; Cen, Yanyan; Qin, Rongxin; Ding, Guofu; Zheng, Jiang; Zhou, Hong

    2016-02-22

    Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

  10. Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides

    Directory of Open Access Journals (Sweden)

    Xichun Pan

    2016-02-01

    Full Text Available Toll-like receptor (TLR 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN. Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS- and lipopeptide (PAM3CSK4-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

  11. Tracking the Interplay between Bound Peptide and the Lid Domain of DnaK, Using Molecular Dynamics

    Directory of Open Access Journals (Sweden)

    Yossi Tsfadia

    2013-06-01

    Full Text Available Hsp70 chaperones consist of two functional domains: the 44 kDa Nucleotide Binding Domain (NBD, that binds and hydrolyses ATP, and the 26 kDa Substrate Binding Domain (SBD, which binds unfolded proteins and reactivates them, utilizing energy obtained from nucleotide hydrolysis. The structure of the SBD of the bacterial Hsp70, DnaK, consists of two sub-domains: A β-sandwich part containing the hydrophobic cavity to which the hepta-peptide NRLLLTG (NR is bound, and a segment made of 5 α-helices, called the “lid” that caps the top of the β-sandwich domain. In the present study we used the Escherichia coli Hsp70, DnaK, as a model for Hsp70 proteins, focusing on its SBD domain, examining the changes in the lid conformation. We deliberately decoupled the NBD from the SBD, limiting the study to the structure of the SBD section, with an emphasis on the interaction between the charges of the peptide with the residues located in the lid. Molecular dynamics simulations of the complex revealed significant mobility within the lid structure; as the structure was released from the forces operating during the crystallization process, the two terminal helices established a contact with the positive charge at the tip of the peptide. This contact is manifested only in the presence of electrostatic attraction. The observed internal motions within the lid provide a molecular role for the function of this sub-domain during the reaction cycle of Hsp 70 chaperones.

  12. Chemoprotective effects of a recombinant protein from Pyropia yezoensis and synthetic peptide against acetaminophen-induced Chang liver cell death.

    Science.gov (United States)

    Choi, Youn Hee; Kim, Eun-Young; Mikami, Koji; Nam, Taek Jeong

    2015-08-01

    In the present study, the chemoprotective effects of recombinant Pyropia yezoensis (P. yezoensis) protein 1 (PYP1) were examined in acetaminophen (APAP)-treated Chang liver cells. The analysis of P. yezoensis revealed the presence of both mature and immature variants of PYP1. PYP1s, designated as PYP1 (15 kDa), PYP1-AC (12 kDa) and PYP1-B (5 kDa), were successfully expressed in Escherichia coli, and their chemoprotective effects were then examined. In addition, a peptide of 11 residues (ALEGGKSSGGG), which is a common sequence at the N-terminus all of the PYP1s, was synthesized and examined. The effects of treatment with PYP1s and the synthetic peptide (SP) on cell proliferation were determined by MTS assay. Our results clearly demonstrated that treatment with all the PYP1s and SP significantly promoted the proliferation of Chang liver cells, protecting them against APAP. Thus, we concluded that recombinant PYP1s exert protective effects against injury to Chang liver cells.

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

    NARCIS (Netherlands)

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

    1990-01-01

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

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jorge Berlanga-Acosta

    2017-02-01

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

  16. Cloning and expression of synthetic genes encoding angiotensin-I converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium pseudocatenulatum.

    Science.gov (United States)

    Losurdo, Luca; Quintieri, Laura; Caputo, Leonardo; Gallerani, Raffaele; Mayo, Baltasar; De Leo, Francesca

    2013-03-01

    A wide range of biopeptides potentially able to lower blood pressure through inhibition of the angiotensin-I converting enzyme (ACE) is produced in fermented foods by proteolytic starter cultures. This work applies a procedure based on recombinant DNA technologies for the synthesis and expression of three ACE-inhibitory peptides using a probiotic cell factory. ACE-inhibitory genes and their pro-active precursors were designed, synthesized by PCR, and cloned in Escherichia coli; after which, they were cloned into the pAM1 E. coli-bifidobacteria shuttle vector. After E. coli transformation, constructs carrying the six recombinant clones were electrotransferred into the Bifidobacterium pseudocatenulatum M115 probiotic strain. Interestingly, five of the six constructs proved to be stable. Their expression was confirmed by reverse transcription PCR. Furthermore, transformed strains displayed ACE-inhibitory activity linearly correlated to increasing amounts of cell-free cellular lysates. In particular, 50 μg of lysates from constructs pAM1-Pro-BP3 and pAM1-BP2 showed a 50% higher ACE-inhibitory activity than that of the controls. As a comparison, addition of 50 ng of Pro-BP1 and Pro-BP3 synthetic peptides to 50 μg of cell-free extracts of B. pseudocatenulatum M115 wild-type strain showed an average of 67% of ACE inhibition; this allowed estimating the amount of the peptides produced by the transformants. Engineering of bifidobacteria for the production of biopeptides is envisioned as a promising cell factory model system. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  17. Β-defensin in Nile tilapia (Oreochromis niloticus): Sequence, tissue expression, and anti-bacterial activity of synthetic peptides.

    Science.gov (United States)

    Dong, Jun-Jian; Wu, Fang; Ye, Xing; Sun, Cheng-Fei; Tian, Yuan-Yuan; Lu, Mai-Xin; Zhang, Rui; Chen, Zhi-Hang

    2015-07-15

    Beta-defensins (β-defensins) are small cationic amphiphilic peptides that are widely distributed in plants, insects, and vertebrates, and are important for their antimicrobial properties. In this study, the β-defensin (Onβ-defensin) gene of the Nile tilapia (Oreochromis niloticus) was cloned from spleen tissue. Onβ-defensin has a genomic DNA sequence of 674 bp and produces a cDNA of 454 bp. Sequence alignments showed that Onβ-defensin contains three exons and two introns. Sequence analysis of the cDNA identified an open reading frame of 201 bp, encoding 66 amino acids. Bioinformatic analysis showed that Onβ-defensin encodes a cytoplasmic protein molecule containing a signal peptide. The deduced amino acid sequence of this peptide contains six conserved cysteine residues and two conserved glycine residues, and shows 81.82% and 78.33% sequence similarities with β-defensin-1 of fugu (Takifugu rubripes) and rainbow trout (Oncorhynchus mykiss), respectively. Real-time quantitative PCR showed that the level of Onβ-defensin expression was highest in the skin (307.1-fold), followed by the spleen (77.3-fold), kidney (17.8-fold), and muscle (16.5-fold) compared to controls. By contrast, low levels of expression were found in the liver, heart, intestine, stomach, and gill (tilapia with Streptococcus agalactiae (group B streptococcus [GBS] strain) resulted in a significantly upregulated expression of Onβ-defensin in the skin, muscle, kidney, and gill. In vitro antimicrobial experiments showed that a synthetic Onβ-defensin polypeptide had a certain degree of inhibitory effect on the growth of Escherichia coli DH5α and S. agalactiae. The results indicate that Onβ-defensin plays a role in immune responses that suppress or kill pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-07-01

    Peptide vaccines are capable of eliciting immune responses targeting tumor-associated antigens such as the Wilms' Tumor 1 (WT1) antigen, often overexpressed in myeloid malignancies. Here, we assessed the safety, tolerability, and immunogenicity of a polyvalent WT1 peptide vaccine. Individuals with WT1-positive acute myeloid leukemia (AML) in first (CR1) or second (CR2) remission or with higher-risk myelodysplastic syndrome (MDS) following at least 1 prior line of therapy were vaccinated with a mixture of peptides derived from the WT1 protein, with sargramostim injections before vaccination to amplify immunogenicity. Six vaccinations were delivered biweekly, continuing then monthly until patients received 12 vaccinations or showed disease relapse or progression. Therapeutic efficacy was evaluated by progression-free and overall survival. Immune responses were evaluated by delayed-type hypersensitivity testing and T-cell IFNγ ELISPOT at specified intervals. In 16 patients who received at least one vaccination, 10 completed the planned course of six vaccinations and six continued for up to six additional monthly vaccinations. Vaccinations were well tolerated, with no patients discontinuing due to toxicity. One of two patients with high-risk MDS experienced a prolonged decrease in transfusion dependence. Two of 14 AML patients demonstrated relapse-free survival >1 year. Both patients were in CR2 at time of vaccination, with duration of their remission exceeding duration of their first remission, suggesting a potential benefit. Our WT1 vaccine was well-tolerated. The clinical benefit that we observed in several patients suggests engagement of a protective immune response, indicating a need for further trials. © 2015 Wiley Periodicals, Inc.

  19. Synthetic antimicrobial peptides of the halictines family disturb the membrane integrity of Candida cells

    Czech Academy of Sciences Publication Activity Database

    Kodedová, Marie; Sychrová, Hana

    2017-01-01

    Roč. 1859, č. 10 (2017), s. 1851-1858 ISSN 0005-2736 R&D Projects: GA TA ČR(CZ) TA04010638; GA ČR(CZ) GA16-03398S; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : antimicrobial peptide * Candida * diS-C3(3) assay * membrane potential * membrane lipids * halictine Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.498, year: 2016

  20. Synthetic aperture radar for maritime domain awareness: Ship detection in a South African context

    CSIR Research Space (South Africa)

    Schwegmann, CP

    2014-10-01

    Full Text Available Maritime Domain Awareness is an initiative started to help each sea-bordering country improve its understanding of its Exclusive Economic Zone. A country that improves its Maritime Domain Awareness ensures that activities such as piracy and Illegal...

  1. Expression and purification of an immunogenic dengue virus epitope using a synthetic consensus sequence of envelope domain III and Saccharomyces cerevisiae.

    Science.gov (United States)

    Nguyen, Ngoc-Luong; Kim, Jung-Mi; Park, Jin-Ah; Park, Seung-Moon; Jang, Yong-Suk; Yang, Moon-Sik; Kim, Dae-Hyuk

    2013-04-01

    A synthetic consensus gene was designed based on residues of the amino acid sequences of dengue envelope domain III (scEDIII) from all four serotypes, and codon optimization for expression was conducted using baker's yeast, Saccharomyces cerevisiae. The synthetic gene was cloned into a yeast episomal expression vector, pYEGPD-TER, which was designed to direct cloned gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator. PCR and back-transformation into Escherichia coli confirmed the presence of the scEDIII gene-containing plasmid in the transformants. Northern blot analysis showed the presence of the scEDIII-specific transcript. Western blot analysis indicated that expressed scEDIII, with mobility similar to purified EDIII from E. coli, was successfully secreted into the culture media. Quantitative ELISA revealed that the recombinant scEDIII comprised approximately 0.1-0.6% of cell-free extract. In addition, 0.1-0.6 mg of scEDIII protein per liter of culture filtrate was detected on day 1 and peaked on day 3 after cultivation. The secreted scEDIII protein can be purified to ≥90% purity with 85% recovery using a simple ion-exchange FPLC followed by molecular weight cut-off. Upon administration of the purified protein to mice, mouse sera contained antibodies that were specific to all four serotypes of dengue virus. Moreover, a balanced immune response against all four serotypes was observed, suggesting that it may be possible to develop an effective tetravalent dengue vaccine using S. cerevisiae. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Frans J. Walther

    2014-05-01

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

  3. A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain

    Directory of Open Access Journals (Sweden)

    Nicolas Panel

    2017-09-01

    Full Text Available PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or “PB/LIE” free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo. The overall performance of the model should allow its use in the design of new PDZ ligands in the future.

  4. A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain.

    Science.gov (United States)

    Panel, Nicolas; Sun, Young Joo; Fuentes, Ernesto J; Simonson, Thomas

    2017-01-01

    PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB) continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or "PB/LIE" free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α 2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo . The overall performance of the model should allow its use in the design of new PDZ ligands in the future.

  5. Crystal structure of hormone-bound atrial natriuretic peptide receptor extracellular domain: rotation mechanism for transmembrane signal transduction.

    Science.gov (United States)

    Ogawa, Haruo; Qiu, Yue; Ogata, Craig M; Misono, Kunio S

    2004-07-02

    A cardiac hormone, atrial natriuretic peptide (ANP), plays a major role in blood pressure and volume regulation. ANP activities are mediated by a single span transmembrane receptor carrying intrinsic guanylate cyclase activity. ANP binding to its extracellular domain stimulates guanylate cyclase activity by an as yet unknown mechanism. Here we report the crystal structure of dimerized extracellular hormone-binding domain in complex with ANP. The structural comparison with the unliganded receptor reveals that hormone binding causes the two receptor monomers to undergo an intermolecular twist with little intramolecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains in the dimer with essentially no change in the interdomain distance. This movement alters the relative orientation of the two domains by a shift equivalent to counterclockwise rotation of each by 24 degrees. These results suggest that transmembrane signaling by the ANP receptor is initiated via a hormone-induced rotation mechanism.

  6. Structural studies and SH3 domain binding properties of a human antiviral salivary proline-rich peptide.

    Science.gov (United States)

    Righino, Benedetta; Pirolli, Davide; Radicioni, Giorgia; Marzano, Valeria; Longhi, Renato; Arcovito, Alessandro; Sanna, Maria Teresa; De Rosa, Maria Cristina; Paoluzi, Serena; Cesareni, Gianni; Messana, Irene; Castagnola, Massimo; Vitali, Alberto

    2016-09-01

    Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016. © 2016 Wiley Periodicals, Inc.

  7. Peptide Based Vaccine Approaches for Cancer—A Novel Approach Using a WT-1 Synthetic Long Peptide and the IRX-2 Immunomodulatory Regimen

    Directory of Open Access Journals (Sweden)

    James E. Egan

    2011-10-01

    Full Text Available Therapeutic cancer vaccines have the potential to generate a long lasting immune response that will destroy tumor cells with specificity and safety, in contrast to many other current cancer therapies. Clinical success to date has been limited by a number of factors including choice of immunogenic cancer rejection antigens, optimization of vaccine platforms and immune adjuvants to effectively polarize the immune response, and incorporation of strategies to reverse cancer mediated immune suppression by utilization of effective adjuvant/immune modulators. WT-1 (Wilms’ tumor gene 1 is a cancer antigen that is required for tumorigenesis, expressed in a high percentage of tumor cells and rarely expressed in adult normal cells. Moreover spontaneous immunity to WT-1 is seen in cancer patients and can be augmented with various therapeutic vaccine approaches. IRX-2 is an immune modulator with demonstrated preclinical and clinical pleiotropic immune activities including enhancement of the immune response to potential tumor antigens. This paper presents the rationale and preclinical data for utilizing the WT-1 tumor antigen in a novel vaccine platform consisting of a synthetic long peptide containing multiple class I and class II epitopes in combination with the IRX-2 immunomodulatory regimen to overcome immuno-suppressive pathways and enhance the anti-tumor response.

  8. Peptide Based Vaccine Approaches for Cancer—A Novel Approach Using a WT-1 Synthetic Long Peptide and the IRX-2 Immunomodulatory Regimen

    Energy Technology Data Exchange (ETDEWEB)

    Naylor, Paul H.; Egan, James E.; Berinstein, Neil L., E-mail: nberinstein@irxtherapeutics.com [IRX Therapeutics, 140 W 57th Street, New York, NY 10019 (United States)

    2011-10-25

    Therapeutic cancer vaccines have the potential to generate a long lasting immune response that will destroy tumor cells with specificity and safety, in contrast to many other current cancer therapies. Clinical success to date has been limited by a number of factors including choice of immunogenic cancer rejection antigens, optimization of vaccine platforms and immune adjuvants to effectively polarize the immune response, and incorporation of strategies to reverse cancer mediated immune suppression by utilization of effective adjuvant/immune modulators. WT-1 (Wilms' tumor gene 1) is a cancer antigen that is required for tumorigenesis, expressed in a high percentage of tumor cells and rarely expressed in adult normal cells. Moreover spontaneous immunity to WT-1 is seen in cancer patients and can be augmented with various therapeutic vaccine approaches. IRX-2 is an immune modulator with demonstrated preclinical and clinical pleiotropic immune activities including enhancement of the immune response to potential tumor antigens. This paper presents the rationale and preclinical data for utilizing the WT-1 tumor antigen in a novel vaccine platform consisting of a synthetic long peptide containing multiple class I and class II epitopes in combination with the IRX-2 immunomodulatory regimen to overcome immuno-suppressive pathways and enhance the anti-tumor response.

  9. Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD.

    Directory of Open Access Journals (Sweden)

    Heng-Li Chen

    Full Text Available The rise of multidrug-resistant (MDR pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc contains an arginine-rich domain (ARD at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV. In this study, we demonstrated that the peptide containing the full-length ARD I-IV (HBc147-183 has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II-IV (HBc153-176 and ARD I-III (HBc147-167 were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS in several in vitro binding assays. Peptide ARD I-IV (HBc147-183 had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p. inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that

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

    Science.gov (United States)

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

    1985-10-01

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

  11. Comparison of radiolabeling efficiency of peptides containing the RGD domain using the Tc-99M and I-131 radioisotopes

    Energy Technology Data Exchange (ETDEWEB)

    Sobral, Danielle V.; Cabral, Francisco Romero; Malavolta, Luciana [Santa Casa de São Paulo, SP (Brazil). Faculdade de Ciências Médicas; Durante, Ana C. Ranucci; Miranda, Ana C. Camargo; Barbosa, Marycel R. F.Figols de [Instituto Israelita de Ensino e Pesquisa Albert Einstein, São Paulo, SP (Brazil)

    2017-07-01

    Full text: Introduction: Radiolabeled peptides have become very important in nuclear medicine and oncology in recent years mainly because they represent the molecular basis for in vivo imaging and radiopharmaceutical therapy with high specificity and affinity for over expressed receptors in tumors (Thno 2(5):481-501, 2012 / Drug Discov. Today. 7:1224-1232, 2012). In this context, peptides containing the RGD domain which possess high affinity for the αvβ3 integrin receptor have become an important tool in a wide variety tumor, including glioblastoma (Exp. Opin. Drug Deliv. 8:1041- 1056, 2011). Objective: The goal of this work was to compare the radiolabeling efficiency of the GRGDYV and GRGDHV peptides when radiolabeled with the {sup 131}I and {sup 99m}Tc radioisotopes, respectively, as well as, to evaluate the features of synthesized complexes. Methods: The GRGDYV and GRGDHV fragments were manually synthesized by peptide synthesis in solid phase accordingly to the Fmoc protocol and purified by preparative HPLC. The GRGDYV and GRGDHV peptides were radiolabeled with the I-131 and Tc-99m radioisotopes respectively, through of the direct method of radiolabeling. The radioiodination was evaluated and optimized using the methodology of Chloramine-T and for the peptide containing the histidine aminoacid the tricarbonyl method was used. Radiochemical yield analyses of [{sup 131}I]-GRGDYV and [{sup 99m}Tc]-GRGDHV peptides were performed by thin layer chromatography on silica gel TLC-SG (Al) in ACN 95%. The radiolabeled peptides were purified by using solid phase extraction (Sep-Pak C18 filter). The stability studies were realized at 2, 24, 48 and 72 hours in room temperature and refrigerate (4 deg C) for [{sup 131}I]-GRGDYV and up to 6 hours for the fragment [{sup 99m}Tc]-GRGDHV. Partition coefficient was determinate for both radiopeptides. Results: The peptides [{sup 131}I]-GRGDYV and [{sup 99m}Tc]-GRGDHV were efficiently synthesized, radiolabeled and showed

  12. Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor*

    Science.gov (United States)

    Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H.; Rudolph, Rainer; Reedtz-Runge, Steffen

    2010-01-01

    GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic β-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9–39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Åresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous α-helix from Thr13 to Val33 when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor. PMID:19861722

  13. Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor.

    Science.gov (United States)

    Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H; Rudolph, Rainer; Reedtz-Runge, Steffen

    2010-01-01

    GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.

  14. Isolation of anti-toxin single domain antibodies from a semi-synthetic spiny dogfish shark display library

    Directory of Open Access Journals (Sweden)

    Goldman Ellen R

    2007-11-01

    Full Text Available Abstract Background Shark heavy chain antibody, also called new antigen receptor (NAR, consists of one single Variable domain (VH, containing only two complementarity-determining regions (CDRs. The antigen binding affinity and specificity are mainly determined by these two CDRs. The good solubility, excellent thermal stability and complex sequence variation of small single domain antibodies (sdAbs make them attractive alternatives to conventional antibodies. In this report, we construct and characterize a diversity enhanced semi-synthetic NAR V display library based on naturally occurring NAR V sequences. Results A semi-synthetic shark sdAb display library with a complexity close to 1e9 was constructed. This was achieved by introducing size and sequence variations in CDR3 using randomized CDR3 primers of three different lengths. Binders against three toxins, staphylococcal enterotoxin B (SEB, ricin, and botulinum toxin A (BoNT/A complex toxoid, were isolated from panning the display library. Soluble sdAbs from selected binders were purified and evaluated using direct binding and thermal stability assays on the Luminex 100. In addition, sandwich assays using sdAb as the reporter element were developed to demonstrate their utility for future sensor applications. Conclusion We demonstrated the utility of a newly created hyper diversified shark NAR displayed library to serve as a source of thermal stable sdAbs against a variety of toxins.

  15. Antiapoptotic effect of a novel synthetic peptide from bovine muscle and MPG peptide on H2O2-induced C2C12 cells.

    Science.gov (United States)

    Sivakumar, Allur Subramaniyan; Ochirbat, Chinzorio; Cho, Soo-Hyun; Yang, Jieun; Hwang, Inho

    2014-08-01

    The objective of this study was to evaluate antioxidant and antiapoptotic effects of a novel peptide (T.peptide) isolated from bovine and commercially available MPG peptide. The amino acid sequences of the T.peptide were (Glu-Val-Pro-Glu-Val-His-Glu-Glu-Val). The antioxidant activities of these peptides were determined by measuring the 1,1-diphenyl-2-picrylhydrazyl (DPPH•) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS+•) radical scavenging assays. The in vitro cytotoxicity of T.peptide and MPG peptide was determined against H2O2-induced C2C12 cells. H2O2-induced apoptosis in C2C12 cells were determined by mRNA expression of caspase-3. Moreover, the mRNA expression of tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NF-κB) were assayed by reverse transcription polymerase chain reaction. The findings of the study indicate that the mRNA expression of TNF-α and NF-κB are significantly (p peptide or MPG peptide. Pretreatment with MPG or T.peptides is also found to significantly (p peptide and MPG peptide could reduce the DPPH• and ABTS+• radical and inhibit cytotoxicity against H2O2-induced injury, resulting in prevention of free radical generation and subsequent apoptotic cell death, which indicates the potential of bovine meat as a source of antioxidant peptides.

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

    Directory of Open Access Journals (Sweden)

    Choi SH

    2016-09-01

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

  17. A synthetic peptide hijacks the catalytic subunit of class I PI3K to suppress the growth of cancer cells.

    Science.gov (United States)

    Guo, Weiwei; You, Xue; Wang, Xiao; Wang, Lin; Chen, Yan

    2017-10-01

    Activation of class I Phosphoinositide 3-kinases (PI3Ks) by mutation or overexpression closely correlates with the development of various human cancers. Class I PI3Ks are heterodimers composed of p110 catalytic subunits and regulatory subunits represented by p85. PAQR3 has been found to inhibit p110α activity by blocking its interaction with p85. In this study, we identified the N-terminal 6-55 amino acid residues of PAQR3 being sufficient for its interaction with p110α. A synthetic peptide, P6-55, that contains the N-terminus of PAQR3 could disrupt the interactions of p110α with both PAQR3 and p85. The activity of PI3K was also inhibited by P6-55, accompanied by significant inhibition of cancer cell proliferation. In a xenograft mouse model, P6-55 was able to reduce tumor growth in vivo. Furthermore, P6-55 was capable of inhibiting the elevated basal PI3K activity of H1047R, a hotspot mutation found in many types of human cancers. The cell proliferation and migration of cancer cells bearing H1047R mutation were also reduced by P6-55. In conclusion, our study provides a proof of concept that blocking the interaction of p110α with p85 by a peptide can serve as a new strategy to inhibit the oncogenic activity of PI3K in cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. A novel fully synthetic and self-assembled peptide solution for endoscopic submucosal dissection-induced ulcer in the stomach.

    Science.gov (United States)

    Uraoka, Toshio; Ochiai, Yasutoshi; Fujimoto, Ai; Goto, Osamu; Kawahara, Yoshiro; Kobayashi, Naoya; Kanai, Takanori; Matsuda, Sachiko; Kitagawa, Yuko; Yahagi, Naohisa

    2016-06-01

    Endoscopic submucosal dissection (ESD) can remove early stage GI tumors of various sizes en bloc; however, success requires reducing the relatively high postprocedure bleeding rate. The aim of this study was to assess the safety and efficacy of a novel, fully synthetic, and self-assembled peptide solution that functions as an extracellular matrix scaffold material to facilitate reconstruction of normal tissues in ESD-induced ulcers. Consecutive patients who underwent gastric ESD were prospectively enrolled. Immediately after the resection, the solution was applied to the site with a catheter. Gastric ulcers were evaluated by endoscopy and classified as active, healing, or scarring stages at weeks 1, 4, and 8 after ESD. Forty-seven patients with 53 lesions, including 14 (29.8%) previously on antithrombotic therapy and 2 (4.3%) requiring heparin bridge therapy, were analyzed; 2 patients were excluded, 1 with perforations and 1 with persistent coagulopathy. The mean size of the en bloc resected specimens was 36.5 ± 11.3 mm. The rate of post-ESD bleeding was 2.0% (1/51; 95% CI, 0.03-10.3). Transitional rate to the healing stage of ESD-induced ulcers at week 1 was 96% (49/51). Subsequent endoscopies demonstrated the scarring stage in 19% (9/48) and 98% (41/42) at weeks 4 and 8, respectively. No adverse effects related to this solution occurred. The use of this novel peptide solution may potentially aid in reducing the delayed bleeding rate by promoting mucosal regeneration and speed of ulcer healing after large endoscopic resections in the stomach. Further studies, particularly randomized controlled studies, are needed to fully evaluate its efficacy. ( 000011548.). Copyright © 2016 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

  19. Random Walks for Synthetic Aperture Radar Image Fusion in Framelet Domain.

    Science.gov (United States)

    Yang, Xiaoyuan; Wang, Jingkai; Zhu, Ridong

    2017-08-30

    A new framelet-based random walks (RW) method is presented for synthetic aperture radar (SAR) image fusion, including SAR-visible images, SAR-infrared images and Multiband SAR images. In this method, we build a novel random walks model based on the statistical characteristics of framelet coefficients to fuse the high-frequency and low-frequency coefficients. This model converts the fusion problem to estimate the probability of each framelet coefficient being assigned each input image. Experimental results show that the proposed approach improves the contrast while preserves the edges simultaneously, and outperforms many traditional and state-of-the-art fusion techniques in both qualitative and quantitative analysis.

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

    Science.gov (United States)

    Choi, Suk Hyun; Lee, Jue Yeon; Suh, Jin Sook; Park, Yoon Shin; Chung, Chong Pyoung; Park, Yoon Jeong

    Angiogenesis plays a critical role in the growth and metastasis of cancer, and growth factors released from cancer promote blood-vessel formation in the tumor microenvironment. The angiogenesis is accelerated via interactions of growth factors with the high-affinity receptors on cancer cells. In particular, heparan sulfate proteoglycans (HSPGs) on the surface of cancer cells have been shown to be important in many aspects of determining a tumor's phenotype and development. Specifically, the regulation of the interactions between HSPGs and growth factors results in changes in tumor progression. A peptide with heparin-binding (HBP) activity has been developed and synthesized to inhibit tumor growth via the prevention of angiogenesis. We hypothesized that HBP could inhibit the interaction of growth factors and HSPGs on the surface of cancer cells, decrease paracrine signaling in endothelial cells (ECs), and finally decrease angiogenesis in the tumor microenvironment. In this study, we found that HBP had antiangiogenic effects in vitro and in vivo. The conditioned media obtained from a breast cancer cell line treated with HBP were used to culture human umbilical vein ECs (HUVECs) to evaluate the antiangiogenic effect of HBP on ECs. HBP effectively inhibited the migration, invasion, and tube formation of HUVECs in vitro. In addition, the expressions of angiogenesis-mediating factors, including ERK, FAK, and Akt, were considerably decreased. HBP also decreased the levels of invasive factors, including MMP2 and MMP9, secreted by the HUVECs. We demonstrated significant suppression of tumor growth in a breast cancer xenograft model and enhanced distribution of HBP at the site of tumors. Taken together, our results show that HBP has antiangiogenic effects on ECs, and suggest that it may serve as a potential antitumor agent through control of the tumor microenvironment.

  1. Synthetic Curcumin Analogs as Inhibitors of β -Amyloid Peptide Aggregation: Potential Therapeutic and Diagnostic Agents for Alzheimer's Disease.

    Science.gov (United States)

    Bukhari, Syed Nasir Abbas; Jantan, Ibrahim

    2015-01-01

    There is a crucial need to develop new effective drugs for Alzheimer's disease (AD) as the currently available AD treatments provide only momentary and incomplete symptomatic relief. Amongst natural products, curcumin, a major constituent of turmeric, has been intensively investigated for its neuroprotective effect against β-amyloid (Aβ)-induced toxicity in cultured neuronal cells. The ability of curcumin to attach to Aβ peptide and prevent its accumulation is attributed to its three structural characteristics such as the presence of two aromatic end groups and their co-planarity, the length and rigidity of the linker region and the substitution conformation of these aromatics. However, curcumin failed to reach adequate brain levels after oral absorption in AD clinical trials due to its low water solubility and poor oral bioavailability. A number of new curcumin analogs that mimic the active site of the compound along with analogs that mimic the curcumin anti-amyloid effect combined with anticholinesterase effect have been developed to enhance the bioavailability, pharmacokinetics, water solubility, stability at physiological conditions and delivery of curcumin. In this article, we have summarized all reported synthetic analogs of curcumin showing effects on β-amyloid and discussed their potential as therapeutic and diagnostic agents for AD.

  2. A novel synthetic peptide polymer with cyclic RGD motifs supports serum-free attachment of anchorage-dependent cells.

    Science.gov (United States)

    Markó, K; Ligeti, M; Mezo, G; Mihala, N; Kutnyánszky, E; Kiss, E; Hudecz, F; Madarász, E

    2008-09-01

    Cell adhesivity is a basic biological principle, which provides mechanisms for construction of multicellular organisms, tissue genesis, migration and individual cell survival. In vivo, the cell adhesive environment is provided by extracellular matrix molecules, neighboring cell surfaces and soluble factors delivered either by tissue cells or by blood circulation. The exact molecular composition of the microenvironment of a cell is not properly understood. The nondefined molecular composition of "native" adhesive components hinders their application when defined culture conditions are necessary, as, for an example, growing human cells for further clinical application. Applying large, substrate-coating molecules as backbones for carrying specific adhesive peptide motifs provides a relatively cheap, reproducible, and chemically defined group of synthetic adhesion molecules. Here, we report on the design, synthesis, and testing of a novel cyclic RGD-containing coating material, which promotes initial attachment, spreading, survival, and proliferation of a number of different cell types. The potent adhesive polypeptide-brush, composed of poly[Lys(DL-Ala(m))] branched chain polypeptide (AK) and multiple copies of cyclic(arginyl-glycyl-aspartyl-D-phenylalanyl-cysteine) pentapeptide prevents anoikis and supports cell attachment in the absence of serum or other biological additives. The defined conditions for cell maintenance make this material a promising candidate for coating artificial cell substrates even for therapeutic applications.

  3. Changes in plasma calcium and in radiocalcium kinetics after termination of 5-week infusions of synthetic parathyroid peptide in dogs

    International Nuclear Information System (INIS)

    Stevenson, R.W.; Parsons, J.A.; Podbesek, R.D.; Reeve, J.

    1983-01-01

    Nine dogs were infused at constant rates with the synthetic parathyroid peptide hPTH 1-34 (initially sc) to produce consistent hypercalcaemia. Over the final week, the infusions were iv. Radioisotopic tracers were injected iv 30 days (5 dogs) and 2 days (9 dogs) before the infusions were suddenly terminated. In 5 dogs, complete urine collections were obtained via a bladder catheter over 8 h beginning 2 h before stopping the infusions. Cessation of treatment caused small rises in the urinary Ca: creatinine ratio. Plasma calcium levels fell by a mean of 0.44 mmol/l, of which total urine calcium excretion only accounted for 55%. Immediately after the PTH infusions were stopped, consistent but transient increases were seen in the ratio of ''new'' 47 Ca to ''old'' 45 Ca label, suggesting inflow of 40 Ca of high 47 Ca specific activity from a fairly rapidly exchangeable bone pool. These data confirm and extend previous evidence that the immediate response of the calcium equilibrium between bone and bloodstream to rapid changes in plasma PTH concentrations in the supra-physiological ranges is paradoxical relative to the classical later response. (author)

  4. A Synthetic Peptide-Acrylate Surface for Production of Insulin-Producing Cells from Human Embryonic Stem Cells

    Science.gov (United States)

    Lin, Pei-Yi; Hung, Shih-Han; Yang, Yao-Chen; Liao, Li-Chuan; Hsieh, Yi-Cheng; Yen, Hsan-Jan; Lu, Huai-En; Lee, Maw-Sheng

    2014-01-01

    Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable β-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. Moreover, we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigel-coated surface. Thus, we provided a totally defined condition from hESC culture to insulin-producing cell differentiation, and the derived cells could be a therapeutic resource for diabetic patients in the future. PMID:24083371

  5. A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts.

    Science.gov (United States)

    Schueler, Ulrike; Kaneski, Christine; Remaley, Alan; Demosky, Stephen; Dwyer, Nancy; Blanchette-Mackie, Joan; Hanover, John; Brady, Roscoe

    2016-01-01

    Fabry disease is an X-linked sphingolipid storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (AGA, EC 3.2.1.22) resulting in the intracellular accumulation of globotriaosylceramide (Gb3). We found that Gb3 storage also correlates with accumulation of endosomal-lysosomal cholesterol in Fabry fibroblasts. This cholesterol accumulation may contribute to the phenotypic pathology of Fabry disease by slowing endosomal-lysosomal trafficking. We found that LDL receptor expression is not downregulated in Fabry fibroblasts resulting in accumulation of both cholesterol and Gb3. 5A-Palmitoyl oleoyl-phosphatidylcholine (5AP) is a phospholipid complex containing a short synthetic peptide that mimics apolipoprotein A1, the main protein component of high-density lipoprotein (HDL) that mediates the efflux of cholesterol from cells via the ATP-binding cassette transporter. We used 5AP and HDL to remove cholesterol from Fabry fibroblasts to examine the fate of accumulated cellular Gb3. Using immunostaining techniques, we found that 5AP is highly effective for depleting cholesterol and Gb3 in these cells. 5AP restores the ApoA-1-mediated cholesterol efflux leading to mobilization of cholesterol and reduction of Gb3 in Fabry fibroblasts.

  6. Apoptosis Activation in Human Lung Cancer Cell Lines by a Novel Synth