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Sample records for chemotactic peptide labeling

  1. Direct and indirect radioiodination of protein: comparative study of chemotactic peptide labeling; Radioiodacao de proteina por via direta e indireta: estudo comparativo da marcacao de peptideo quimiotatico

    Energy Technology Data Exchange (ETDEWEB)

    Lavinas, Tatiana

    2004-07-01

    The development of simple methods for protein radioiodination have stimulated the use of radioiodinated peptides in vivo. There are two basic methods for labeling proteins with radioiodine: direct labeling, reaction of an electrophilic radioiodine with functional activated groups on protein, like the phenol ring in the tyrosine residue, and the conjugation of a previously radioiodinated molecule to the protein, referred as indirect method. The great problem related to the direct radioiodination of proteins is the in vivo dehalogenation. This problem can be minimized if a non-phenolic prosthetic group is used in the indirect radioiodination of the peptide. The ATE prosthetic group, N-succinimidyl 3-(tri-n-butylstannyl) benzoate, when radioiodinated by electrophilic iododestannilation produces N-succinimidyl 3-[{sup 123}l/{sup 131}l] iodine benzoate (SIB) that is subsequently conjugated to the protein by the acylation of the lysine group. There are many radiopharmaceuticals employed in scintigraphic images of infection and inflammation used with some limitations. These limitations stimulated the improvement of a new class of radiopharmaceuticals, the receptor-specific related labeled peptides, as the mediators of the inflammatory response, that presents high affinity by receptors expressed in the inflammation process, and fast clearance from blood and non-target tissues. One of these molecules is the synthetic chemotactic peptide fNleLFNIeYK that presents potent chemotaxis for leukocytes, with high affinity by the receptors presented in polymorphonuclear leukocytes and mononuclear phagocytes. The objective of this work included the synthesis of ATE prosthetic group and comparative radioiodination of the chemotactic peptide fNleLFNIeYK by direct and indirect methods, with radiochemical purity determination and evaluation of in vivo and in vitro stability of the compounds. This work presented an original contribution in the comparative biological distribution studies

  2. Boc SPPS of two hydrophobic peptides using a ''solubilising tail'' strategy : Dodecaalanine and chemotactic protein 10(42-55)

    NARCIS (Netherlands)

    Englebretsen, DR; Alewood, PF

    1996-01-01

    The solid phase syntheses of the hydrophobic peptides dodecaalanine and chemotactic protein-10(42-55) were achieved using a ''solubilising tail'' strategy. Peptide constructs of the form H-hydrophobic peptide-glycolamide ester-(Gly-Arg)(4)-Gly-OH were synthesised by Boc SPPS. The peptide-constructs

  3. Down-regulation of the chemokine receptor CCR5 by activation of chemotactic formyl peptide receptor in human monocytes.

    Science.gov (United States)

    Shen, W; Li, B; Wetzel, M A; Rogers, T J; Henderson, E E; Su, S B; Gong, W; Le, Y; Sargeant, R; Dimitrov, D S; Oppenheim, J J; Wang, J M

    2000-10-15

    Interactions between cell surface receptors are important regulatory elements in the complex host responses to infections. In this study, it is shown that a classic chemotactic factor, the bacterial chemotactic peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), rapidly induced a protein-kinase-C-mediated serine phosphorylation and down-regulation of the chemokine receptor CCR5, which serves as a major human immunodeficiency virus (HIV)-1 coreceptor. The fMLF binding to its receptor, formyl peptide receptor (FPR), resulted in significant attenuation of cell responses to CCR5 ligands and in inhibition of HIV-1-envelope-glycoprotein-mediated fusion and infection of cells expressing CD4, CCR5, and FPR. The finding that the expression and function of CCR5 can be regulated by peptides that use an unrelated receptor may provide a novel approach to the design of anti-inflamatory and antiretroviral agents. (Blood. 2000;96:2887-2894)

  4. Chemical labeling of electrochemically cleaved peptides

    NARCIS (Netherlands)

    Roeser, Julien; Alting, Niels F. A.; Permentier, Hjalmar P.; Bruins, Andries P.; Bischoff, Rainer P. H.

    2013-01-01

    RATIONALE Cleavage of peptide bonds C-terminal to tyrosine and tryptophan after electrochemical oxidation may become a complementary approach to chemical and enzymatic cleavage. A chemical labeling approach specifically targeting reactive cleavage products is presented here and constitutes a promisi

  5. Neutron encoded labeling for peptide identification.

    Science.gov (United States)

    Rose, Christopher M; Merrill, Anna E; Bailey, Derek J; Hebert, Alexander S; Westphall, Michael S; Coon, Joshua J

    2013-05-21

    Metabolic labeling of cells using heavy amino acids is most commonly used for relative quantitation; however, partner mass shifts also detail the number of heavy amino acids contained within the precursor species. Here, we use a recently developed metabolic labeling technique, NeuCode (neutron encoding) stable isotope labeling with amino acids in cell culture (SILAC), which produces precursor partners spaced ~40 mDa apart to enable amino acid counting. We implement large scale counting of amino acids through a program, "Amino Acid Counter", which determines the most likely combination of amino acids within a precursor based on NeuCode SILAC partner spacing and filters candidate peptide sequences during a database search using this information. Counting the number of lysine residues for precursors selected for MS/MS decreases the median number of candidate sequences from 44 to 14 as compared to an accurate mass search alone (20 ppm). Furthermore, the ability to co-isolate and fragment NeuCode SILAC partners enables counting of lysines in product ions, and when the information is used, the median number of candidates is reduced to 7. We then demonstrate counting leucine in addition to lysine results in a 6-fold decrease in search space, 43 to 7, when compared to an accurate mass search. We use this scheme to analyze a nanoLC-MS/MS experiment and demonstrate that accurate mass plus lysine and leucine counting reduces the number of candidate sequences to one for ~20% of all precursors selected, demonstrating an ability to identify precursors without MS/MS analysis.

  6. Peptide-membrane Interactions by Spin-labeling EPR

    Science.gov (United States)

    Smirnova, Tatyana I.; Smirnov, Alex I.

    2016-01-01

    Site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spin-labeling methods and preparation of membrane mimetic systems are also described. PMID:26477253

  7. Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs.

    Science.gov (United States)

    Froyen, G; Proost, P; Ronsse, I; Mitera, T; Haelens, A; Wuyts, A; Opdenakker, G; Van Damme, J; Billiau, A

    1997-02-01

    Human osteosarcoma cells secrete a novel C-X-C chemokine called granulocyte chemotactic protein-2 (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin-1 beta and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP-2 was isolated. This sequence was cloned into the bacterial expression vector pHEN1 and, after induction, GCP-2 was secreted into the periplasm of Escherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characterized by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal sequencing. The chemoattractive potency of GCP-2 for neutrophilic granulocytes was about 10-times less than that of interleukin-8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial-cell-derived neutrophil-activating peptide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell lines tested. In addition, GCP-2 and ENA-78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin-1 was demonstrated to be a general inducer for both chemokines, while interferon-gamma down-regulates their mRNA expression. The

  8. New Methods for Labeling RGD Peptides with Bromine-76

    Directory of Open Access Journals (Sweden)

    Lixin Lang, Weihua Li, Hong-Mei Jia, De-Cai Fang, Shushu Zhang, Xilin Sun, Lei Zhu, Ying Ma, Baozhong Shen, Dale O. Kiesewetter, Gang Niu, Xiaoyuan Chen

    2011-01-01

    Full Text Available Direct bromination of the tyrosine residues of peptides and antibodies with bromine-76, to create probes for PET imaging, has been reported. For peptides that do not contain tyrosine residues, however, a prosthetic group is required to achieve labeling via conjugation to other functional groups such as terminal α-amines or lysine ε-amines. The goal of this study was to develop new strategies for labeling small peptides with Br-76 using either a direct labeling method or a prosthetic group, depending on the available functional group on the peptides. A new labeling agent, N-succinimidyl-3-[76Br]bromo-2,6-dimethoxybenzoate ([76Br]SBDMB was prepared for cyclic RGD peptide labeling. N-succinimidyl-2, 6-dimethoxybenzoate was also used to pre-attach a 2, 6-dimethoxybenzoyl (DMB moiety to the peptide, which could then be labeled with Br-76. A competitive cell binding assay was performed to determine the binding affinity of the brominated peptides. PET imaging of U87MG human glioblastoma xenografted mice was performed using [76Br]-BrE[c(RGDyK]2 and [76Br]-BrDMB-E[c(RGDyK]2. An ex vivo biodistribution assay was performed to confirm PET quantification. The mechanisms of bromination reaction between DMB-c(RGDyK and the brominating agent CH3COOBr were investigated with the SCRF-B3LYP/6-31G* method with the Gaussian 09 program package. The yield for direct labeling of c(RGDyK and E[c(RGDyK]2 using chloramine-T and peracetic acid at ambient temperature was greater than 50%. The yield for [76Br]SBDMB was over 60% using peracetic acid. The conjugation yields for labeling c(RGDfK and c(RGDyK were over 70% using the prosthetic group at room temperature. Labeling yield for pre-conjugated peptides was over 60%. SDMB conjugation and bromination did not affect the binding affinity of the peptides with integrin receptors. Both [76Br]Br-E[c(RGDyK]2 and [76Br]BrDMB-E[c(RGDyK]2 showed high tumor uptake in U87MG tumor bearing mice. The specificity of the imaging tracers

  9. Secondary structure of fluorescence labelled synthetic peptides

    CERN Document Server

    Martin, A S

    2000-01-01

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

  10. Labeled Antimicrobial Peptides for Detection of Microorganisms

    Science.gov (United States)

    2008-12-01

    Beer , C, Koka, R. and Wang, X., 2001: Solid-Phase Capture of Proteins, Spores, and Bacteria. Applied and Environmental Microbiology , 67: 1300-1307. Yu...detection but often lack stability and sensitivity. Current biosensor technologies using fluorescently labeled antibodies are generally capable of...biosensor. J. ofRapid Methods and Automation in Microbiology , 9: 241- 257. Demarco, D. R. and Lim, D. Y., 2002: Detection of Escherichia coli 0157:H7

  11. Feasibility and availability of {sup 68}Ga-labelled peptides

    Energy Technology Data Exchange (ETDEWEB)

    Decristoforo, Clemens [Innsbruck Medical University, Department of Nuclear Medicine, Innsbruck (Austria); European Directorate of Quality of Medicines, Group 14, Radioactive Compounds, The European Pharmacopeia, Strasbourg (France); Pickett, Roger D. [GE Healthcare, Little Chalfont (United Kingdom); European Directorate of Quality of Medicines, Group 14, Radioactive Compounds, The European Pharmacopeia, Strasbourg (France); Verbruggen, Alfons [University of Leuven, Laboratory of Radiopharmacy, Department of Pharmaceutical Sciences, Leuven (Belgium); European Directorate of Quality of Medicines, Group 14, Radioactive Compounds, The European Pharmacopeia, Strasbourg (France)

    2012-02-15

    {sup 68}Ga has attracted tremendous interest as a radionuclide for PET based on its suitable half-life of 68 min, high positron emission yield and ready availability from {sup 68}Ge/{sup 68}Ga generators, making it independent of cyclotron production. {sup 68}Ga-labelled DOTA-conjugated somatostatin analogues, including DOTA-TOC, DOTA-TATE and DOTA-NOC, have driven the development of technologies to provide such radiopharmaceuticals for clinical applications mainly in the diagnosis of somatostatin receptor-expressing tumours. We summarize the issues determining the feasibility and availability of {sup 68}Ga-labelled peptides, including generator technology, {sup 68}Ga generator eluate postprocessing methods, radiolabelling, automation and peptide developments, and also quality assurance and regulatory aspects. {sup 68}Ge/{sup 68}Ga generators based on SnO{sub 2}, TiO{sub 2} or organic matrices are today routinely supplied to nuclear medicine departments, and a variety of automated systems for postprocessing and radiolabelling have been developed. New developments include improved chelators for {sup 68}Ga that could open new ways to utilize this technology. Challenges and limitations in the on-site preparation and use of {sup 68}Ga-labelled peptides outside the marketing authorization track are also discussed. (orig.)

  12. Lutetium-177 Labeled Peptides: The European Institute of Oncology Experience.

    Science.gov (United States)

    Carollo, Angela; Papi, Stefano; Chinol, Marco

    2016-01-01

    Peptide receptor radionuclide therapy (PRRT) using radiolabeled somatostatin analogues has shown encouraging results in various somatostatin receptor positive tumors. Partial remission rates up to 30% have been documented as well as significant improvements in quality of life and survival. This treatment takes advantage of the high specific binding of the radiolabeled peptide to somatostatin receptors overexpressed by the tumors thus being more effective on the tumor cells with less systemic side-effects. The development of macrocyclic chelators conjugated to peptides made possible the stable binding with various radionuclides. In particular 177Lu features favourable physical characteristics with a half-life of 6.7 days, emission of β- with energy of 0.5 MeV for treatment and γ-emissions suitable for imaging. The present contribution describes the learning process achieved at the European Institute of Oncology (IEO) since the first application of 90Y labeled peptides to the therapy of neuroendocrine tumors back in 1997. Continuous improvements led to the preparation of a safe 177Lu labeled peptide for human use. Our learning curve began with the identification of the optimal characteristics of the isotope paying attention to its chemical purity and specific activity along with the optimization of the parameters involved in the radiolabeling procedure. Also the radiation protection issues have been improved along the years and recently more and more attention has been devoted to the pharmaceutical aspects involved in the preparation. The overall issue of the quality has now been completed by drafting an extensive documentation with the goal to deliver a safe and reliable product to our patients.

  13. Efficient and Selective Chemical Labeling of Electrochemically Generated Peptides Based on Spirolactone Chemistry

    NARCIS (Netherlands)

    Zhang, Tao; Niu, Xiaoyu; Yuan, Tao; Tessari, Marco; de Vries, Marcel P.; Permentier, Hjalmar P.; Bischoff, Rainer

    2016-01-01

    Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Cleavage of the peptide bond following electrochemical oxidation of Tyr or Trp r

  14. Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry.

    Science.gov (United States)

    Shabanpoor, Fazel; Gait, Michael J

    2013-11-11

    We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.

  15. Monitoring membrane binding and insertion of peptides by two-color fluorescent label.

    Science.gov (United States)

    Postupalenko, V Y; Shvadchak, V V; Duportail, G; Pivovarenko, V G; Klymchenko, A S; Mély, Y

    2011-01-01

    Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide-membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.

  16. 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 hy...... hydrogenation chemistry with the use of a heterogeneous palladium catalyst and carrier-free tritium gas on a tritium manifold system....

  17. Full automation of {sup 68}Ga labelling of DOTA-peptides including cation exchange prepurification

    Energy Technology Data Exchange (ETDEWEB)

    Ocak, M. [Clinical Department of Nuclear Medicine, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Department of Pharmaceutical Technology, Pharmacy Faculty, Istanbul University, Istanbul (Turkey); Antretter, M. [Clinical Department of Nuclear Medicine, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Knopp, R.; Kunkel, F. [Eckert and Ziegler Eurotope GmbH, Berlin (Germany); Petrik, M. [Clinical Department of Nuclear Medicine, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Bergisadi, N. [Department of Pharmaceutical Technology, Pharmacy Faculty, Istanbul University, Istanbul (Turkey); Decristoforo, C. [Clinical Department of Nuclear Medicine, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)], E-mail: Clemens.Decristoforo@uki.at

    2010-02-15

    Here we describe a fully automated approach for the synthesis of {sup 68}Ga-labelled DOTA-peptides based on pre-concentration and purification of the generator eluate by using a cation exchange-cartridge and its comparison with fully automated direct labelling applying fractionated elution. Pre-concentration of the eluate on a cation exchange cartridge both using a resin-based and a disposable cation-exchange cartridge efficiently removed {sup 68}Ge as well as major metal contaminations with Fe and Zn. This resulted in a high labelling efficiency of DOTA-peptides at high specific activity (SA) with short synthesis times.

  18. Label-free peptide profiling of Orbitrap™ full mass spectra

    Directory of Open Access Journals (Sweden)

    Titulaer Mark K

    2011-01-01

    Full Text Available Abstract Background We developed a new version of the open source software package Peptrix that can yet compare large numbers of Orbitrap™ LC-MS data. The peptide profiling results for Peptrix on MS1 spectra were compared with those obtained from a small selection of open source and commercial software packages: msInspect, Sieve™ and Progenesis™. The properties compared in these packages were speed, total number of detected masses, redundancy of masses, reproducibility in numbers and CV of intensity, overlap of masses, and differences in peptide peak intensities. Reproducibility measurements were taken for the different MS1 software applications by measuring in triplicate a complex peptide mixture of immunoglobulin on the Orbitrap™ mass spectrometer. Values of peptide masses detected from the high intensity peaks of the MS1 spectra by peptide profiling were verified with values of the MS2 fragmented and sequenced masses that resulted in protein identifications with a significant score. Findings Peptrix finds about the same number of peptide features as the other packages, but peptide masses are in some cases approximately 5 to 10 times less redundant present in the peptide profile matrix. The Peptrix profile matrix displays the largest overlap when comparing the number of masses in a pair between two software applications. The overlap of peptide masses between software packages of low intensity peaks in the spectra is remarkably low with about 50% of the detected masses in the individual packages. Peptrix does not differ from the other packages in detecting 96% of the masses that relate to highly abundant sequenced proteins. MS1 peak intensities vary between the applications in a non linear way as they are not processed using the same method. Conclusions Peptrix is capable of peptide profiling using Orbitrap™ files and finding differential expressed peptides in body fluid and tissue samples. The number of peptide masses detected in

  19. Spasmogenic activity of chemotactic N-formylated oligopeptides: identity of structure--function relationships for chemotactic and spasmogenic activities.

    OpenAIRE

    Marasco, W. A.; Fantone, J. C.; Ward, P. A.

    1982-01-01

    The chemotactic N-formylated oligopeptides are potent spasmogenic agents for guinea pig ileum. Structure-activity studies with various N-formylated peptides suggest the presence of a specific receptor that resembles in specificity the formyl peptide receptor on leukocytes. A competitive antagonist of the formyl peptide receptor on leukocytes also inhibits formyl peptide-induced ileum contraction, whereas the antihistamine diphenhydramine is without effect. The contractile response caused by t...

  20. Chemotactic Maneuverability of Sperm

    CERN Document Server

    Guasto, Jeffrey S; Zimmer, Richard K; Stocker, Roman

    2011-01-01

    In this fluid mechanics video, we explore the kinematics of chemotaxing sperm cells (sea urchin, \\textit{Arbacia punctulata}) swimming in a chemoattractant gradient. We demonstrate that the complex swimming trajectories resulting in chemotactic behavior (`turn-and-run' motility) are comprised of several distinct flagellar maneuvers. These motility patterns likely play an important role optimizing chemotaxic motility and navigation, when the sperm cells are subjected external fluid flows.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-01-01

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

  3. Targeted therapy of colorectal neoplasia with rapamycin in peptide-labeled pegylated octadecyl lithocholate micelles.

    Science.gov (United States)

    Khondee, Supang; Rabinsky, Emily F; Owens, Scott R; Joshi, Bishnu P; Qiu, Zhen; Duan, Xiyu; Zhao, Lili; Wang, Thomas D

    2015-02-10

    Many powerful drugs have limited clinical utility because of poor water solubility and high systemic toxicity. Here, we formulated a targeted nanomedicine, rapamycin encapsulated in pegylated octadecyl lithocholate micelles labeled with a new ligand for colorectal neoplasia, LTTHYKL peptide. CPC;Apc mice that spontaneously develop colonic adenomas were treated with free rapamycin, plain rapamycin micelles, and peptide-labeled rapamycin micelles via intraperitoneal injection for 35days. Endoscopy was performed to monitor adenoma regression in vivo. We observed complete adenoma regression at the end of therapy. The mean regression rate for peptide-labeled rapamycin micelles was significantly greater than that for plain rapamycin micelles, Prapamycin micelles compared to that of free drug, and no other toxicities were found on chemistries. Together, this unique targeted micelle represents a potential therapeutic for colorectal neoplasia with comparable therapeutic efficacy to rapamycin free drug and significantly less systemic toxicity.

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

    Directory of Open Access Journals (Sweden)

    Zhongqiu Ni

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

  5. Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: improved labeling and structural implications.

    Science.gov (United States)

    Kyro, Kelly; Manandhar, Surya P; Mullen, Daniel; Schmidt, Walter K; Distefano, Mark D

    2011-12-15

    Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-l-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design.

  6. Molecular level studies on binding modes of labeling molecules with polyalanine peptides

    Science.gov (United States)

    Mao, Xiaobo; Wang, Chenxuan; Ma, Xiaojing; Zhang, Min; Liu, Lei; Zhang, Lan; Niu, Lin; Zeng, Qindao; Yang, Yanlian; Wang, Chen

    2011-04-01

    In this work, the binding modes of typical labeling molecules (thioflavin T (ThT), Congo red (CR) and copper(ii) phthalocyanine tetrasulfonic acid tetrasodium salt (PcCu(SO3Na)4)) on pentaalanine, which is a model peptide segment of amyloidpeptides, have been resolved at the molecular level by using scanning tunneling microscopy (STM). In the STM images, ThT molecules are predominantly adsorbed parallel to the peptide strands and two binding modes could be identified. It was found that ThT molecules are preferentially binding on top of the peptide strand, and the mode of intercalated between neighboring peptides also exists. The parallel binding mode of CR molecules can be observed with pentaalaninepeptides. Besides the binding modes of labeling molecules, the CR and PcCu(SO3Na)4 display different adsorption affinity with the pentaalaninepeptides. The results could be beneficial for obtaining molecular level insight of the interactions between labeling molecules and peptides.In this work, the binding modes of typical labeling molecules (thioflavin T (ThT), Congo red (CR) and copper(ii) phthalocyanine tetrasulfonic acid tetrasodium salt (PcCu(SO3Na)4)) on pentaalanine, which is a model peptide segment of amyloidpeptides, have been resolved at the molecular level by using scanning tunneling microscopy (STM). In the STM images, ThT molecules are predominantly adsorbed parallel to the peptide strands and two binding modes could be identified. It was found that ThT molecules are preferentially binding on top of the peptide strand, and the mode of intercalated between neighboring peptides also exists. The parallel binding mode of CR molecules can be observed with pentaalaninepeptides. Besides the binding modes of labeling molecules, the CR and PcCu(SO3Na)4 display different adsorption affinity with the pentaalaninepeptides. The results could be beneficial for obtaining molecular level insight of the interactions between labeling molecules and peptides. Electronic

  7. Gallium-labelled peptides for imaging of inflammation

    Energy Technology Data Exchange (ETDEWEB)

    Roivainen, Anne [University of Turku and Turku University Hospital, Turku PET Centre, Turku (Finland); University of Turku, Turku Center for Disease Modeling, Turku (Finland); Jalkanen, Sirpa [University of Turku, MediCity Research Laboratory, Turku (Finland); University of Turku, Department of Medical Microbiology and Immunology, Turku (Finland); Nanni, Cristina [Azienda Ospedaliero-Universitaria S.Orsola Malpighi, UO Medicina Nucleare, Bologna (Italy)

    2012-02-15

    Inflammation plays a major role in the development of many diseases. This review article summarizes recent research in the field of in vivo imaging of inflammation. Novel methodologies using PET with {sup 68}Ga peptides targeting, for example, vascular adhesion protein 1 are discussed. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Gerrit Volkmann

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

  9. Rapid biosynthesis of stable isotope-labeled peptides from a reconstituted in vitro translation system for targeted proteomics.

    Science.gov (United States)

    Xian, Feng; Li, Shuwei; Liu, Siqi

    2015-01-01

    Stable isotope-labeled peptides are routinely used as internal standards (a.k.a. reference peptides) for absolute quantitation of proteins in targeted proteomics. These peptides can either be synthesized chemically on solid supports or expressed biologically by concatenating multiple peptides together to a large protein. Neither method, however, has required versatility, convenience, and economy for making a large number of reference peptides. Here, we describe the biosynthesis of stable isotope-labeled peptides from a reconstituted Escherichia coli in vitro translation system. We provide a detailed protocol on how to express these peptides with high purity and how to determine their concentrations with easiness. Our strategy offers a general, fast, and scalable approach for the easy preparation of labeled reference peptides, which will have broad application in both basic research and translational medicine.

  10. Metabolic flux analysis using 13C peptide label measurements

    Science.gov (United States)

    13C metabolic flux analysis (MFA) has become the experimental method of choice to investigate cellular metabolism. MFA has established flux maps of central metabolism for dozens of microbes, cell cultures, and plant seeds. Steady-state MFA utilizes isotopic labeling measurements of amino acids obtai...

  11. RGDC Peptide Modified Quantum Dots Labelling and Imaging of Tumor Cells

    Institute of Scientific and Technical Information of China (English)

    GUO Yi; LI Chun-rong; SHEN Huai-bin; ZHANG Xue-zhong; LI Lin-song; YU Qian; XU Li

    2011-01-01

    The labelling and imaging of tumor cells were investigated via arginine-glycine-aspartic acidcysteine(RGDC) peptide-labelled quantum dots(QDs).The results show that RGDC modified QDs can label SMMC-7721 tumor cells and adhere to cellular membrane.In constrast,the unmodified QDs are mainly dispersed around the cell.We also found that the RGDC-QDs can penetrate into the cell at 2 h of incubation.After 6 h of incubation,RGDC-QDs can accumulate in a unique intracellular region.

  12. Recommended administered activities for {sup 68}Ga-labelled peptides in paediatric nuclear medicine

    Energy Technology Data Exchange (ETDEWEB)

    Machado, J.S.; Beykan, S.; Lassmann, M. [University Hospital Wuerzburg, Department of Nuclear Medicine, Wuerzburg (Germany); Herrmann, K. [University Hospital Wuerzburg, Department of Nuclear Medicine, Wuerzburg (Germany); David Geffen School of Medicine at UCLA, Department of Molecular and Medical Pharmacology, Los Angeles, CA (United States)

    2016-10-15

    The aim of this study was to establish a method for determining administered activities for {sup 68}Ga-labelled peptides. Dose calculations were based on the weight-independent effective dose model proposed by the EANM paediatric dosage card for use in paediatric nuclear medicine. Previously published time-integrated activity coefficients for {sup 68}Ga-DOTATATE, {sup 68}Ga-DOTATOC and {sup 68}Ga-pentixafor were used to calculate age-independent effective doses. Consequently, the corresponding weight-dependent effective dose coefficients were rescaled according to the formalism of the EANM dosage card to determine the radiopharmaceutical class of {sup 68}Ga-labelled peptides (''multiples'') and to calculate the baseline activities based on an upper limit for administered activity (185 MBq) in an adult. All calculated normalization factors suggest that the {sup 68}Ga-labelled peptides are class ''B'' radiopharmaceuticals. The baseline activity for all compounds is 12.8 MBq. In analogy to {sup 18}F-fluoride, we recommend a minimum activity of 14 MBq. For paediatric nuclear medicine applications involving {sup 68}Ga-labelled peptides, we suggest determining administered activities based on the formalism proposed in this work. The corresponding effective doses from these procedures will remain age-independent. (orig.)

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    The aim of the present work was to demonstrate how selenium labelling of a synthetic cell-penetrating peptide may be employed in evaluation of stability and quantitative estimation of cellular uptake by inductively coupled plasma mass spectrometry (ICP-MS). Two analogues of the cell-penetrating p...

  14. Nephrotoxicity in mice after repeated imaging using 111In-labeled peptides.

    NARCIS (Netherlands)

    Melis, M.; Vegt, E.; Konijnenberg, M.W.; Visser, M. de; Bijster, M.; Vermeij, M.; Krenning, E.P.; Boerman, O.C.; Jong, M. de

    2010-01-01

    We determined the renal radiation dose of a series of (111)In-labeled peptides using animal SPECT. Because the animals' health deteriorated, renal toxicity was assessed. METHODS: Wild-type and megalin-deficient mice were imaged repeatedly at 3- to 6-wk intervals to quantify renal retention after inj

  15. Interaction of short peptides with FITC-labeled wheat histones and their complexes with deoxyribooligonucleotides.

    Science.gov (United States)

    Fedoreyeva, L I; Smirnova, T A; Kolomijtseva, G Ya; Khavinson, V Kh; Vanyushin, B F

    2013-02-01

    Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2B, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind predominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation.

  16. Label-free discrimination of membrane-translocating peptides on porous silicon microfluidic biosensors.

    Science.gov (United States)

    Li, Zhen; Luo, Qiaohui; Wu, Jianmin

    2016-11-01

    A label-free optical sensor was constructed by integrating electrochemically etched porous silicon (pSi) and supported phospholipid bilayers in a microfluidic chip. The translocation of peptides through the phospholipid bilayers could induce a red shift in effective optical thickness of the pSi layer, which could be monitored by reflective interferometric Fourier transform spectroscopy. By measuring the kinetic data of membrane translocating on the phospholipid bilayers/pSi chip, the relationship between the behavior of membrane-translocating peptides (MTPs) and translocating mechanism was established. With these optical data, MTPs with different action modes on the cell membrane can be correctly discriminated. The bio-functionalized microfluidic sensor will provide a reliable and cost-effective platform to study the transmembrane behavior of peptides, which is of great importance in the MTP screening and peptide function study.

  17. Peptide tag/probe pairs based on the coordination chemistry for protein labeling.

    Science.gov (United States)

    Uchinomiya, Shohei; Ojida, Akio; Hamachi, Itaru

    2014-02-17

    Protein-labeling methods serve as essential tools for analyzing functions of proteins of interest under complicated biological conditions such as in live cells. These labeling methods are useful not only to fluorescently visualize proteins of interest in biological systems but also to conduct protein and cell analyses by harnessing the unique functions of molecular probes. Among the various labeling methods available, an appropriate binding pair consisting of a short peptide and a de novo designed small molecular probe has attracted attention because of its wide utility and versatility. Interestingly, most peptide tag/probe pairs exploit metal-ligand coordination interactions as the main binding force responsible for their association. Herein, we provide an overview of the recent progress of these coordination-chemistry-based protein-labeling methods and their applications for fluorescence imaging and functional analysis of cellular proteins, while highlighting our originally developed labeling methods. These successful examples clearly exemplify the utility and versatility of metal coordination chemistry in protein functional analysis.

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

    Directory of Open Access Journals (Sweden)

    Nattawut Yotapan

    2014-09-01

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

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

    Science.gov (United States)

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong; Wagenknecht, Hans-Achim; Vilaivan, Tirayut

    2014-01-01

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

  20. Hydrogen atom scrambling in selectively labeled anionic peptides upon collisional activation by MALDI tandem time-of-flight mass spectrometry

    DEFF Research Database (Denmark)

    Bache, Nicolai; Rand, Kasper Dyrberg; Roepstorff, Peter;

    2008-01-01

    have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens ((1)H...

  1. Metabolic flux analysis using ¹³C peptide label measurements.

    Science.gov (United States)

    Mandy, Dominic E; Goldford, Joshua E; Yang, Hong; Allen, Doug K; Libourel, Igor G L

    2014-02-01

    ¹³C metabolic flux analysis (MFA) has become the experimental method of choice to investigate the cellular metabolism of microbes, cell cultures and plant seeds. Conventional steady-state MFA utilizes isotopic labeling measurements of amino acids obtained from protein hydrolysates. To retain spatial information in conventional steady-state MFA, tissues or subcellular fractions must be dissected or biochemically purified. In contrast, peptides retain their identity in complex protein extracts, and may therefore be associated with a specific time of expression, tissue type and subcellular compartment. To enable 'single-sample' spatially and temporally resolved steady-state flux analysis, we investigated the suitability of peptide mass distributions (PMDs) as an alternative to amino acid label measurements. PMDs are the discrete convolution of the mass distributions of the constituent amino acids of a peptide. We investigated the requirements for the unique deconvolution of PMDs into amino acid mass distributions (AAMDs), the influence of peptide sequence length on parameter sensitivity, and how AAMD and flux estimates that are determined through deconvolution compare to estimates from a conventional GC-MS measurement-based approach. Deconvolution of PMDs of the storage protein β-conglycinin of soybean (Glycine max) resulted in good AAMD and flux estimates if fluxes were directly fitted to PMDs. Unconstrained deconvolution resulted in inferior AAMD and flux estimates. PMD measurements do not include amino acid backbone fragments, which increase the information content in GC-MS-derived analyses. Nonetheless, the resulting flux maps were of comparable quality due to the precision of Orbitrap quantification and the larger number of peptide measurements.

  2. Preparation of ⁶⁸Ga-labelled DOTA-peptides using a manual labelling approach for small-animal PET imaging.

    Science.gov (United States)

    Romero, Eduardo; Martínez, Alfonso; Oteo, Marta; García, Angel; Morcillo, Miguel Angel

    2016-01-01

    (68)Ga-DOTA-peptides are a promising PET radiotracers used in the detection of different tumours types due to their ability for binding specifically receptors overexpressed in these. Furthermore, (68)Ga can be produced by a (68)Ge/(68)Ga generator on site which is a very good alternative to cyclotron-based PET isotopes. Here, we describe a manual labelling approach for the synthesis of (68)Ga-labelled DOTA-peptides based on concentration and purification of the commercial (68)Ga/(68)Ga generator eluate using an anion exchange-cartridge. (68)Ga-DOTA-TATE was used to image a pheochromocytoma xenograft mouse model by a microPET/CT scanner. The method described provides satisfactory results, allowing the subsequent (68)Ga use to label DOTA-peptides. The simplicity of the method along with its implementation reduced cost, makes it useful in preclinical PET studies.

  3. Spasmogenic activity of chemotactic N-formylated oligopeptides: identity of structure--function relationships for chemotactic and spasmogenic activities.

    Science.gov (United States)

    Marasco, W A; Fantone, J C; Ward, P A

    1982-12-01

    The chemotactic N-formylated oligopeptides are potent spasmogenic agents for guinea pig ileum. Structure-activity studies with various N-formylated peptides suggest the presence of a specific receptor that resembles in specificity the formyl peptide receptor on leukocytes. A competitive antagonist of the formyl peptide receptor on leukocytes also inhibits formyl peptide-induced ileum contraction, whereas the antihistamine diphenhydramine is without effect. The contractile response caused by the synthetic N-formylated peptides differs from those induced by acetylcholine, histamine, and substance P. In particular, a latent period after treatment with the N-formyl peptides is seen before the onset of the response, and a sustained contractile response is not maintained. In addition, tachyphylaxis does occur, but complete recovery of activity is seen after a 20- to 30-min rest period. These observations suggest broad biological roles of prokaryotic signal peptides from bacteria as acute inflammatory mediators.

  4. Psoriasin: a novel chemotactic protein

    DEFF Research Database (Denmark)

    Jinquan, T; Vorum, H; Larsen, C G;

    1996-01-01

    calcium-binding protein (psoriasin, molecular mass 11,457 Da, pI 6.77) belonging to the S1OO family that is highly upregulated in psoriatic keratinocytes and whose expression patterns implied a role in the inflammatory response. Here we report that human psoriasin is a potent and selective chemotactic...... inflammatory protein for CD4+ T lymphocytes and neutrophils at concentrations of about 10(-11) M. Psoriasin is not structurally related to the alpha or the beta chemokine subfamilies or to lymphotactin, a member of a newly described class of chemokines. Thus, we have observed a chemotactic protein outside...

  5. Exploring the Potential of (99m)Tc(CO)3-Labeled Triazolyl Peptides for Tumor Diagnosis.

    Science.gov (United States)

    Gaonkar, Raghuvir H; Ganguly, Soumya; Baishya, Rinku; Dewanjee, Saikat; Sinha, Samarendu; Gupta, Amit; Ganguly, Shantanu; Debnath, Mita C

    2016-04-01

    In recent years the authors have reported on (99m)Tc(CO)3-labeled peptides that serve as carriers for biomolecules or radiopharmaceuticals to the tumors. In continuation of that work they report the synthesis of a pentapeptide (Met-Phe-Phe-Gly-His; pep-1), a hexapeptide (Met-Phe-Phe-Asp-Gly-His; pep-2), and a tetrapeptide (Asp-Gly-Arg-His; pep-3) and the attachment of 3-amino-1,2,4-triazole to the β carboxylic function of the aspartic acid unit of pep-2 and pep-3. The pharmacophores were radiolabeled in high yields with [(99m)Tc(CO)3(H2O)3](+) metal aqua ion, characterized for their stability in serum and saline, as well as in His solution, and found to be substantially stable. B16F10 cell line binding studies showed favorable uptake and internalization. In vivo behavior of the radiolabeled triazolyl peptides was assessed in mice bearing induced tumor. The (99m)Tc(CO)3-triazolyl pep-3 demonstrated rapid urinary clearance and comparatively better tumor uptake. Imaging studies showed visualization of the tumor using (99m)Tc(CO)3-triazolyl pep-3, but due to high abdominal background, low delineation occurred. Based on the results further experiments will be carried out for targeting tumor with triazolyl peptides.

  6. Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

    Science.gov (United States)

    Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

    2011-11-01

    Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

  7. 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.

  8. Optimization of labelling PSMA-HBED-CC peptide with {sup 68}Ga

    Energy Technology Data Exchange (ETDEWEB)

    Alcarde, Lais F.; Dias, Luis A.P.; Massicano, Adriana V.F.; Mengatti, Jair; Araujo, Elaine B. de, E-mail: lais_alcarde@hotmail.com.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2015-07-01

    Early detection of metastases or recurrent prostate cancer (PC) lesions is of clinical relevance in terms of clinical staging, prognosis and therapy management. When PC is not treated, it is potentially lethal. Clinical methods for diagnosis of PC include the dosage of prostatic specific antigen (PSA) and the rectal touch. Unfortunately, these initial procedures are not specific for PC detection. The level of PSA, in about 20 to 30% of the cases is high, due to benign pathologies, that result in false positive and unneeded biopsy. The prostatic specific membrane antigen (PSMA) is a type II transmembrane glycoprotein and differs from the PSA that is a free protein in blood. High levels of PSMA are observed in almost all prostatic pathologies and low levels were observed in brain, kidneys, salivary glands and small intestine. This fact stimulated the development of PSMA inhibitor molecules that could be used as a vector for imaging tumor agents and that could perfuse in the tumor microvasculature. Recent studies suggest that the chelator HBED-CC contributes intrinsically for the labelling of the PSMA inhibitor peptide based in urea - Glu-urea-Lys (Ahx) – to the pharmacophore group. This work describes the study of labelling conditions of PSMA-HBED-CC with {sup 68}Ga and determined the ideal conditions to obtaining the high radiochemical purity (≥ 95%) and stability, without final purification, and stimulates the in vitro and in vivo evaluation to determine the potential of the radiopharmaceutical for clinical application. (author)

  9. Stable isotope N-phosphorylation labeling for Peptide de novo sequencing and protein quantification based on organic phosphorus chemistry.

    Science.gov (United States)

    Gao, Xiang; Wu, Hanzhi; Lee, Kim-Chung; Liu, Hongxia; Zhao, Yufen; Cai, Zongwei; Jiang, Yuyang

    2012-12-04

    In this paper, we describe the development of a novel stable isotope N-phosphorylation labeling (SIPL) strategy for peptide de novo sequencing and protein quantification based on organic phosphorus chemistry. The labeling reaction could be performed easily and completed within 40 min in a one-pot reaction without additional cleanup procedures. It was found that N-phosphorylation labeling reagents were activated in situ to form labeling intermediates with high reactivity targeting on N-terminus and ε-amino groups of lysine under mild reaction conditions. The introduction of N-terminal-labeled phosphoryl group not only improved the ionization efficiency of peptides and increased the protein sequence coverage for peptide mass fingerprints but also greatly enhanced the intensities of b ions, suppressed the internal fragments, and reduced the complexity of the tandem mass spectrometry (MS/MS) fragmentation patterns of peptides. By using nano liquid chromatography chip/time-of-flight mass spectrometry (nano LC-chip/TOF MS) for the protein quantification, the obtained results showed excellent correlation of the measured ratios to theoretical ratios with relative errors ranging from 0.5% to 6.7% and relative standard deviation of less than 10.6%, indicating that the developed method was reproducible and precise. The isotope effect was negligible because of the deuterium atoms were placed adjacent to the neutral phosphoryl group with high electrophilicity and moderately small size. Moreover, the SIPL approach used inexpensive reagents and was amenable to samples from various sources, including cell culture, biological fluids, and tissues. The method development based on organic phosphorus chemistry offered a new approach for quantitative proteomics by using novel stable isotope labeling reagents.

  10. Preprocessing significantly improves the peptide/protein identification sensitivity of high-resolution isobarically labeled tandem mass spectrometry data.

    Science.gov (United States)

    Sheng, Quanhu; Li, Rongxia; Dai, Jie; Li, Qingrun; Su, Zhiduan; Guo, Yan; Li, Chen; Shyr, Yu; Zeng, Rong

    2015-02-01

    Isobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994.

  11. Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

    Directory of Open Access Journals (Sweden)

    Cai-Xia Zhuo

    2016-11-01

    Full Text Available Trypsin is important during the regulation of pancreatic exocrine function. The detection of trypsin activity is currently limited because of the need for the substrate to be labeled with a fluorescent tag. A label-free fluorescent method has been developed to monitor trypsin activity. The designed peptide probe consists of six arginine molecules and a cysteine terminus and can be conjugated to DNA-stabilized silver nanoclusters (DNA-AgNCs by Ag-S bonding to enhance fluorescence. The peptide probe can also be adsorbed to the surface of graphene oxide (GO, thus resulting in the fluorescence quenching of DNA-AgNCs-peptide conjugate because of Förster resonance energy transfer. Once trypsin had degraded the peptide probe into amino acid residues, the DNA-AgNCs were released from the surface of GO, and the enhanced fluorescence of DNA-AgNCs was restored. Trypsin can be determined with a linear range of 0.0–50.0 ng/mL with a concentration as low as 1 ng/mL. This label-free method is simple and sensitive and has been successfully used for the determination of trypsin in serum. The method can also be modified to detect other proteases.

  12. Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling.

    Science.gov (United States)

    Dong, Maoqing; Miller, Laurence J

    2013-08-01

    Direct analysis of mode of peptide docking using intrinsic photoaffinity labeling has provided detailed insights for the molecular basis of cholecystokinin (CCK) interaction with the type 1 CCK receptor. In the current work, this technique has been applied to the closely related type 2 CCK receptor that also binds the natural full agonist peptide, CCK, with high affinity. A series of photolabile CCK analog probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogs that possessed full biological activity utilized in photoaffinity labeling. The position 29 probe, incorporating a photolabile benzoyl-phenylalanine in that position, was shown to bind with high affinity and to be a full agonist, with potency not different from that of natural CCK, and to covalently label the type 2 CCK receptor in a saturable, specific and efficient manner. Using proteolytic peptide mapping, mutagenesis, and radiochemical Edman degradation sequencing, this probe was shown to establish a covalent bond with type 2 CCK receptor residue Phe¹²⁰ in the first extracellular loop. This was in contrast to its covalent attachment to Glu³⁴⁵ in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors.

  13. Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate.

    Science.gov (United States)

    Kyro, Kelly; Manandhar, Surya P; Mullen, Daniel; Schmidt, Walter K; Distefano, Mark D

    2010-08-01

    Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras converting enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site.

  14. SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides

    Institute of Scientific and Technical Information of China (English)

    ZHAI Xiao-hui; LIU Min; GUO Xiao-juan; WANG Si-cen; ZHANG Hong-xia; GUO You-min

    2011-01-01

    Background The hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines,SKOV-3.Methods hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid gadolinium (Ⅲ) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM).Results The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r=0.990, P <0.01) and concentration-dependent manner (r=0.964, P <0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium.Conclusions hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

  15. A strategy with label-free quantification of the targeted peptides for quantitative peptidome analysis of human serum

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease. However, there is lack of efficient quantification strategy in peptidome analysis. In this study, a strategy with label-free quantification of the targeted endogenous peptides based on peak intensity using μUPLC-Q-TOF-MS/MS was developed for quantitative peptidome analysis of human serum. Different amounts of standard BSA tryptic digesting peptides were added into the same serum extracts for evaluation of the developed strategy, and it was observed that the average relative error of the targeted peptides was 6.42%, which was superior to the result obtained directly by commercially available software PLGS. It was also demonstrated that this quantification strategy could obviously increase the detection sensitivity of the peptide by DDA analysis. Then, this strategy was applied to comparatively analyze the peptides extracted from the serum of HCC or breast cancer patients and healthy individuals, respectively. Peptides with charge states up to 5 and molecular weight over 4000 can be reliably identified and quantified. This quantitative analysis method based on μUPLC-Q-TOF-MS/MS exhibited superior sensitivity than that by MALDI-TOF-MS commonly used in peptidome analysis. Finally, some interesting endogenous peptides related to corresponding diseases were successfully obtained.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Technetium-99m labelled fluconazole and antimicrobial peptides for imaging of Candida albicans and Aspergillus fumigatus infections

    Energy Technology Data Exchange (ETDEWEB)

    Lupetti, Antonella [Department of Infectious Diseases, Leiden University Medical Center (LUMC), Leiden (Netherlands); Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Univ. di Pisa (Italy); Welling, Mick M. [Department of Radiology, Division of Nuclear Medicine, LUMC, Leiden (Netherlands); Mazzi, Ulderico [Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Padova (Italy); Nibbering, Peter H. [Department of Infectious Diseases, Leiden University Medical Center (LUMC), Leiden (Netherlands); Pauwels, Ernest K.J. [Department of Radiology, Division of Nuclear Medicine, LUMC, Leiden (Netherlands); Department of Radiology, Leiden University Medical Center (LUMC) (Netherlands)

    2002-05-01

    The aim of this study was to investigate whether technetium-99m labelled fluconazole can distinguish fungal from bacterial infections. Fluconazole was labelled with {sup 99m}Tc and radiochemical analysis showed less than 5% impurities. The labelling solution was injected into animals with experimental infections. For comparison, we used two peptides for infection detection, i.e. UBI 29-41 and hLF 1-11, and human IgG, all labelled with {sup 99m}Tc. Mice were infected with Candida albicans or injected with heat-killed C. albicans or lipopolysaccharides to induce sterile inflammation. Also, mice were infected with Staphylococcus aureus or Klebsiella pneumoniae. Next, accumulation of {sup 99m}Tc-fluconazole and {sup 99m}Tc-labelled peptides/IgG at affected sites was determined scintigraphically. {sup 99m}Tc-fluconazole detected C. albicans infections (T/NT ratio=3.6{+-}0.47) without visualising bacterial infections (T/NT ratio=1.3{+-}0.04) or sterile inflammatory processes (heat-killed C. albicans: T/NT ratio=1.3{+-}0.2; lipopolysaccharide: T/NT ratio=1.4{+-}0.1). C. albicans infections were already seen within the first hour after injection of {sup 99m}Tc-fluconazole (T/NT ratio=3.1{+-}0.2). A good correlation (R{sup 2}=0.864; P<0.05) between T/NT ratios for this tracer and the number of viable C. albicans was found. Although {sup 99m}Tc-UBI 29-41 and {sup 99m}Tc-hLF 1-11 were able to distinguish C. albicans infections from sterile inflammatory processes in mice, these {sup 99m}Tc-labelled peptides did not distinguish these fungal infections from bacterial infections. It is concluded that {sup 99m}Tc-fluconazole distinguishes infections with C. albicans from bacterial infections and sterile inflammations. (orig.)

  18. PET imaging of CXCR4 using copper-64 labeled peptide antagonist

    Directory of Open Access Journals (Sweden)

    Orit Jacobson, Ido D. Weiss, Lawrence P. Szajek, Gang Niu, Ying Ma, Dale O. Kiesewetter, Joshua M. Farber, Xiaoyuan Chen

    2011-01-01

    Full Text Available Expression of CXCR4 in cancer has been found to correlate with poor prognosis and resistance to chemotherapy. In this study we developed a derivative of the CXCR4 peptide antagonist, T140-2D, that can be labeled easily with the PET isotope copper-64, and thereby enable in vivo visualization of CXCR4 in tumors. T140 was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester (DOTA-NHS to give T140-2D, which contains a DOTA molecule on each of the two lysine residues. 64Cu-T140-2D was evaluated in vitro by migration and binding experiments, and in vivo by microPET imaging and biodistribution, in mice bearing CXCR4-positive and CXCR4-negative tumor xenografts. T140-2D was labeled with copper-64 to give 64Cu-T140-2D in a high radiochemical yield of 86 ± 3% (not decay-corrected and a specific activity of 0.28 - 0.30 mCi/µg (10.36 - 11.1 MBq/µg. 64Cu-T140-2D had antagonistic and binding characteristics to CXCR4 that were similar to those of T140. In vivo, 64Cu-T140-2D tended to bind to red blood cells and had to be used in a low specific activity form. In this new form 64Cu-T140-2D enabled specific imaging of CXCR4-positive, but not CXCR4-negative tumors. Undesirably, however, 64Cu-T140-2D also displayed high accumulation in the liver and kidneys. In conclusion, 64Cu-T140-2D was easily labeled and, in its low activity form, enabled imaging of CXCR4 in tumors. It had high uptake, however, in metabolic organs. Further research with imaging tracers targeting CXCR4 is required.

  19. PET imaging of CXCR4 using copper-64 labeled peptide antagonist.

    Science.gov (United States)

    Jacobson, Orit; Weiss, Ido D; Szajek, Lawrence P; Niu, Gang; Ma, Ying; Kiesewetter, Dale O; Farber, Joshua M; Chen, Xiaoyuan

    2011-01-01

    Expression of CXCR4 in cancer has been found to correlate with poor prognosis and resistance to chemotherapy. In this study we developed a derivative of the CXCR4 peptide antagonist, T140-2D, that can be labeled easily with the PET isotope copper-64, and thereby enable in vivo visualization of CXCR4 in tumors. T140 was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) to give T140-2D, which contains a DOTA molecule on each of the two lysine residues. (64)Cu-T140-2D was evaluated in vitro by migration and binding experiments, and in vivo by microPET imaging and biodistribution, in mice bearing CXCR4-positive and CXCR4-negative tumor xenografts. T140-2D was labeled with copper-64 to give (64)Cu-T140-2D in a high radiochemical yield of 86 ± 3% (not decay-corrected) and a specific activity of 0.28 - 0.30 mCi/µg (10.36 - 11.1 MBq/µg). (64)Cu-T140-2D had antagonistic and binding characteristics to CXCR4 that were similar to those of T140. In vivo, (64)Cu-T140-2D tended to bind to red blood cells and had to be used in a low specific activity form. In this new form (64)Cu-T140-2D enabled specific imaging of CXCR4-positive, but not CXCR4-negative tumors. Undesirably, however, (64)Cu-T140-2D also displayed high accumulation in the liver and kidneys. In conclusion, (64)Cu-T140-2D was easily labeled and, in its low activity form, enabled imaging of CXCR4 in tumors. It had high uptake, however, in metabolic organs. Further research with imaging tracers targeting CXCR4 is required.

  20. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    Science.gov (United States)

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-02-07

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  1. Therapeutic Efficacy with Treatment-related Toxicities of {sup 177}Lu-labeled Bombesin Derivative for the Peptide Receptor Radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Jae Cheong; Cho, Eun Ha; Lee, So Young [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-05-15

    The gastrin-releasing peptide receptor (GRPR) has been shown to be overexpressed in many human tumours, including breast cancer, prostate cancer, small cell lung cancer, ovarian cancers, endometrial cancers, and gastrointestinal stromal tumors. In particular, GRPR expression is high in 83 % of invasive primary prostatic carcinomas. These results suggest that {sup 177}Lu-labeled bombesin derivative has promising characteristics as a novel nuclear medicine, especially for the treatment of GRPR over-expressing prostate tumors.

  2. [The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

    Science.gov (United States)

    Zolotarev, A; Dadayan, A K; Kost, N V; Voevodina, M E; Sokolov, O Y; Kozik, V S; Shram, S I; Azev, V N; Bocharov, E V; Bogachouk, A P; Lipkin, V M; Myasoedov, N F

    2015-01-01

    The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

  3. The effect of superparamagnetic iron oxide with iRGD peptide on the labeling of pancreatic cancer cells in vitro: a preliminary study.

    Science.gov (United States)

    Zuo, Hou Dong; Yao, Wei Wu; Chen, Tian Wu; Zhu, Jiang; Zhang, Juan Juan; Pu, Yu; Liu, Gang; Zhang, Xiao Ming

    2014-01-01

    The iRGD peptide loaded with iron oxide nanoparticles for tumor targeting and tissue penetration was developed for targeted tumor therapy and ultrasensitive MR imaging. Binding of iRGD, a tumor homing peptide, is mediated by integrins, which are widely expressed on the surface of cells. Several types of small molecular drugs and nanoparticles can be transfected into cells with the help of iRGD peptide. Thus, we postulate that SPIO nanoparticles, which have good biocompatibility, can also be transfected into cells using iRGD. Despite the many kinds of cell labeling studies that have been performed with SPIO nanoparticles and RGD peptide or its analogues, only a few have applied SPIO nanoparticles with iRGD peptide in pancreatic cancer cells. This paper reports our preliminary findings regarding the effect of iRGD peptide (CRGDK/RGPD/EC) combined with SPIO on the labeling of pancreatic cancer cells. The results suggest that SPIO with iRGD peptide can enhance the positive labeling rate of cells and the uptake of SPIO. Optimal functionalization was achieved with the appropriate concentration or concentration range of SPIO and iRGD peptide. This study describes a simple and economical protocol to label panc-1 cells using SPIO in combination with iRGD peptide and may provide a useful method to improve the sensitivity of pancreatic cancer imaging.

  4. The Effect of Superparamagnetic Iron Oxide with iRGD Peptide on the Labeling of Pancreatic Cancer Cells In Vitro: A Preliminary Study

    Directory of Open Access Journals (Sweden)

    Hou Dong Zuo

    2014-01-01

    Full Text Available The iRGD peptide loaded with iron oxide nanoparticles for tumor targeting and tissue penetration was developed for targeted tumor therapy and ultrasensitive MR imaging. Binding of iRGD, a tumor homing peptide, is mediated by integrins, which are widely expressed on the surface of cells. Several types of small molecular drugs and nanoparticles can be transfected into cells with the help of iRGD peptide. Thus, we postulate that SPIO nanoparticles, which have good biocompatibility, can also be transfected into cells using iRGD. Despite the many kinds of cell labeling studies that have been performed with SPIO nanoparticles and RGD peptide or its analogues, only a few have applied SPIO nanoparticles with iRGD peptide in pancreatic cancer cells. This paper reports our preliminary findings regarding the effect of iRGD peptide (CRGDK/RGPD/EC combined with SPIO on the labeling of pancreatic cancer cells. The results suggest that SPIO with iRGD peptide can enhance the positive labeling rate of cells and the uptake of SPIO. Optimal functionalization was achieved with the appropriate concentration or concentration range of SPIO and iRGD peptide. This study describes a simple and economical protocol to label panc-1 cells using SPIO in combination with iRGD peptide and may provide a useful method to improve the sensitivity of pancreatic cancer imaging.

  5. The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aβ peptide.

    Directory of Open Access Journals (Sweden)

    Jitka Petrlova

    Full Text Available BACKGROUND: The deposition and oligomerization of amyloid β (Aβ peptide plays a key role in the pathogenesis of Alzheimer's disease (AD. Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species. METHODOLOGY/PRINCIPAL FINDINGS: To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species. CONCLUSIONS: Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they possess both antioxidant properties and the ability to disrupt AβO species.

  6. Method for the Determination of ¹⁵N Incorporation Percentage in Labeled Peptides and Proteins.

    Science.gov (United States)

    Kilpatrick, Eric L

    2016-01-01

    Use of labeled (15)N proteins and peptides as internal standards in isotope-dilution mass spectrometry for the quantification of proteins has been increasing and is now accepted as a gold standard for this analysis. As a necessary reagent in this process, stable heavy isotope-labeled internal standards must be rigorously characterized in a number of ways including identity, concentration, purity, and structure. Additionally, the degree of the incorporation of the heavy isotope is a critical feature to consider. For proteins that are (15)N labeled, the percentage of incorporation is a valid measurement used to assess the fitness-to-purpose of the material. This measurement should be objective, repeatable, and based on empirical analysis. One means of assigning this value is to compare a mass spectrum of the isotopic profile of a peptide against a series of theoretical profiles containing different enrichment rates. This comparison can be made using the Pearson product-moment correlation coefficient (r) to find the best match between the empirical and theoretical profiles. Theoretical profiles can be generated using probability multinomial analysis but are computationally intensive and require the use of computers for practical use. The method described in this chapter describes the development and use of a computer program to calculate the percentage of (15)N enrichment of a labeled internal standard. Additionally, methods will be described for the empirical determination of an isotopic profile using a variety of mass spectrometry techniques.

  7. Functional endothelial cells derived from embryonic stem cells labeled with HIV transactivator peptide-conjugated superparamagnetic nanoparticles

    Institute of Scientific and Technical Information of China (English)

    GAO Bin; FU Wei-guo; DONG Zhi-hui; FANG Zheng-dong; LIU Zhen-jie; SI Yi; ZHANG Xiang-man; WANG Yu-qi

    2011-01-01

    Background The development of regenerative therapies using derivatives of embryonic stem (ES) cells would be facilitated by a non-invasive method to monitor transplanted cells in vivo,for example,magnetic resonance imaging of cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles.Although ES cells have been labeled with SPIO particles,the potential adverse effects of the label have not been fully examined.The objective of this study was to determine whether SPIO labeling affects murine ES cell viability,proliferation,or ability to differentiate into functional endothelial cells (ECs).Methods Cross-linked iron oxide (CLIO,an SPIO) was conjugated with human immunodeficiency virus transactivator of transcription (HIV-Tat) peptides,and murine ES cells were labeled with either CLiO-Tat,CLIO,or HIV-Tat.After labeling,ES cells were cultured for 4 days and FIk-1+ ES cells identified and sorted by immunocytochemistry and fluorescence activated cell sorting (FACS).FIk-1+ cells were raplated on fibronectin-coated dishes,and ECs were obtained by culturing these for 4 weeks in endothelial cell growth medium supplemented with vascular endothelial growth factor (VEGF).ES cell viability was determined using trypan blue exclusion,and the proportion of SPIO+ cells was evaluated using Prussian blue staining and transmission electron microscopy.After differentiation,the behavior and phenotype of ECs were analyzed by reverse transcription-polymerase chain reaction,flow cytometry,immunocytochemistry,Dil-labeled acetylated low-density lipoprotein (AcLDL) uptake,and Matrigel tube formation assay.Results CLIO-Tat was a highly effective label for ES cells,with >96% of cells incorporating the particles,and it did not alter the viability of the labeled cells.ECs derived from CLIO-Tat+ ES cells were very similar to murine aortic ECs in their morphology,expression of endothelial cell markers,ability to form vascular-like channels,and scavenging of AcLDL from the culture medium

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

    Science.gov (United States)

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

    2003-12-30

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

  9. Imaging integrin alpha-v-beta-3 expression in tumors with an 18F-labeled dimeric RGD peptide

    Science.gov (United States)

    Dijkgraaf, Ingrid; Terry, Samantha; McBride, William J.; Goldenberg, David M.; Laverman, Peter; Franssen, Gerben M.; Oyen, Wim J. G.; Boerman, Otto C.

    2014-01-01

    Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine-glycine-aspartatic acid (RGD) peptide and integrin αvβ3, but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of 18F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with 18F in a simple one-pot process with a radiolabeling yield of 20%; the whole process lasting only 45 min. NODAGA-E-[c(RGDfK)]2 labeled with 18F at a specific activity of 1.8 MBq/nmol and a radiochemical purity of 100% could be achieved. Log P value of 18F-labeled NODAGA-E-[c(RGDfK)]2 was −4.26 ± 0.02. In biodistribution studies, 18F-NODAGA-E-[c(RGDfK)]2 cleared rapidly from the blood with 0.03 ± 0.01 %ID/g in the blood at 2 h p.i., mainly via the kidneys and showed good in vivo stability. Tumor uptake of 18F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID/g, 2 h p.i.) was significantly lower than that of reference compounds 68Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID/g; P <0.001) and 111In-labeled NODAGA-E-[c(RGDfK)]2 (4.99 ± 0.64 %ID/g; P < 0.01). Co-injection of an excess of unlabeled NODAGA-E-[c(RGDfK)]2 along with 18F-NODAGA-E-[c(RGDfK)]2 resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID/g). The αvβ3 integrin-expressing SK-RC-52 tumor could be successfully visualized by microPET with 18F-labeled NODAGA-E-[c(RGDfK)]2. In conclusion, NODAGA-E-[c(RGDfK)]2 could be labeled rapidly with 18F using a direct aqueous, one-pot method and it accumulated specifically in αvβ3 integrin-expressing SK-RC-52 tumors, allowing for visualization by microPET. PMID:23606427

  10. Label-free SPR detection of gluten peptides in urine for non-invasive celiac disease follow-up.

    Science.gov (United States)

    Soler, Maria; Estevez, M-Carmen; Moreno, Maria de Lourdes; Cebolla, Angel; Lechuga, Laura M

    2016-05-15

    Motivated by the necessity of new and efficient methods for dietary gluten control of celiac patients, we have developed a simple and highly sensitive SPR biosensor for the detection of gluten peptides in urine. The sensing methodology enables rapid and label-free quantification of the gluten immunogenic peptides (GIP) by using G12 mAb. The overall performance of the biosensor has been in-depth optimized and evaluated in terms of sensitivity, selectivity and reproducibility, reaching a limit of detection of 0.33 ng mL(-1). Besides, the robustness and stability of the methodology permit the continuous use of the biosensor for more than 100 cycles with excellent repeatability. Special efforts have been focused on preventing and minimizing possible interferences coming from urine matrix enabling a direct analysis in this fluid without requiring extraction or purification procedures. Our SPR biosensor has proven to detect and identify gluten consumption by evaluating urine samples from healthy and celiac individuals with different dietary gluten conditions. This novel biosensor methodology represents a novel approach to quantify the digested gluten peptides in human urine with outstanding sensitivity in a rapid and non-invasive manner. Our technique should be considered as a promising opportunity to develop Point-of-Care (POC) devices for an efficient, simple and accurate gluten free diet (GFD) monitoring as well as therapy follow-up of celiac disease patients.

  11. Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure

    Energy Technology Data Exchange (ETDEWEB)

    Altman, Robin [Univ. of California, Davis, CA (United States). Dept. of Biochemistry and Molecular Medicine; Ly, Sonny [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Physical and Life Science Directorate; Hilt, Silvia [Univ. of California, Davis, CA (United States). Dept. of Biochemistry and Molecular Medicine; Petrlova, Jitka [Univ. of California, Davis, CA (United States). Dept. of Biochemistry and Molecular Medicine; Maezawa, Izumi [Univ. of California Davis, Sacramento, CA (United States). MIND Inst. and Dept. of Pathology and Laboratory Medicine; Kálai, Tamás [Univ. of Pecs (Hungary). Inst. of Organic and Medicinal Chemistry; Hideg, Kálmán [Univ. of Pecs (Hungary). Inst. of Organic and Medicinal Chemistry; Jin, Lee-Way [Univ. of California Davis, Sacramento, CA (United States). MIND Inst. and Dept. of Pathology and Laboratory Medicine; Laurence, Ted A. [Univ. of California, Davis, CA (United States). Dept. of Biochemistry and Molecular Medicine; Voss, John C. [Univ. of California, Davis, CA (United States). Dept. of Biochemistry and Molecular Medicine

    2015-12-01

    Alzheimer’s disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aβ) peptides. Soluble oligomers of the Aβ peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aβ oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aβ) prevent and disrupt oligomeric assemblies of Aβ in solution. The FCS results, combined with electron paramagnetic resonance spectroscopy and circular dichroism spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers while retaining Aβ in a largely disordered state. Furthermore, while the ability of SLF to block Aβ toxicity correlates with a reduction in oligomer size, our results suggest the conformation of Aβ within the oligomer determines the toxicity of the species. Attenuation of Aβ toxicity, which has been associated primarily with the soluble oligomeric form, can be achieved through redistribution of the peptides into smaller oligomers and arrest of the fractional increase in beta secondary structure.

  12. An (125)I-labeled octavalent peptide fluorescent nanoprobe for tumor-homing imaging in vivo.

    Science.gov (United States)

    Luo, Haiming; Shi, Jiyun; Jin, Honglin; Fan, Di; Lu, Lisen; Wang, Fan; Zhang, Zhihong

    2012-06-01

    Targeting radiopeptides are promising agents for radio-theranostics. However, in vivo evaluation of their targeting specificity is often obscured by their short biologic half-lives and low binding affinities. Here, we report an approach to efficiently examine targeting radiopeptides with a new class of octavalent peptide fluorescent nanoprobe (Octa-FNP) platform, which is composed of candidate targeting peptides and a tetrameric far-red fluorescent protein (tfRFP) scaffold. To shed light on this process, (125)I-Octa-FNP, (125)I-tfRFP and (125)I-peptide were synthesized, and their targeting functionalities were compared. Both fluorescence imaging and radioactive quantification results confirmed that (125)I-Octa-FNP had a significantly higher cellular binding capability than (125)I-tfRFP. In vivo biodistribution studies show that at 6 h post-injection, (125)I-Octa-FNP had 2-fold and 30-fold higher tumor uptake than that of (125)I-tfRFP and (125)I-peptide, respectively. Moreover, γ-imaging at 24 h post-injection revealed a remarkable accumulation of (125)I-Octa-FNP in the tumor while maintaining an extremely low background contrast, which was further confirmed by immunofluorescence analysis. These data suggested that, as an engineered and multivalent platform, Octa-FNP could enhance the tumor targeting of a designed peptide and provide excellent contrast radioimaging, making it a valuable tool for the evaluation of the targeting ability of specifically designed radiopeptides for cancer theranostics.

  13. Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe

    Directory of Open Access Journals (Sweden)

    Lei Zhu, Ning Guo, Quanzheng Li, Ying Ma, Orit Jacboson, Seulki Lee, Hak Soo Choi, James R. Mansfield, Gang Niu, Xiaoyuan Chen

    2012-01-01

    Full Text Available Purpose: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/64Cu dual-labeled cyclic RGD peptide.Methods: The integrin αvβ3 binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data.Results: The dual-labeled probe 64Cu-RGD-C(DOTA-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp derived from dynamic optical imaging (1.762 ± 0.020 is comparable to that from dynamic PET (1.752 ± 0.026.Conclusion: The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models.

  14. DIVERSE System: De Novo Creation of Peptide Tags for Non-enzymatic Covalent Labeling by In Vitro Evolution for Protein Imaging Inside Living Cells.

    Science.gov (United States)

    Kawakami, Takashi; Ogawa, Koji; Goshima, Naoki; Natsume, Tohru

    2015-12-17

    Polypeptide-tag/small-molecule pairs for specific cellular protein labeling are useful for visualizing cellular proteins and controlling their activity. Here, we report the development of an in vitro evolution-based (poly)peptide tag identification system named the DIVERSE (Directed In Vitro Evolution of Reactive peptide tags via Sequential Enrichment) system. In this system, an extremely diverse (10(14)) library of peptide tags, displayed by covalent attachment to their encoding cDNAs, is continuously prepared from the DNA library in a one-pot approach. Using this system, we demonstrated de novo creation of non-enzymatically covalent-labeling peptide tags for a synthetic small-molecule target from a random peptide library. Protein labeling with these tags was applicable to N- and C-terminal fusions, multiple different proteins and fluorophores, and intracellular labeling. The DIVERSE system can be used not only for the de novo creation of polypeptide tags but also sequence optimization of existing polypeptide tags from extremely diverse libraries.

  15. Label-free detection of biomolecular interaction — DNA — Antimicrobial peptide binding

    DEFF Research Database (Denmark)

    Fojan, Peter; Jensen, Kasper Risgaard; Gurevich, Leonid

    2011-01-01

    an interest in Antimicrobial peptides that are active against broad range of infections including bacteria, fungi and viruses and were shown to be capable of treating multi-resistant infection either alone or in combination with the conventional antibiotics. In this paper , we demonstrate an application...

  16. Nephrotoxicity in mice after repeated imaging using 111In- labeled peptides

    NARCIS (Netherlands)

    M.L. Melis (Marleen); E. Vegt (Erik); M.W. Konijnenberg (Mark); M. de Visser (Monique); M. Bijster (Magda); M. Vermeij (Marcel); E.P. Krenning (Eric); O.C. Boerman (Otto); M. de Jong (Marion)

    2010-01-01

    textabstractWe determined the renal radiation dose of a series of111Inlabeled peptides using animal SPECT. Because the animals' health deteriorated, renal toxicity was assessed. Methods: Wild-type and megalin-deficient mice were imaged repeatedly at 3- to 6-wk intervals to quantify renal retention a

  17. Fluorescently labeled peptide increases identification of degenerated facial nerve branches during surgery and improves functional outcome.

    Directory of Open Access Journals (Sweden)

    Timon Hussain

    Full Text Available Nerve degeneration after transection injury decreases intraoperative visibility under white light (WL, complicating surgical repair. We show here that the use of fluorescently labeled nerve binding probe (F-NP41 can improve intraoperative visualization of chronically (up to 9 months denervated nerves. In a mouse model for the repair of chronically denervated facial nerves, the intraoperative use of fluorescent labeling decreased time to nerve identification by 40% compared to surgeries performed under WL alone. Cumulative functional post-operative recovery was also significantly improved in the fluorescence guided group as determined by quantitatively tracking of the recovery of whisker movement at time intervals for 6 weeks post-repair. To our knowledge, this is the first description of an injectable probe that increases visibility of chronically denervated nerves during surgical repair in live animals. Future translation of this probe may improve functional outcome for patients with chronic denervation undergoing surgical repair.

  18. Rapid 'de novo' peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer.

    Science.gov (United States)

    Shevchenko, A; Chernushevich, I; Ens, W; Standing, K G; Thomson, B; Wilm, M; Mann, M

    1997-01-01

    Protein microanalysis usually involves the sequencing of gel-separated proteins available in very small amounts. While mass spectrometry has become the method of choice for identifying proteins in databases, in almost all laboratories 'de novo' protein sequencing is still performed by Edman degradation. Here we show that a combination of the nanoelectrospray ion source, isotopic end labeling of peptides and a quadrupole/ time-of-flight instrument allows facile read-out of the sequences of tryptic peptides. Isotopic labeling was performed by enzymatic digestion of proteins in 1:1 16O/18O water, eliminating the need for peptide derivatization. A quadrupole/time-of-flight mass spectrometer was constructed from a triple quadrupole and an electrospray time-of-flight instrument. Tandem mass spectra of peptides were obtained with better than 50 ppm mass accuracy and resolution routinely in excess of 5000. Unique and error tolerant identification of yeast proteins as well as the sequencing of a novel protein illustrate the potential of the approach. The high data quality in tandem mass spectra and the additional information provided by the isotopic end labeling of peptides enabled automated interpretation of the spectra via simple software algorithms. The technique demonstrated here removes one of the last obstacles to routine and high throughput protein sequencing by mass spectrometry.

  19. Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry.

    Science.gov (United States)

    Niu, Mingming; Cho, Ji-Hoon; Kodali, Kiran; Pagala, Vishwajeeth; High, Anthony A; Wang, Hong; Wu, Zhiping; Li, Yuxin; Bi, Wenjian; Zhang, Hui; Wang, Xusheng; Zou, Wei; Peng, Junmin

    2017-02-22

    Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from coisolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection, and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT)-labeled Escherichia coli peptides at 1:3:10 ratios and added in ∼20-fold more rat peptides as background, followed by the analysis of two-dimensional liquid chromatography (LC)-MS/MS. Systematic investigation shows that quantitative interference was impacted by LC fractionation depth, MS isolation window, and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10 000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  1. Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye.

    Science.gov (United States)

    Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks

    2016-09-15

    Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.

  2. A photoaffinity analogue of discodermolide specifically labels a peptide in beta-tubulin.

    Science.gov (United States)

    Xia, Shujun; Kenesky, Craig S; Rucker, Paul V; Smith, Amos B; Orr, George A; Horwitz, Susan Band

    2006-10-01

    Discodermolide is a potentially important antitumor agent that stabilizes microtubules and blocks cells at the G2/M phase of the cell cycle in a manner similar to that of Taxol. Discodermolide also has unique properties that distinguish it from Taxol. In the present study, photoaffinity-labeled discodermolide analogues are used to investigate their binding site in tubulin. Three photoaffinity-labeled discodermolide analogues were synthesized, all of which promoted microtubule polymerization in the absence of GTP. The analogue, C19-[4-(4-(3)H-benzoyl-phenyl)-carbamate]-discodermolide (C19-[3H]BPC-discodermolide), was selected for photolabeling studies because it had the highest extent of photoincorporation, approximately 1%, of the three radiolabeled discodermolide analogues explored. Although compared to discodermolide, C19-BPC-discodermolide revealed no hypernucleation effect in the in vitro microtubule polymerization assay, it was more cytotoxic than discodermolide, and, like discodermolide, demonstrated synergism with Taxol. These results suggest that the hypernucleation effect of discodermolide is not involved in its cytotoxic activity. Similar to discodermolide, C19-BPC-discodermolide can effectively displace [3H]Taxol from microtubules, but Taxol cannot effectively displace C19-[3H]BPC-discodermolide binding. Discodermolide can effectively displace C19-[3H]BPC-discodermolide binding. Formic acid hydrolysis, immunoprecipitation experiments, and subtilisin digestion indicate that C19-BPC-discodermolide labels amino acid residues 305-433 in beta-tubulin. Further digestion with Asp-N and Arg-C enzymes suggested that C19-BPC-discodermolide binds to amino acid residues, 355-359, in beta-tubulin, which is in close proximity to the Taxol binding site. Molecular modeling guided by the above evidence led to a putative binding model for C19-BPC-discodermolide in tubulin.

  3. Synthesis and evaluation of a technetium-99m labeled cytotoxic bombesin peptide conjugate for targeting bombesin receptor-expressing tumors

    Energy Technology Data Exchange (ETDEWEB)

    Okarvi, Subhani M. [Cyclotron and Radiopharmaceuticals Department, King Faisal Specialist Hospital and Research Centre, MBC-03, PO Box 3354, Riyadh 11211 (Saudi Arabia)], E-mail: sokarvi@kfshrc.edu.sa; Al Jammaz, Ibrahim [Cyclotron and Radiopharmaceuticals Department, King Faisal Specialist Hospital and Research Centre, MBC-03, PO Box 3354, Riyadh 11211 (Saudi Arabia)

    2010-04-15

    Conjugation of the cytotoxic drugs to receptor-binding peptides is an attractive approach for the targeted delivery of cytotoxic peptide conjugates to tumor cells. In an attempt to develop an efficient peptide-based radiopharmaceutical for targeting bombesin (BN) receptor-expressing tumors (i.e., breast and prostate), we have prepared by solid-phase peptide synthesis, a novel BN analog derived from the universal sequence of BN and conjugated to a widely characterized antineoplastic agent, methotrexate (MTX). MTX-BN, after radiolabeling with {sup 99m}Tc via stannous-tartrate exchange, showed a good stability against cysteine and histidine transchelation as well as a high in vitro metabolic stability in human plasma. In vitro cell-binding and internalization on MDA-MB-231, MCF-7, T47-D breast cancer and PC-3 prostate cancer cell lines demonstrated high affinity and specificity of {sup 99m}Tc-MTX-BN towards both human breast and prostate cancer cells (binding affinities in nanomolar range). In addition, the radioconjugate displayed a significant internalization (values ranged between 19-35%) into the tumor cells. In vivo biodistribution and clearance kinetics in Balb/c mice are characterized by an efficient clearance from the blood and excretion mainly through the renal-urinary pathway with some elimination via the hepatobiliary system. In vivo tumor uptake in nude mice bearing MDA-MB-231 cells was 2.70{+-}0.44% ID/g at 1 h, whereas in nude mice with human epidermoid KB cells the accumulation in the tumor was found to be 1.48{+-}0.31% ID/g at 1 h post injection. The tumor uptake was always higher than in the blood and muscle, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. The accumulation/retention in the major organs (i.e., lungs, stomach, liver, intestines, etc.) was low to moderate (<6% ID/g) in both healthy and tumor-bearing mice. However, the uptake/retention in the kidneys was rather high (up to 11.05{+-}1.80% ID/g), which is of a

  4. Kinetic and hydrodynamic models of chemotactic aggregation

    CERN Document Server

    Chavanis, Pierre-Henri

    2007-01-01

    We derive general kinetic and hydrodynamic models of chemotactic aggregation that describe certain features of the morphogenesis of biological colonies (like bacteria, amoebae, endothelial cells or social insects). Starting from a stochastic model defined in terms of N coupled Langevin equations, we derive a nonlinear mean field Fokker-Planck equation governing the evolution of the distribution function of the system in phase space. By taking the successive moments of this kinetic equation and using a local thermodynamic equilibrium condition, we derive a set of hydrodynamic equations involving a damping term. In the limit of small frictions, we obtain a hyperbolic model describing the formation of network patterns (filaments) and in the limit of strong frictions we obtain a parabolic model which is a generalization of the standard Keller-Segel model describing the formation of clusters (clumps). Our approach connects and generalizes several models introduced in the chemotactic literature. We discuss the anal...

  5. Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography–tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jingshun [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China); Lai, Shiyun [Beingmate Research Institute, Beingmate Baby and Child Food Co., Ltd., Hangzhou 310007 (China); Cai, Zengxuan [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China); Chen, Qi [Beingmate Research Institute, Beingmate Baby and Child Food Co., Ltd., Hangzhou 310007 (China); Huang, Baifen [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China); Ren, Yiping, E-mail: renyiping@263.net [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China)

    2014-06-01

    Highlights: • A UHPLC–MS/MS method for quantification of bovine lactoferrin was developed. • Tryptic fragment LRPVAAEIYGTK was chosen as signature peptide of bovine lactoferrin. • A winged peptide containing isotopically-labeled signature peptide was designed as internal standard. • The method for determining lactoferrin does not discriminate between the different forms of lactoferrin. • Meet the growing demand to quantify bovine lactoferrin in different dairy products. Abstract: A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10–1000 nmol L⁻¹ showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD < 6.5% and RSD < 7.1%, respectively. Excellent repeatability (RSD < 6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method

  6. Self-propelled chemotactic ionic liquid droplets

    OpenAIRE

    Francis, Wayne; Fay, Cormac; Florea, Larisa; Diamond, Dermot

    2015-01-01

    Herein we report the chemotactic behaviour of self-propelled droplets composed solely of the ionic liquid trihexyl(tetradecyl)phosphonium chloride ([P6,6,6,14][Cl]). These droplets spontaneously move along an aqueous-air boundary in the direction of chloride gradients to specific destinations due to asymmetric release of [P6,6,6,14]+ cationic surfactant from the droplet into the aqueous phase.

  7. Peptide mapping of /sup 125/I-labelled membrane protein of influenza viruses by reverse-phase high-performance liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Darveau, A.; Lecomte, J. (Centre de Recherche en Virologie, Institut Armand-Frappier, Laval, Quebec (Canada)); Seidah, N.G.; Chretien, M. (Institut de Recherches Cliniques de Montreal, Montreal (Canada))

    1982-03-01

    The resolution potential of reverse-phase high-performance liquid chromatography (HPLC) for peptide analysis of hydrophobic viral membranes has been investigated, using as a model the membrane (M) protein of influenza virus. Proteolytic digests of /sup 125/I-labelled M protein CNBr fragments, extracted from radioiodinated whole virus, have been separated on a uBondapak C/sub 18/ column with an isopropanol or acetonitrile solvent system. Peptide mapping of trypsin digests of M protein from A/PR/8/34 (H1N1) and A/chicken/Germany/N/49 (H10N7) viruses was identical, whereas Staphylococcus aureus V8 protease digests showed minor differences in at least two peptides. The results also show that HPLC is a powerful tool for the separation of proteolytic digests of viral proteins, since the peptide maps are highly reproducible and recovery was always greater than 85%.

  8. Cellular penetration and nuclear importation properties of {sup 111}In-labeled and {sup 123}I-labeled HIV-1 tat peptide immunoconjugates in BT-474 human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cornelissen, Bart [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Hu, Meiduo [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); McLarty, Kristin [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Costantini, Dan [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Reilly, Raymond M. [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada) and Department of Medical Imaging, University of Toronto, Toronto, ON, M5S 3M2 (Canada) and Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada) and Toronto General Research Institute, University Health Network, Toronto, ON, M5S 3M2 (Canada)]. E-mail: raymond.reilly@utoronto.ca

    2007-01-15

    Introduction: Our objective was to compare the cell penetration and nuclear importation properties of {sup 111}In-labeled and {sup 123}I-labeled immunoconjugates (ICs) composed of 16-mer peptides (GRKKRRQRRRPPQGYG) derived from HIV-1 transactivator of transcription (tat) protein and anti-mouse IgG (mIgG) in BT-474 breast cancer (BC) cells. Methods: [{sup 111}In]tat ICs were constructed by site-specific conjugation of tat peptides to NaIO{sub 4} {sup -}-oxidized carbohydrates in the Fc domain of diethylenetriaminepentaacetic-acid-modified anti-mIgG antibodies. Immunoreactivity against mIgG was assessed in a competition assay. The kinetics of the accumulation of [{sup 111}In]anti-mIgG-tat IC and [{sup 123}I]anti-mIgG-tat ICs in BT-474 cells and the elimination of radioactivity from cells, cytoplasm or nuclei were determined. The effects of excess tat peptides or NH{sub 4}Cl (an inhibitor of endosomal acidification) on cellular uptake and nuclear importation of [{sup 111}In]anti-mIgG-tat were measured. Results: [{sup 111}In]anti-mIgG-tat was >97% radiochemically pure and exhibited preserved immunoreactivity with mIgG epitopes. [{sup 123}I]Anti-mIgG-tat penetrated BT-474 cells more rapidly than [{sup 111}In]anti-mIgG-tat ICs and achieved a 1.5-fold to a 2-fold higher uptake in cells and nuclei. Cell penetration and nuclear uptake of [{sup 111}In]anti-mIgG-tat were inhibited by excess tat peptides and NH{sub 4}Cl. Elimination of radioactivity from BT-474 cells and nuclei was more rapid and complete for {sup 123}I-labeled than for {sup 111}In-labeled anti-mIgG-tat ICs. Conclusion: Tat peptides derived from HIV-1 tat protein promoted the penetration and nuclear uptake of radioactivity following the incubation of {sup 111}In-labeled and {sup 123}I-labeled anti-mIgG antibodies with BT-474 human BC cells. {sup 111}In-labeled tat ICs are feasible for inserting radionuclides into cancer cells with potential for targeting intracellular and, particularly, nuclear epitopes for

  9. A comparative analysis of computational approaches to relative protein quantification using peptide peak intensities in label-free LC-MS proteomics experiments.

    Science.gov (United States)

    Matzke, Melissa M; Brown, Joseph N; Gritsenko, Marina A; Metz, Thomas O; Pounds, Joel G; Rodland, Karin D; Shukla, Anil K; Smith, Richard D; Waters, Katrina M; McDermott, Jason E; Webb-Robertson, Bobbie-Jo

    2013-02-01

    Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred from one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.

  10. A Comparative Analysis of Computational Approaches to Relative Protein Quantification Using Peptide Peak Intensities in Label-free LC-MS Proteomics Experiments

    Energy Technology Data Exchange (ETDEWEB)

    Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.; Metz, Thomas O.; Pounds, Joel G.; Rodland, Karin D.; Shukla, Anil K.; Smith, Richard D.; Waters, Katrina M.; McDermott, Jason E.; Webb-Robertson, Bobbie-Jo M.

    2013-02-01

    Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to identify and quantify peptides in complex biological samples. In particular, label-free shotgun proteomics is highly effective for the identification of peptides and subsequently obtaining a global protein profile of a sample. As a result, this approach is widely used for discovery studies. Typically, the objective of these discovery studies is to identify proteins that are affected by some condition of interest (e.g. disease, exposure). However, for complex biological samples, label-free LC-MS proteomics experiments measure peptides and do not directly yield protein quantities. Thus, protein quantification must be inferred from one or more measured peptides. In recent years, many computational approaches to relative protein quantification of label-free LC-MS data have been published. In this review, we examine the most commonly employed quantification approaches to relative protein abundance from peak intensity values, evaluate their individual merits, and discuss challenges in the use of the various computational approaches.

  11. Peptide receptor radionuclide therapy with {sup 90}Y/{sup 177}Lu-labelled peptides for inoperable head and neck paragangliomas (glomus tumours)

    Energy Technology Data Exchange (ETDEWEB)

    Puranik, Ameya D.; Kulkarni, Harshad R.; Singh, Aviral; Baum, Richard P. [Zentralklinik Bad Berka, THERANOSTICS Centre for Molecular Radiotherapy and Molecular Imaging, ENETS Center of Excellence, Bad Berka (Germany)

    2015-07-15

    Head and neck paragangliomas (HNPGLs) are rare tumours arising from autonomic nervous system ganglia. Although surgery offers the best chance of complete cure, there is associated morbidity due to the crucial location of these tumours. Radiotherapy arrests tumour growth and provides symptomatic improvement, but has long-term consequences. These tumours express somatostatin receptors (SSTR) and hence peptide receptor radionuclide therapy (PRRT) is now a treatment option. We assessed the molecular, morphological and clinical responses of inoperable HNPGLs to PRRT. Nine patients with inoperable HNPGL assessed between June 2006 and June 2014 were included. Four patients had a solitary lesion, four had multifocal involvement and one had distant metastases (bone and lungs). The patients were treated with PRRT using {sup 90}Y/{sup 177}Lu-labelled peptides after positive confirmation of SSTR expression on {sup 68}Ga-DOTATOC PET/CT. All patients received two to four courses of PRRT. Subsequent serial imaging with {sup 68}Ga-DOTATOC PET/CT was carried out every 6 months to assess response to treatment. Clinical (symptomatic) response was also assessed. Based on molecular response (EORTC) criteria, four of the nine patients showed a partial molecular response to treatment seen as significant decreases in SUV{sub max}, accompanied by a reduction in tumour size. Five patients showed stable disease on both molecular and morphological criteria. Six out of nine patients were symptomatic at presentation with manifestations of cranial nerve involvement, bone destruction at the primary site and metastatic bone pain. Molecular responses were correlated with symptomatic improvement in four out of these six patients; while two patients showed small reductions in tumour size and SUV{sub max}. The three asymptomatic patients showed no new lesions or symptomatic worsening. PRRT was effective in all patients, with no disease worsening seen, either in the form of neurological symptoms or

  12. Sulfonation of Tyrosine as a Method to Improve Biodistribution of Peptide-Based Radiotracers: Novel (18)F-Labelled Cyclic RGD Analogues.

    Science.gov (United States)

    Haskali, Mohammad Baqir; Denoyer, Delphine; Noonan, Wayne; Cullinane, Carleen; Rangger, Christine; Pouliot, Normand; Haubner, Roland; Roselt, Peter D; Hicks, Rodney J; Hutton, Craig A

    2017-02-13

    The labeling of peptides with positron emitting radionuclides has long held the promise of a wide range of PET agents possessing high affinity and selectivity. Not surprisingly, controlling the biodistribution of these agents has proven to be a major challenge in their successful application. Modification of peptide hydrophilicity in order to increase renal clearance has been a common endeavor to improve overall biodistribution. Herein, we examine the effect of site-specific sulfonation of tyrosine moieties in cyclic(RGDyK) peptides as a means to enhance their hydrophilicity and improve their biodistribution. The novel sulfonated cyclic(RGDyK) peptides were conjugated directly to 4-nitrophenyl 2-[18F]fluoropropionate and the biodistribution of the radiolabeled peptides was compared with that of their non-sulfonated, clinically relevant counterparts, [18F]GalactoRGD and [18F]FPPRGD2. Site-specific sulfonation of the tyrosine residues was shown to increase hydrophilicity and improve biodistribution of the RGD peptides, despite contributing just 79 Da towards the MW, compared with 189 Da for both the 'Galacto' and mini-PEG moieties, suggesting this may be a broadly applicable approach to enhancing biodistribution of radiolabelled peptides.

  13. {sup 68}Ga-labelled peptides in the management of neuroectodermal tumours

    Energy Technology Data Exchange (ETDEWEB)

    Naji, Meeran [Maidstone and Tunbridge Wells NHS Trust, Departments of Nuclear Medicine and Radiology, Maidstone (United Kingdom); Al-Nahhas, Adil [Hammersmith Hospital, Imperial College NHS Trust, Department of Nuclear Medicine, London (United Kingdom)

    2012-02-15

    Neuroectodermal tumours arise from chromaffin cells and possess the ability to secrete catecholamines. They are generally rare and may occur in association with a variety of hereditary syndromes such as MEN-2A and 2B, neurofibromatosis type 1 and von Hippel-Lindau disease. The most common types are phaeochromocytoma arising from the adrenal medulla and paraganglioma of extra-adrenal origin. Phaeochromocytomas tend to be benign and are often associated with a gene mutation if the disease is bilateral, while paragangliomas are often malignant, have a more aggressive nature and tend to metastasize. There are no specific histological or immunohistochemical features that indicate the malignant potential and the diagnosis of malignancy can only be established by the presence of distant metastases. Therefore, imaging can play a vital role in the diagnosis, localization, staging and assessment of spread. Traditionally, this is achieved with a combination of cross-sectional (CT and MRI) and functional ({sup 123}I-MIBG or {sup 111}In-octreotide) imaging. However, these modalities are not adequate and achieve moderate sensitivity. The introduction of {sup 68}Ga-DOTA peptide in PET/CT imaging has led to improved receptor targeting and superb PET resolution, as well as accurate localization of lesions. The use of this technique in neuroectodermal tumours has been shown to be superior to all available modalities, but the available data are limited and larger studies are awaited to establish its role in the management of these tumours. (orig.)

  14. Automated Chemotactic Sorting and Single-cell Cultivation of Microbes using Droplet Microfluidics

    Science.gov (United States)

    Dong, Libing; Chen, Dong-Wei; Liu, Shuang-Jiang; Du, Wenbin

    2016-04-01

    We report a microfluidic device for automated sorting and cultivation of chemotactic microbes from pure cultures or mixtures. The device consists of two parts: in the first part, a concentration gradient of the chemoeffector was built across the channel for inducing chemotaxis of motile cells; in the second part, chemotactic cells from the sample were separated, and mixed with culture media to form nanoliter droplets for encapsulation, cultivation, enumeration, and recovery of single cells. Chemotactic responses were assessed by imaging and statistical analysis of droplets based on Poisson distribution. An automated procedure was developed for rapid enumeration of droplets with cell growth, following with scale-up cultivation on agar plates. The performance of the device was evaluated by the chemotaxis assays of Escherichia coli (E. coli) RP437 and E. coli RP1616. Moreover, enrichment and isolation of non-labelled Comamonas testosteroni CNB-1 from its 1:10 mixture with E. coli RP437 was demonstrated. The enrichment factor reached 36.7 for CNB-1, based on its distinctive chemotaxis toward 4-hydroxybenzoic acid. We believe that this device can be widely used in chemotaxis studies without necessarily relying on fluorescent labelling, and isolation of functional microbial species from various environments.

  15. PET imaging of {alpha}{sub v}{beta}{sub 3} integrin expression in tumours with {sup 68}Ga-labelled mono-, di- and tetrameric RGD peptides

    Energy Technology Data Exchange (ETDEWEB)

    Dijkgraaf, Ingrid; Franssen, Gerben M.; Oyen, Wim J.G.; Boerman, Otto C. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, P.O. Box 9101, Nijmegen (Netherlands); Yim, Cheng-Bin [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, P.O. Box 9101, Nijmegen (Netherlands); Utrecht University, Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht (Netherlands); Schuit, Robert C. [VU University Medical Centre, Department of Nuclear Medicine and PET Research, P.O. Box 7057, Amsterdam (Netherlands); Luurtsema, Gert [University Medical Center Groningen, Department of Nuclear Medicine and Molecular Imaging, Hanzeplein 1, P.O. Box 30.001, Groningen (Netherlands); Liu, Shuang [Purdue University, School of Health Sciences, West Lafayette, IN (United States)

    2011-01-15

    Due to the restricted expression of {alpha}{sub v}{beta}{sub 3} in tumours, {alpha}{sub v}{beta}{sub 3} is considered a suitable receptor for tumour targeting. In this study the {alpha}{sub v}{beta}{sub 3}-binding characteristics of {sup 68}Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their {sup 111}In-labelled counterparts. A monomeric (E-c(RGDfK)), a dimeric (E-[c(RGDfK)]{sub 2}) and a tetrameric (E{l_brace}E[c(RGDfK)]{sub 2}{r_brace}{sub 2}) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with {sup 68}Ga. In vitro {alpha}{sub v}{beta}{sub 3}-binding characteristics were determined in a competitive binding assay. In vivo {alpha}{sub v}{beta}{sub 3}-targeting characteristics of the compounds were assessed in mice with subcutaneously growing SK-RC-52 xenografts. In addition, microPET images were acquired using a microPET/CT scanner. The IC{sub 50} values for the Ga(III)-labelled DOTA-E-c(RGDfK), DOTA-E-[c(RGDfK)]{sub 2} and DOTA-E{l_brace}E[c(RGDfK)]{sub 2}{r_brace}{sub 2} were 23.9 {+-} 1.22, 8.99 {+-} 1.20 and 1.74 {+-} 1.18 nM, respectively, and were similar to those of the In(III)-labelled mono-, di- and tetrameric RGD peptides (26.6 {+-} 1.15, 3.34 {+-} 1.16 and 1.80 {+-} 1.37 nM, respectively). At 2 h post-injection, tumour uptake of the {sup 68}Ga-labelled mono-, di- and tetrameric RGD peptides (3.30 {+-} 0.30, 5.24 {+-} 0.27 and 7.11 {+-} 0.67%ID/g, respectively) was comparable to that of their {sup 111}In-labelled counterparts (2.70 {+-} 0.29, 5.61 {+-} 0.85 and 7.32 {+-} 2.45%ID/g, respectively). PET scans were in line with the biodistribution data. On all PET scans, the tumour could be clearly visualised. The integrin affinity and the tumour uptake followed the order of DOTA-tetramer > DOTA-dimer > DOTA-monomer. The {sup 68}Ga-labelled tetrameric RGD peptide has excellent characteristics for imaging of {alpha}{sub v} {beta}{sub 3} expression with PET. (orig.)

  16. Identification of a chemotactic sensitivity in a coupled system

    CERN Document Server

    Fister, K Renee

    2007-01-01

    Chemotaxis is the process by which cells behave in a way that follows the chemical gradient. Applications to bacteria growth, tissue inflammation, and vascular tumors provide a focus on optimization strategies. Experiments can characterize the form of possible chemotactic sensitivities. This paper addresses the recovery of the chemotactic sensitivity from these experiments while allowing for nonlinear dependence of the parameter on the state variables. The existence of solutions to the forward problem is analyzed. The identification of a chemotactic parameter is determined by inverse problem techniques. Tikhonov regularization is investigated and appropriate convergence results are obtained. Numerical results of concentration dependent chemotactic terms are explored.

  17. Therapeutic efficacy and toxicity of {sup 225}Ac-labelled vs. {sup 213}Bi-labelled tumour-homing peptides in a preclinical mouse model of peritoneal carcinomatosis

    Energy Technology Data Exchange (ETDEWEB)

    Essler, Markus; Gaertner, Florian C.; Blechert, Birgit; Senekowitsch-Schmidtke, Reingard; Seidl, Christof [Technische Universitaet Muenchen, Department of Nuclear Medicine, Munich (Germany); Neff, Frauke [Helmholtz Zentrum Muenchen, Institute of Pathology, Neuherberg (Germany); Bruchertseifer, Frank; Morgenstern, Alfred [Institute for Transuranium Elements, European Commission, Joint Research Centre, Karlsruhe (Germany)

    2012-04-15

    Targeted delivery of alpha-particle-emitting radionuclides is a promising novel option in cancer therapy. We generated stable conjugates of the vascular tumour-homing peptide F3 both with {sup 225}Ac and {sup 213}Bi that specifically bind to nucleolin on the surface of proliferating tumour cells. The aim of our study was to determine the therapeutic efficacy of {sup 225}Ac-DOTA-F3 in comparison with that of {sup 213}Bi-DTPA-F3. ID{sub 50} values of {sup 213}Bi-DTPA-F3 and {sup 225}Ac-DOTA-F3 were determined via clonogenic assays. The therapeutic efficacy of both constructs was assayed by repeated treatment of mice bearing intraperitoneal MDA-MB-435 xenograft tumours. Therapy was monitored by bioluminescence imaging. Nephrotoxic effects were analysed by histology. ID{sub 50} values of {sup 213}Bi-DTPA-F3 and {sup 225}Ac-DOTA-F3 were 53 kBq/ml and 67 Bq/ml, respectively. The median survival of control mice treated with phosphate-buffered saline was 60 days after intraperitoneal inoculation of 1 x 10{sup 7} MDA-MB-435 cells. Therapy with 6 x 1.85 kBq of {sup 225}Ac-DOTA-F3 or 6 x 1.85 MBq of {sup 213}Bi-DTPA-F3 prolonged median survival to 95 days and 97 days, respectively. While F3 labelled with short-lived {sup 213}Bi (t{sub 1/2} 46 min) reduced the tumour mass at early time-points up to 30 days after treatment, the antitumour effect of {sup 225}Ac-DOTA-F3 (t{sub 1/2} 10 days) increased at later time-points. The difference in the fraction of necrotic cells after treatment with {sup 225}Ac-DOTA-F3 (43%) and with {sup 213}Bi-DTPA-F3 (36%) was not significant. Though histological analysis of kidney samples revealed acute tubular necrosis and tubular oedema in 10-30% of animals after treatment with {sup 225}Ac-DOTA-F3 or {sup 213}Bi-DTPA-F3, protein casts were negligible (2%), indicating only minor damage to the kidney. Therapy with both {sup 225}Ac-DOTA-F3 and {sup 213}Bi-DTPA-F3 increased survival of mice with peritoneal carcinomatosis. Mild renal toxicity of both

  18. Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

    Directory of Open Access Journals (Sweden)

    Kazuma Ogawa

    Full Text Available (68Ga (T 1/2 = 68 min, a generator-produced nuclide has great potential as a radionuclide for clinical positron emission tomography (PET. Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Aspn (n = 2, 5, 8, 11, or 14 with easy-to-handle (67Ga, with the previously described (67Ga-DOTA complex conjugated bisphosphonate, (67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Aspn by a Fmoc-based solid-phase method, complexes were formed with (67Ga, resulting in (67Ga-DOTA-(Aspn with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67Ga-DOTA-(Aspn increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67Ga-DOTA-(Asp8, (67Ga-DOTA-(Asp11, and (67Ga-DOTA-(Asp14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67Ga-DOTA-(Aspn was lower than that of (67Ga-DOTA-Bn-SCN-HBP, blood clearance of (67Ga-DOTA-(Aspn was more rapid. Accordingly, the bone/blood ratios of (67Ga-DOTA-(Asp11 and (67Ga-DOTA-(Asp14 were comparable with those of (67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.

  19. Human recombinant macrophage inflammatory protein-1 alpha and -beta and monocyte chemotactic and activating factor utilize common and unique receptors on human monocytes.

    Science.gov (United States)

    Wang, J M; Sherry, B; Fivash, M J; Kelvin, D J; Oppenheim, J J

    1993-04-01

    The human macrophage inflammatory proteins-1 alpha and -beta (MIP-1 alpha and -beta), which are also known as LD78 and ACT2, respectively, are distinct but highly related members of the chemoattractant cytokine (chemokine) family. rMIP-1 alpha and -beta labeled with 125I specifically bind to human peripheral blood monocytes, the monocytic cell line THP-1, peripheral blood T cells, and the YT cell line. Steady state binding experiments revealed approximately 3000 high affinity binding sites/cell for MIP-1 alpha on human monocytes and on THP-1 cells, with Kd values of 383 pM and 450 pM, respectively. Human MIP-1 alpha and -beta had nearly identical affinities for the binding sites and each competed equally well for binding. Human monocyte chemotactic and activating factor (MCAF), a member of the same chemokine family, consistently displaced about 25% of human MIP-1 alpha and -beta binding on monocytes but not on YT cells, which did not bind MCAF. On the other hand, human rMIP-1 alpha and -beta partially inhibited binding of radiolabeled MCAF to monocytes. Both MIP-1 alpha and -beta were chemotactic for human monocytes. Preincubation of monocytes with human rMIP-1 alpha or -beta markedly reduced cell migration towards the other cytokine, whereas preincubation with human rMCAF only partially desensitized the monocyte chemotaxis response to human rMIP-1 alpha or -beta. These data suggest the existence of three subtypes of receptors, i.e., one unique receptor shared by MIP-1 alpha and -beta, a second unique receptor for MCAF, and a third species that recognizes both MCAF and MIP-1 peptides.

  20. {sup 99m}Tc-labelled gold nanoparticles capped with HYNIC-peptide/mannose for sentinel lymph node detection

    Energy Technology Data Exchange (ETDEWEB)

    Ocampo-Garcia, Blanca E. [Instituto Nacional de Investigaciones Nucleares, Estado de Mexico (Mexico); Universidad Autonoma del Estado de Mexico, Estado de Mexico (Mexico); Ramirez, Flor de M. [Instituto Nacional de Investigaciones Nucleares, Estado de Mexico (Mexico); Ferro-Flores, Guillermina, E-mail: ferro_flores@yahoo.com.m [Instituto Nacional de Investigaciones Nucleares, Estado de Mexico (Mexico); De Leon-Rodriguez, Luis M. [Universidad de Guanajuato, Guanajuato (Mexico); Santos-Cuevas, Clara L.; Morales-Avila, Enrique [Instituto Nacional de Investigaciones Nucleares, Estado de Mexico (Mexico); Universidad Autonoma del Estado de Mexico, Estado de Mexico (Mexico); Arteaga de Murphy, Consuelo; Pedraza-Lopez, Martha [Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City (Mexico); Medina, Luis A. [Universidad Nacional Autonoma de Mexico, Mexico City (Mexico); Instituto Nacional de Cancerologia, Mexico City (Mexico); Camacho-Lopez, Marco A. [Universidad Autonoma del Estado de Mexico, Estado de Mexico (Mexico)

    2011-01-15

    The aim of this research was to prepare a multifunctional system of technetium-99m-labelled gold nanoparticles conjugated to HYNIC-GGC/mannose and to evaluate its biological behaviour as a potential radiopharmaceutical for sentinel lymph node detection (SLND). Methods: Hydrazinonicotinamide-Gly-Gly-Cys-NH{sub 2} (HYNIC-GGC) peptide and a thiol-triazole-mannose derivative were synthesized, characterized and conjugated to gold nanoparticles (AuNP, 20 nm) to prepare a multifunctional system of HYNIC-GGC-AuNP-mannose by means of spontaneous reaction of the thiol (Cys) present in HYNIC-GGC sequence and in the thiol-mannose derivative. The nanoconjugate was characterized by transmission electron microscopy (TEM), IR, UV-Vis, Raman, fluorescence and X-ray photoelectron spectroscopy (XPS). Technetium-99m labelling was carried out using EDDA/tricine as coligands and SnCl{sub 2} as reducing agent with further size-exclusion chromatography purification. Radiochemical purity was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in rat liver homogenized tissue (mannose-receptor positive tissue). Biodistribution studies were accomplished in Wistar rats and images obtained using a micro-SPECT/CT system. Results: TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with HYNIC-GGC-NH{sub 2} and thiol-mannose through interactions with thiol groups and the N-terminal amine of cysteine. Radio-chromatograms showed radiochemical purity higher than 95%. {sup 99m}Tc-EDDA/HYNIC-GGC-AuNP-mannose ({sup 99m}Tc-AuNP-mannose) showed specific recognition for mannose receptors in rat liver tissue. After subcutaneous administration of {sup 99m}Tc-AuNP-mannose in rats (footpad), radioactivity levels in the popliteal and inguinal lymph nodes revealed that 99% of the activity was extracted by the first lymph node (popliteal extraction). Biodistribution studies and in vivo micro-SPECT/CT images in Wistar rats showed an evident

  1. Dynamics of Chemotactic Droplets in Salt Concentration Gradients

    DEFF Research Database (Denmark)

    Cejkova, J.; Novak, M.; Stepanek, F.;

    2014-01-01

    The chemotactic movement of decanol droplets in aqueous solutions of sodium decanoate in response to concentration gradients of NaCl has been investigated. Key parameters of the chemotactic response, namely the induction time and the migration velocity, have been evaluated as a function of the so...

  2. Eosinophil chemotactic factors from cysticercoids of Hymenolepis nana.

    Science.gov (United States)

    Niwa, A; Asano, K; Ito, A

    1998-09-01

    A comparative study of eosinophil chemotactic factors was carried out using cysticercoids and oncospheres of Hymenolepis nana. Cysticercoids showed twice the chemotactic activity for eosinophils than the oncospheres. Eosinophilia induced by oncospheres and cysticercoids observed in secondary and primary infections, respectively, were discussed from the view point of the immunobiology of this parasite.

  3. Rapid Generation of a Nanocrystal-Labeled Peptide Library for Specific Identification of the Bacterium Clostrium Botulinum

    Energy Technology Data Exchange (ETDEWEB)

    Tok, J B

    2004-11-11

    Several peptide libraries containing up to 2 million unique peptide ligands have been synthesized. The peptides are attached onto a 80 micron resin and the length of these peptide ligands ranges from 5 to 9 amino acid residues. Using a novel calorimetric assay, the libraries were screened for binding to the ganglioside-binding domain of Clostridium Tetanus Toxin, a structural similar analog of the Clostridium Botulinum toxin. Several binding peptide sequences were identified, in which the detailed binding kinetics are currently underway using the Surface Plasmon Resonance (SPR) technique.

  4. Preparation of 18F-labeled peptides using the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition.

    Science.gov (United States)

    Gill, Herman S; Marik, Jan

    2011-10-13

    An optimized procedure for preparing fluorine-18 ((18)F)-labeled peptides by the copper-catalyzed azide-alkyne 1,3-dipolar cyloaddition (CuAAC) is presented here. The two-step radiosynthesis begins with the microwave-assisted nucleophilic (18)F-fluorination of a precursor containing a terminal p-toluenesulfonyl, terminal azide and polyethylene glycol backbone. The resulting (18)F-fluorinated azide-containing building block is coupled to an alkyne-decorated peptide by the CuAAC. The reaction is accelerated by the copper(I)-stabilizing ligand bathophenanthroline disulfonate and can be performed in either reducing or nonreducing conditions (e.g., to preserve disulfide bonds). After an HPLC purification, (18)F-labeled peptide can be obtained with a 31 ± 6% radiochemical yield (n = 4, decay-corrected from (18)F-fluoride elution) and a specific activity of 39.0 ± 12.4 Ci μmol(-1) within 77 ± 4 min.

  5. Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affinity Tag Method

    Directory of Open Access Journals (Sweden)

    Young Ah Goo

    2008-01-01

    Full Text Available Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.

  6. Preclinical Pharmacokinetic Studies of the Tritium Labelled D-Enantiomeric Peptide D3 Developed for the Treatment of Alzheimer´s Disease.

    Science.gov (United States)

    Jiang, Nan; Leithold, Leonie H E; Post, Julia; Ziehm, Tamar; Mauler, Jörg; Gremer, Lothar; Cremer, Markus; Schartmann, Elena; Shah, N Jon; Kutzsche, Janine; Langen, Karl-Josef; Breitkreutz, Jörg; Willbold, Dieter; Willuweit, Antje

    2015-01-01

    Targeting toxic amyloid beta (Aβ) oligomers is currently a very attractive drug development strategy for treatment of Alzheimer´s disease. Using mirror-image phage display against Aβ1-42, we have previously identified the fully D-enantiomeric peptide D3, which is able to eliminate Aβ oligomers and has proven therapeutic potential in transgenic Alzheimer´s disease animal models. However, there is little information on the pharmacokinetic behaviour of D-enantiomeric peptides in general. Therefore, we conducted experiments with the tritium labelled D-peptide D3 (3H-D3) in mice with different administration routes to study its distribution in liver, kidney, brain, plasma and gastrointestinal tract, as well as its bioavailability by i.p. and p.o. administration. In addition, we investigated the metabolic stability in liver microsomes, mouse plasma, brain, liver and kidney homogenates, and estimated the plasma protein binding. Based on its high stability and long biological half-life, our pharmacokinetic results support the therapeutic potential of D-peptides in general, with D3 being a new promising drug candidate for Alzheimer´s disease treatment.

  7. Preclinical Pharmacokinetic Studies of the Tritium Labelled D-Enantiomeric Peptide D3 Developed for the Treatment of Alzheimer´s Disease.

    Directory of Open Access Journals (Sweden)

    Nan Jiang

    Full Text Available Targeting toxic amyloid beta (Aβ oligomers is currently a very attractive drug development strategy for treatment of Alzheimer´s disease. Using mirror-image phage display against Aβ1-42, we have previously identified the fully D-enantiomeric peptide D3, which is able to eliminate Aβ oligomers and has proven therapeutic potential in transgenic Alzheimer´s disease animal models. However, there is little information on the pharmacokinetic behaviour of D-enantiomeric peptides in general. Therefore, we conducted experiments with the tritium labelled D-peptide D3 (3H-D3 in mice with different administration routes to study its distribution in liver, kidney, brain, plasma and gastrointestinal tract, as well as its bioavailability by i.p. and p.o. administration. In addition, we investigated the metabolic stability in liver microsomes, mouse plasma, brain, liver and kidney homogenates, and estimated the plasma protein binding. Based on its high stability and long biological half-life, our pharmacokinetic results support the therapeutic potential of D-peptides in general, with D3 being a new promising drug candidate for Alzheimer´s disease treatment.

  8. [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.

  9. Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards

    DEFF Research Database (Denmark)

    Mirgorodskaya, O A; Kozmin, Y P; Titov, M I;

    2000-01-01

    A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards...... for the tested sample by enzymatic hydrolysis of the same sample (with known concentration) in (18)O-water. A mathematical algorithm was developed which uses the isotopic patterns of the substance, the internal standard, and the substance/internal standard mixture for accurate quantitation of the substance...

  10. Modification of β-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro.

    Directory of Open Access Journals (Sweden)

    Janna G Kiselar

    Full Text Available Beta-defensins (hBDs provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human β-defensin-2 (hBD-2 acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO and glyoxal (GO.The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2 to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg22 and Arg23 near the C-terminus, and the N-terminal glycine (Gly1. We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.

  11. Synthesis, {sup 68}Ga labeling and preliminary evaluation of DOTA peptide binding vascular adhesion protein-1: a potential PET imaging agent for diagnosing osteomyelitis

    Energy Technology Data Exchange (ETDEWEB)

    Ujula, Tiina [Turku PET Center, Turku University Hospital, Turku (Finland); Salomaeki, Satu [Turku PET Center, Turku University Hospital, Turku (Finland); Department of Chemistry, University of Turku, Turku (Finland); Virsu, Pauliina [Turku PET Center, Turku University Hospital, Turku (Finland); Lankinen, Petteri; Maekinen, Tatu J. [Orthopedic Research Unit, University of Turku, Turku (Finland); Autio, Anu [Turku PET Center, Turku University Hospital, Turku (Finland); Yegutkin, Gennady G. [MediCity Research Laboratory, University of Turku, Turku (Finland); Knuuti, Juhani [Turku PET Center, Turku University Hospital, Turku (Finland); Jalkanen, Sirpa [MediCity Research Laboratory, University of Turku, Turku (Finland); National Public Health Institute, Turku (Finland); Roivainen, Anne [Turku PET Center, Turku University Hospital, Turku (Finland); Turku Center for Disease Modeling, University of Turku, Turku (Finland)], E-mail: anne.roivainen@utu.fi

    2009-08-15

    Introduction: Vascular adhesion protein-1 (VAP-1) is an infection/inflammation-inducible endothelial glycoprotein. Based on our previous studies, the most VAP-1-selective peptide (VAP-P1) was 1,4,7,10-tetraazacyclododecane-N',N'',N''',N,,,,-tetraacetic acid (DOTA)-conjugated, {sup 68}gallium ({sup 68}Ga)-labeled (named [{sup 68}Ga]DOTAVAP-P1) and evaluated preliminarily. Methods: Targeting was evaluated by using VAP-1-transfected cells. Biodistribution of [{sup 68}Ga]DOTAVAP-P1 was studied by positron emission tomography imaging of healthy rats and rats with bone inflammation caused by Staphylococcus aureus infection. Uptake of [{sup 68}Ga]DOTAVAP-P1 in osteomyelitis was compared with negative control peptide and competition with an excess of unlabeled DOTAVAP-P1. Results: [{sup 68}Ga]DOTAVAP-P1 bound more efficiently to VAP-1-transfected cells than to controls. In rats, [{sup 68}Ga]DOTAVAP-P1 cleared rapidly from blood circulation, excreted quickly in urine and showed an in vivo half-life of 26{+-}2.3 min. Imaging of osteomyelitis demonstrated modest target-to-background ratio. Studies with the negative control peptide and competitors revealed a significantly lower uptake at the infection site compared to [{sup 68}Ga]DOTAVAP-P1. Conclusions: The results represent a proof-of-concept that infection-induced VAP-1 can be targeted by [{sup 68}Ga]DOTA peptide. [{sup 68}Ga]DOTAVAP-P1 is just the first candidate peptide and an essential opening for developing VAP-1-specific imaging agents.

  12. Biological characterization of purified macrophage-derived neutrophil chemotactic factor

    Directory of Open Access Journals (Sweden)

    M. Dias-Baruffi

    1995-01-01

    Full Text Available We have recently described the purification of a 54 kDa acidic protein, identified as macrophage-derived neutrophil chemotactic factor (MNCF. This protein causes in vitro chemotaxis as well as in vivo neutrophil migration even in animals treated with dexamethasone. This in vivo chemotactic activity of MNCF in animals pretreated with dexamethasone is an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. MNCF is released in the supernatant by macrophage monolayers stimulated with lipopolysaccharide (LPS. In the present study, we describe some biological characteristics of homogenous purified MNCF. When assayed in vitro, MNCF gave a bell-shaped dose–response curve. This in vitro activity was shown to be caused by haptotaxis. Unlike N-formyl-methionylleucyl- phenylalanine (FMLP or interleukin 8 (IL-8, the chemotactic activity of MNCF in vivo and in vitro, was inhibited by preincubation with D-galactose but not with D-mannose. In contrast with IL-8, MNCF did not bind to heparin and antiserum against IL-8 was ineffective in inhibiting its chemotactic activity. These data indicate that MNCF induces neutrophil migration through a carbohydrate recognition property, but by a mechanism different from that of the known chemokines. It is suggested that MNCF may be an important mediator in the recruitment of neutrophils via the formation of a substrate bound chemotactic gradient (haptotaxis in the inflamed tissues.

  13. Cleavable ester linked magnetic nanoparticles for labeling of solvent exposed primary amine groups of peptides/proteins

    Science.gov (United States)

    In order to study the solvent exposed lysine residues of peptides/proteins, we previously reported disulfide linked N-hydrosuccinimide ester modified silica coated iron oxide magnetic nanoparticles (NHS-SS-SiO2@Fe3O4 MNPs). The presence of a disulfide bond in the linker limits the use of disulfide r...

  14. Electron transfer dissociation facilitates the measurement of deuterium incorporation into selectively labeled peptides with single residue resolution

    DEFF Research Database (Denmark)

    Zehl, Martin; Rand, Kasper D; Jensen, Ole N;

    2008-01-01

    show in the present study that electron transfer dissociation in a 3D-quadrupole ion trap retains the site-specific solution-phase deuterium incorporation pattern and allows for localization of incorporated deuterium with single residue resolution. Furthermore, we exploit this finding to monitor how...... collisional activation induces proton mobility in a gaseous peptide ion at various levels of vibrational excitation....

  15. High sensitive and selective electrochemical biosensor: Label-free detection of human norovirus using affinity peptide as molecular binder.

    Science.gov (United States)

    Hwang, Hye Jin; Ryu, Myung Yi; Park, Chan Young; Ahn, Junki; Park, Hyun Gyu; Choi, Changsun; Ha, Sang-Do; Park, Tae Jung; Park, Jong Pil

    2017-01-15

    Norovirus is known as the major cause of highly infection for gastrointestinal tracts. In this study, robust and highly sensitive biosensors for detecting human norovirus by employing a recognition affinity peptide-based electrochemical platform were described. A series of amino acid-substituted and cysteine-incorporated recognition peptides isolated from evolutionary phage display technique was chemically synthesized and immobilized to a gold sensor layer, the detection performance of the gold-immobilized synthetic peptide-based sensor system was assessed using QCM, CV and EIS. Using EIS, the limit of detection with Noro-1 as a molecular binder was found to be 99.8nM for recombinant noroviral capsid proteins (rP2) and 7.8copies/mL for human norovirus, thereby demonstrating a high degree of sensitivity for their corresponding targets. These results suggest that a biosensor which consists of affinity peptides as a molecular binder and miniaturized microdevices as diagnostic tool could be served as a new type of biosensing platform for point-of-care testing.

  16. Improved labelling of DTPA- and DOTA-conjugated peptides and antibodies with 111In in HEPES and MES buffer.

    OpenAIRE

    Brom, Maarten; Joosten, Lieke; Oyen, Wim JG; Gotthardt, Martin; Boerman, Otto C.

    2012-01-01

    ABSTRACT: BACKGROUND: In single photon emission computed tomography [SPECT], high specific activity of 111In-labelled tracers will allow administration of low amounts of tracer to prevent receptor saturation and/or side effects. To increase the specific activity, we studied the effect of the buffer used during the labelling procedure: NaAc, NH4Ac, HEPES and MES buffer. The effect of the ageing of the 111InCl3 stock and cadmium contamination, the decay product of 111In, was also examined in th...

  17. Improved labelling of DTPA- and DOTA-conjugated peptides and antibodies with 111In in HEPES and MES buffer.

    NARCIS (Netherlands)

    Brom, M.; Joosten, L.; Oyen, W.J.G.; Gotthardt, M.; Boerman, O.C.

    2012-01-01

    ABSTRACT: BACKGROUND: In single photon emission computed tomography [SPECT], high specific activity of 111In-labelled tracers will allow administration of low amounts of tracer to prevent receptor saturation and/or side effects. To increase the specific activity, we studied the effect of the buffer

  18. Imaging of urokinase-type plasminogen activator receptor expression using a 64Cu-labeled linear peptide antagonist by microPET

    DEFF Research Database (Denmark)

    Li, Z.B.; Niu, G.; Wang, H.;

    2008-01-01

    for positron emission tomography (PET) imaging. A linear, high-affinity uPAR-binding peptide antagonist AE105 was conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled with (64)Cu for microPET imaging of mice bearing U87MG human glioblastoma (uPAR positive) and MDA-MB-435...... human breast cancer (uPAR negative). RESULTS: Surface plasmon resonance measurements show that AE105 with DOTA conjugated at the alpha-amino group (DOTA-AE105) has high affinity toward uPAR. microPET imaging reveals a rapid and high accumulation of (64)Cu-DOTA-AE105 in uPAR-positive U87MG tumors (10...... translation of this class of radiopharmaceuticals for uPAR-positive cancer detection and patient stratification for uPA/uPAR system-based cancer therapy Udgivelsesdato: 2008/8/1...

  19. Helix-helix interconversion rates of short 13C-labeled helical peptides as measured by dynamic NMR spectroscopy.

    Science.gov (United States)

    Kubasik, Matthew; Kotz, James; Szabo, Christopher; Furlong, Theresa; Stace, Justin

    2005-06-05

    The rates at which a peptide hexamer and a peptide octamer interconvert between left- and right-handed helical forms in CD2Cl2 solution have been characterized by 13C dynamic NMR (DNMR) spectroscopy. The peptide esters studied are Fmoc-(Aib)n-OtBu (n = 6 and 8), where Fmoc is 9-fluorenylmethyoxycarbonyl and Aib is the strongly helix-forming residue alpha-aminoisobutyric acid. Because the Aib residue is itself achiral, homooligomers of this residue form a 50/50 mixture of enantiomeric 3(10)-helices in solution. It has been demonstrated (R.-P. Hummel, C. Toniolo, and G. Jung, Angewandte Chemie International Edition, 1987, Vol. 26, pp. 1150-1152) that oligomers of Aib interconvert on the millisecond timescale. We have performed lineshape analysis of 13C-NMR spectra collected for our peptides enriched with 13C at a single residue. Rate constants for the octamer range from 6 s(-1) at 196 K to about 56,500 s(-1) at 320 K. At all temperatures, the hexamer interconverts about three times faster than the octamer. Eyring plots of the data reveal experimentally indistinguishable DeltaH++ values for the hexamer and octamer of 37.8 +/- 0.6 and 37.6 +/- 0.4 kJ mol(-1) respectively. The difference in the rates of interconversion is dictated by entropic factors. The hexamer and octamer exhibit negative DeltaS++ values of -29.0(-1) +/- 2.5 and -37.3 +/- 1.7 J K(-1) mol(-1), respectively. A mechanism for the helix-helix interconversion is proposed. and calculated DeltaG++ values are compared to the estimate for a decamer undergoing a helix-helix interconversion.

  20. Fischer carbene mediated covalent grafting of a peptide nucleic acid on gold surfaces and IR optical detection of DNA hybridization with a transition metalcarbonyl label

    Science.gov (United States)

    Srivastava, Pratima; Ghasemi, Mahsa; Ray, Namrata; Sarkar, Amitabha; Kocabova, Jana; Lachmanova, Stepanka; Hromadova, Magdalena; Boujday, Souhir; Cauteruccio, Silvia; Thakare, Pramod; Licandro, Emanuela; Fosse, Céline; Salmain, Michèle

    2016-11-01

    Amine-reactive surfaces comprising N-hydroxysuccinimide ester groups as well as much more unusual Fischer alkoxymetallocarbene groups were generated on gold-coated surfaces via self-assembled monolayers of carboxy- and azido-terminated thiolates, respectively. These functions were further used to immobilize homothymine peptide nucleic acid (PNA) decamer in a covalent fashion involving the primary amine located at its N-terminus. These stepwise processes were monitored by polarization modulation reflection - absorption infrared spectroscopy (PM-RAIRS) that gave useful information on the molecular composition of the organic layers. PNA grafting and hybridization with complementary DNA strand were successfully transduced by quartz crystal microbalance (QCM) measurements. Unfortunately, attempts to transduce the hybridization optically by IR in a label-free fashion were inconclusive. Therefore we undertook to introduce an IR reporter group, namely a transition metalcarbonyl (TMC) entity at the 5‧ terminus of complementary DNA. Evidence for the formation of PNA-DNA heteroduplex was brought by the presence of ν(Ctbnd O) bands in the 2000 cm-1 region of the IR spectrum of the gold surface owing to the metalcarbonyl label.

  1. THE ISOLATION OF NOVEL MESENCHYMAL STROMAL CELL CHEMOTACTIC FACTORS FROM THE CONDITIONED MEDIUM OF TUMOR CELLS

    Science.gov (United States)

    Lin, Siang-Yo; Yang, Jun; Everett, Allen D.; Clevenger, Charles V.; Koneru, Mythili; Mishra, Pravin J.; Kamen, Barton; Banerjee, Debabrata; Glod, John

    2008-01-01

    Bone marrow-derived mesenchymal stromal cells (MSCs) localize to solid tumors. Defining the signaling mechanisms that regulate this process is important to understanding the role of MSCs in tumor growth. Using a combination of chromatography and electrospray tandem mass spectrometry we have identified novel soluble signaling molecules that induce MSC chemotaxis present in conditioned medium of the breast carcinoma cell line MDA-MB231. Previous work has employed survey strategies using ELISA assay to identify known chemokines that promote MSC chemotaxis. While these studies provide valuable insights into the intercellular signals that impact MSC behavior, many less well-described, but potentially important soluble signaling molecules could be overlooked using these methods. Through the less directed method of column chromatography we have identified novel candidate MSC chemotactic peptides. Two proteins, cyclophilin B and hepatoma-derived growth factor were then further characterized and shown to promote MSC chemotaxis. PMID:18722367

  2. Evaluation of two novel {sup 64}Cu-labeled RGD peptide radiotracers for enhanced PET imaging of tumor integrin α{sub v}β{sub 3}

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez, Reinier; Graves, Stephen A.; Nickles, Robert J. [University of Wisconsin, Department of Medical Physics, Madison, WI (United States); Czerwinski, Andrzej; Valenzuela, Francisco [Peptides International, Inc., Louisville, KY (United States); Chakravarty, Rubel; Yang, Yunan; England, Christopher G. [University of Wisconsin, Department of Radiology, Madison, WI (United States); Cai, Weibo [University of Wisconsin, Department of Medical Physics, Madison, WI (United States); University of Wisconsin, Department of Radiology, Madison, WI (United States); University of Wisconsin Carbone Cancer Center, Madison, WI (United States)

    2015-11-15

    Our goal was to demonstrate that suitably derivatized monomeric RGD peptide-based PET tracers, targeting integrin α{sub v}β{sub 3}, may offer advantages in image contrast, time for imaging, and low uptake in nontarget tissues. Two cyclic RGDfK derivatives, (PEG){sub 2}-c(RGDfK) and PEG{sub 4}-SAA{sub 4}-c(RGDfK), were constructed and conjugated to NOTA for {sup 64}Cu labeling. Their integrin α{sub v}β{sub 3}-binding properties were determined via a competitive cell binding assay. Mice bearing U87MG tumors were intravenously injected with each of the {sup 64}Cu-labeled peptides, and PET scans were acquired during the first 30 min, and 2 and 4 h after injection. Blocking and ex vivo biodistribution studies were carried out to validate the PET data and confirm the specificity of the tracers. The IC{sub 50} values of NOTA-(PEG){sub 2}-c(RGDfK) and NOTA-PEG{sub 4}-SAA{sub 4}-c(RGDfK) were 444 ± 41 nM and 288 ± 66 nM, respectively. Dynamic PET data of {sup 64}Cu-NOTA-(PEG){sub 2}-c(RGDfK) and {sup 64}Cu-NOTA-PEG{sub 4}-SAA{sub 4}-c(RGDfK) showed similar circulation t{sub 1/2} and peak tumor uptake of about 4 %ID/g for both tracers. Due to its marked hydrophilicity, {sup 64}Cu-NOTA-PEG{sub 4}-SAA{sub 4}-c(RGDfK) provided faster clearance from tumor and normal tissues yet maintained excellent tumor-to-background ratios. Static PET scans at later time-points corroborated the enhanced excretion of the tracer, especially from abdominal organs. Ex vivo biodistribution and receptor blocking studies confirmed the accuracy of the PET data and the integrin α{sub v}β{sub 3}-specificity of the peptides. Our two novel RGD-based radiotracers with optimized pharmacokinetic properties allowed fast, high-contrast PET imaging of tumor-associated integrin α{sub v}β{sub 3}. These tracers may facilitate the imaging of abdominal malignancies, normally precluded by high background uptake. (orig.)

  3. A novel Tc-99 m and fluorescence labeled peptide as a multimodal imaging agent for targeting angiogenesis in a murine tumor model.

    Science.gov (United States)

    Kim, Myoung Hyoun; Kim, Chang Guhn; Kim, Seul-Gi; Kim, Dae-Weung

    2016-11-01

    The serine-aspartic acid-valine (SDV) peptide binds specifically to integrin αV β3 . In the present study, we successfully developed a TAMRA-GHEG-ECG-SDV peptide labeled with both Tc-99 m and TAMRA to target the integrin αV β3 of tumor cells; furthermore, we evaluated the diagnostic performance of Tc-99 m TAMRA-GHEG-ECG-SDV as a dual-modality imaging agent for tumor of the murine model. TAMRA-GHEG-ECG-SDV was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-SDV with Tc-99 m was done using ligand exchange methods. Labeling stability and cytotoxicity studies were performed. Gamma camera imaging, biodistribution and ex vivo imaging studies were performed in murine models with HT-1080 and HT-29 tumors. A tumor tissue slide was prepared and analyzed using confocal microscopy. After radiolabeling procedures with Tc-99 m, the Tc-99 m TAMRA-GHEG-ECG-SDV complexes were prepared in high yield (>99%). In the gamma camera imaging study, a substantial uptake of Tc-99 m TAMRA-GHEG-ECG-SDV into HT-1080 tumor (integrin αV β3 positive) and low uptake of Tc-99 m TAMRA-GHEG-ECG-SDV into HT-29 tumor (integrin αV β3 negative) were demonstrated. A competition study revealed that HT-1080 tumor uptake was effectively blocked by the co-injection of an excess concentration of SDV. Specific uptake of Tc-99 m TAMRA-GHEG-ECG-SDV was confirmed by biodistribution, ex vivo imaging and confocal microscopy studies. Our in vivo and in vitro studies revealed substantial uptake of Tc-99 m TAMRA-GHEG-ECG-SDV in the integrin αV β3 -positive tumor. Tc-99 m TAMRA-GHEG-ECG-SDV could be a good candidate for a dual-modality imaging agent targeting tumor angiogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

  4. Radio-labeling of T7 Peptide with 99mTc and Its Biodistribution 
in Nude Mice Bearing Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Yumei HAO

    2014-03-01

    Full Text Available Background and objective Lung cancer is a malignant tumor with high mortality rates. This study aims to develop potential candidates of integrin αvβ3 imaging agents, which can facilitate the diagnosis and treatment of lung cancer. Methods The T7 peptide was labeled with carbonyl technetium. The thin layer chromatography with acetone as the development system was performed to investigate the purity and stability of 99mTc-T7. The binding affinity of 99mTc-T7 with NCI-H157 tumor cells was determined. The biodistribution of 99mTc-T7 in nude mice bearing non-small cell lung carcinoma was observed after injection of 99mTc-T7 at 0.5 h, 1 h, 2 h, 4 h, and 8 h, and the radioactive ratio of tumor (T and non-tumor tissues (NT was calculated. Results 99mTc labeled T7 had high radiochemical purity of more than 90%, which does not require further purification, with good stability in vitro. The association and dissociation constant (KD of 99mTc-T7 with NCI-H157 tumor cells was 196.1 nM. 99mTc-T7 was mainly metabolism through the internal organs with rapid blood removal. Moreover, the uptake in tumor tissue was significantly higher than the muscle with tumor/muscle ratio of 5.8. In addition, the 99mTc-T7 exhibited a transient accumulation in the lungs. Conclusion The 99mTc-T7 could be prepared using a simple method, had high labeling rate and good stability, and could be accumulated at tumor site. Thus, 99mTc-T7 is a potential lung cancer SPECT/CT imaging agent.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-06

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

  6. Monocyte chemotactic protein-1 gene polymorphism and spontaneous bacterial peritonitis

    Institute of Scientific and Technical Information of China (English)

    Levent; Filik

    2010-01-01

    I read with great interest the article by Gbele et al published in issue 44 of World J Gastroenterol 2009.The results of their study indicate that-2518 Monocyte chemotactic protein-1(MCP-1)genotype AA is a risk factor for spontaneous bacterial peritonitis in patients with alcoholic cirrhosis.However,there are some items that need to be discussed.

  7. Sensory Adaptation of Dictyostelium discoideum Cells to Chemotactic Signals

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1983-01-01

    Postvegetative Dictyostelium discoideum cells react chemotactically to gradients of cAMP, folic acid, and pterin. In the presence of a constant concentration of 10-5 M cAMP cells move at random. They still are able to respond to superimposed gradients of cAMP, although the response is less efficient

  8. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

    Science.gov (United States)

    Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

    2016-02-01

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 ‑ 2.0 × 108 cells mL‑1), a low limit of detection (79 cells mL‑1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  9. A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library.

    Science.gov (United States)

    Han, Lei; Liu, Pei; Petrenko, Valery A; Liu, Aihua

    2016-02-24

    One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 10(2) - 2.0 × 10(8) cells mL(-1)), a low limit of detection (79 cells mL(-1), S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

  10. PNA Peptide chimerae

    DEFF Research Database (Denmark)

    Koch, T.; Næsby, M.; Wittung, P.;

    1995-01-01

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

  11. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

    Science.gov (United States)

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

    2016-01-01

    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

  12. Imaging Tumor Vasculature Noninvasively with Positron Emission Tomography and RGD Peptides Labeled with Copper 64 Using the Bifunctonal Chelates DOTA, Oxo-DO3A. and PCTA

    Directory of Open Access Journals (Sweden)

    Donald T.T. Yapp

    2013-06-01

    Full Text Available Two novel bifunctional chelates, 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15,11,13-triene-3,6,9-triacetic acid (PCTA and 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (Oxo-DO3A, were found to radiolabel antibodies with copper 64 (64Cu well for positron emission tomography (PET. In this study, the same chelators were used to radiolabel peptides with 64Cu for PET imaging of angiogenesis. PCTA, Oxo-DO3A, and 1,4,7,10-tetraazacyclododecane-N,N‘,N“,N”’-tetraacetic acid (DOTA were conjugated to cyclic-(RGDyK, and their binding affinities were confirmed. Conditions for 64Cu radiolabeling were optimized for maximum yield and specific activity. The in vitro stability of the radiolabeled compounds was challenged with serum incubation. PET studies were carried out in a non-αvβ3-expressing tumor model to evaluate the compounds' specificity for proliferating tumor vasculature and their in vivo pharmacokinetics. The PCTA and Oxo-DO3A bioconjugates were labeled with 64Cu at higher effective specific activity and radiochemical yield than the DOTA bioconjugate. In the imaging studies, all the 64Cu bioconjugates could be used to visualize the tumor and the radiotracer uptake was blocked with cyclic-(RGDyK. Target uptake of each bioconjugate was similar, but differences in other tissues were observed. 64Cu-PCTA-RGD showed the best clearance from nontarget tissue and the highest tumor to nontarget ratios. PCTA was the most promising bifunctional chelate for 64Cu peptide imaging and warrants further investigation.

  13. Influence of biological assay conditions on stability assessment of radiometal-labelled peptides exemplified using a {sup 177}Lu-DOTA-minigastrin derivative

    Energy Technology Data Exchange (ETDEWEB)

    Ocak, Meltem [Clinical Department of Nuclear Medicine, Medical University Innsbruck, A-6020, Innsbruck (Austria); Department of Pharmaceutical Technology, Pharmacy Faculty, Istanbul University, 34116, Istanbul (Turkey); Helbok, Anna; Guggenberg, Elisabeth von [Clinical Department of Nuclear Medicine, Medical University Innsbruck, A-6020, Innsbruck (Austria); Ozsoy, Y. [Department of Pharmaceutical Technology, Pharmacy Faculty, Istanbul University, 34116, Istanbul (Turkey); Kabasakal, Levent [Department of Nuclear Medicine, Cerrahpasa Medical Faculty, 34098, Istanbul (Turkey); Kremser, Leopold [Division of Clinical Biochemistry, Protein Micro-Analysis Facility, Biocenter, Medical University Innsbruck, A-6020, Innsbruck (Austria); Decristoforo, Clemens, E-mail: clemens.decristoforo@uki.a [Clinical Department of Nuclear Medicine, Medical University Innsbruck, A-6020, Innsbruck (Austria)

    2011-02-15

    Introduction: Lack of correlation between in vitro and in vivo stability is a general problem for the development of radiopeptides especially in the case of minigastrin derivatives for therapeutic applications. In this study, we compared the influence of experimental conditions on radiopeptide stability results in vitro using a model Minigastrin (MG) analogue labelled with Lu-177. Additionally, we attempted to characterize the main serum enzymatic cleavage sites by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) analysis. Methods: In vitro stability of a DOTA-minigastrin derivative ({sup 177}Lu-DOTA-His-His-Glu-Ala-Tyr-Gly-Trp-NIe-Asp-Phe-NH{sub 2}) was tested in serum, rat tissue homogenates and two different standardised enzymatic mixtures. Quantification of the metabolised radiopeptides at different time intervals was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). Metabolites were characterised by MALDI-TOF-MS. Urine was collected after 15 min p.i. into the mice and compared with in vitro metabolites by RP-HPLC. Results: Faster degradation of the radiopeptide was found in blood in comparison with plasma and serum incubation and in components from rats faster than from human origin. Fast degradation was observed in kidney and liver homogenates as well as in standardised enzymatic mixtures, also revealing variations in the metabolic profile. In urine, no intact peptide was detected already 5 min post injection. MALDI-TOF-MS revealed major cleavage sites at the carboxy terminus of the peptide. Conclusion: Very variable results may be found when different kind of incubation media for testing radiopeptide stabilities is used. Serum incubation studies may overestimate stability; therefore, results should be interpreted with care and combined with alternative in vitro and in vivo investigations.

  14. Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.

    Science.gov (United States)

    Koehler, Sybille; Brähler, Sebastian; Braun, Fabian; Hagmann, Henning; Rinschen, Markus M; Späth, Martin R; Höhne, Martin; Wunderlich, F Thomas; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

    2017-02-07

    Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type) mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type)-mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type) and hNphs2.Cre mice to Phb2(fl/fl) mice. The podocyte-specific Phb2 knockout by Nphs2(pod.T2A.ciCre.T2A.mTomato/wild-type) mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines.

  15. The efficacy of {sup 177}Lu-labelled peptide receptor radionuclide therapy in patients with neuroendocrine tumours: a meta-analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seong-Jang; Pak, Kyoungjune [Pusan National University Hospital, Department of Nuclear Medicine and Biomedical Research Institute, Busan (Korea, Republic of); Koo, Phillip J.; Kwak, Jennifer J.; Chang, Samuel [University of Colorado School of Medicine, Department of Radiology, Aurora, CO (United States)

    2015-12-15

    This study was performed to evaluate the efficacy of {sup 177}Lu-labelled peptide receptor radionuclide therapy (PRRT) in patients with inoperable or metastatic neuroendocrine tumours (NETs). Systematic searches of MEDLINE and EMBASE databases were performed using the keywords of ''neuroendocrine'', ''{sup 177}Lu'' and ''prognosis''. All published studies of neuroendocrine tumours treated with {sup 177}Lu-labelled radiopharmaceuticals and evaluated with either Response Evaluation Criteria in Solid Tumours (RECIST) 1.0 or Southwest Oncology Group (SWOG) criteria or both were included. If there was more than one published study from the same institution, only one report with the information most relevant to this study was included. Each response criteria group was analysed for disease response rates and disease control rates, defined as the percentages of patients with complete response (CR) + partial response (PR), and CR + PR + stable disease (SD), respectively, to a therapeutic intervention in clinical trials of anticancer agents. The pooled proportions are presented with both a fixed-effects model and random-effects model. Six studies with 473 patients (4 in RECIST criteria group with 356 patients, 3 in SWOG criteria group with 375 patients and 1 in both groups) were included. The RECIST criteria group demonstrated disease response rates ranging between 17.6 and 43.8 % with a pooled effect of 29 % [95 % confidence interval (CI) 24-34 %]. Disease control rates ranged from 71.8 to 100 %. The random-effects model showed an average disease control rate of 81 % (95 % CI 71-91 %). The SWOG criteria group demonstrated disease response rates ranging between 7.0 and 36.5 % with a pooled effect of 23 % (95 % CI 11-38 %). Disease control rates ranged from 73.9 to 89.1 %. The random-effects model showed an average disease control rate of 82 % (95 % CI 71-91 %). {sup 177}Lu-labelled PRRT is an effective treatment

  16. Synthesis and evaluation of diverse analogs of amygdalin as potential peptidomimetics of peptide T.

    Science.gov (United States)

    Araya, Eyleen; Rodriguez, Alex; Rubio, Jaime; Spada, Alessandro; Joglar, Jesus; Llebaria, Amadeu; Lagunas, Carmen; Fernandez, Andres G; Spisani, Susanna; Perez, Juan J

    2005-03-01

    Peptide T (ASTTTNYT) is a promising molecule to prevent the neuropsychometric symptoms of patients suffering AIDS and for the treatment of psoriasis. In order to fully prove its therapeutic benefits, efforts were put forward to design peptidomimetics of the peptide. In this direction, in a recent computational study the natural product amygdalin was identified as a prospective peptidomimetic of the peptide and later proved to exhibit a similar chemotactic profile to the peptide. However, the cyanide moiety of amygdalin provides to the molecule a toxic profile. The present study reports the synthesis of a set of amygdalin analogs lacking the cyanide group with improved chemotactic profiles.

  17. Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

    Science.gov (United States)

    Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda-Yamamoto, Mari

    2016-08-01

    Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/μL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods.

  18. 68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers

    DEFF Research Database (Denmark)

    Persson, Morten; Madsen, Jacob; Østergaard, Søren;

    2012-01-01

    INTRODUCTION: The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH(2) labeled with (64)Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using...... positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) and evaluate their imaging properties using small-animal PET. METHODS: Synthesis of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) was based on solid-phase peptide......, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft. RESULTS: In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated...

  19. Analysis of bacterial chemotactic response using dynamic laser speckle

    Science.gov (United States)

    Murialdo, Silvia E.; Sendra, Gonzalo H.; Passoni, Lucía I.; Arizaga, Ricardo; Gonzalez, J. Froilán; Rabal, Héctor; Trivi, Marcelo

    2009-11-01

    Chemotaxis has a meaningful role in several fields, such as microbial physiology, medicine and biotechnology. We present a new application of dynamic laser speckle (or biospeckle) to detect different degrees of bacterial motility during chemotactic response experiments. Encouraging results showed different bacterial dynamic responses due to differences in the hardness of the support in the swarming plates. We compare this method to a conventional technique that uses white light. Both methods showed to be analogous and, in some cases, complementary. The results suggest that biospeckle processed images can be used as an alternative method to evaluate bacterial chemotactic response and can supply additional information about the bacterial motility in different areas of the swarm plate assay that might be useful for biological analysis.

  20. Stability and dynamics of anisotropically-tumbling chemotactic swimmers

    CERN Document Server

    Lushi, Enkeleida

    2016-01-01

    Micro-swimmers such as bacteria E. coli are known to perform random walks known as run-and-tumbles to move up chemo-attractant gradients and as a result aggregate. It is also known that such micro-swimmers can self-organize into macroscopic patterns due to interactions with neighboring cells through the fluidic environment they live in. While the pattern formation resulting from chemotactic and hydrodynamic interactions separately and together have been previously investigated, the effect of the tumbling anisotropy in micro-swimmers has been unexplored. Here we show through linear analysis and full nonlinear simulations that the slight anisotropy in the individual swimmer tumbles can alter the collective pattern formation in non-trivial ways. We show that the tumbling anisotropy diminishes the magnitude of the chemotactic aggregates but may result in more such aggregation peaks.

  1. Novel targeted nuclear imaging agent for gastric cancer diagnosis: glucose-regulated protein 78 binding peptide-guided 111In-labeled polymeric micelles

    Directory of Open Access Journals (Sweden)

    Cheng CC

    2013-04-01

    Full Text Available Chun-Chia Cheng,1,2,* Chiung-Fang Huang,3,4,* Ai-Sheng Ho,5 Cheng-Liang Peng,6 Chun-Chao Chang,7,8 Fu-Der Mai,1,9 Ling-Yun Chen,10 Tsai-Yueh Luo,2 Jungshan Chang1,11,121Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, 2Institute of Nuclear Energy Research, Atomic Energy Council, Taoyuan, 3School of Dental Technology, Taipei Medical University, Taipei, 4Division of Family and Operative Dentistry, Department of Dentistry, Taipei Medical University Hospital, Taipei, 5Division of Gastroenterology, Cheng Hsin General Hospital, Taipei, 6Institute of Biomedical Engineering, National Taiwan University, Taipei, 7Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, 8Department of Internal Medicine, Taipei Medical University, Taipei, 9Department of Biochemistry, Taipei Medical University, Taipei, 10Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, 11Neuroscience Research Center, Taipei Medical University Hospital, Taipei, 12Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei, Taiwan*These authors contributed equally to this workAbstract: Increased expression of cellular membrane bound glucose-regulated protein 78 (GRP78 is considered to be one of the biomarkers for gastric cancers. Therefore, peptides or molecules with specific recognition to GRP78 can act as a guiding probe to direct conjugated imaging agents to localized cancers. Based on this rationale, GRP78-guided polymeric micelles were designed and manufactured for nuclear imaging detection of tumors. Thiolated GRP78 binding peptide (GRP78BP was first labeled with maleimide-terminated poly(ethylene glycol–poly(ε-caprolactone and then mixed with diethylenetriaminepentaacetic acid (DTPA-linked poly(ethylene glycol–poly(ε-caprolactone to form DTPA/GRP78BP-conjugated micelles. The coupling efficiency of micelles with

  2. Polarised clathrin-mediated endocytosis of EGFR during chemotactic invasion.

    Science.gov (United States)

    Mutch, Laura Jane; Howden, Jake Davey; Jenner, Emma Poppy Louise; Poulter, Natalie Sarah; Rappoport, Joshua Zachary

    2014-06-01

    Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin-mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA-MB-231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin-mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin-mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin-mediated endocytosis to directed cell motility.

  3. Organ-specific chemotactic factors present in lung extracellular matrix.

    Science.gov (United States)

    Cerra, R F; Nathanson, S D

    1989-05-01

    The preferential colonization of a distant organ by a circulating tumor cell (organ specific metastasis) may be regulated by chemotactic factors present within the extracellular matrix of the host organ. Organ-specific extracellular matrix was prepared from murine kidney and lung by high salt extraction and DNAase/RNAase digestion. A soluble protein fraction (S2) from each of the matricies was obtained by 4 M guanidine extraction and was tested for organ-specific chemotactic activity in a modified Boyden chamber. The lung colonizing B16-F10 and B16-BL6 tumor cell lines demonstrated organ-specific motility only toward the lung extract. The low metastasizing B16 parental line and liver colonizing B16-L4b line showed no preference for either lung or kidney. The lung activity resolves into five fractions by gel filtration chromatography, with the highest activity eluting at Mr approximately 71,000. Chemotactic factors present in lung extracellular matrix may regulate the preferential colonization of an organ by stimulating the migration of tumor cells in a specific manner. These factors may be released during the degradation of the extracellular matrix.

  4. Mytilus galloprovincialis myticin C: a chemotactic molecule with antiviral activity and immunoregulatory properties.

    Science.gov (United States)

    Balseiro, Pablo; Falcó, Alberto; Romero, Alejandro; Dios, Sonia; Martínez-López, Alicia; Figueras, Antonio; Estepa, Amparo; Novoa, Beatriz

    2011-01-01

    Previous research has shown that an antimicrobial peptide (AMP) of the myticin class C (Myt C) is the most abundantly expressed gene in cDNA and suppressive subtractive hybridization (SSH) libraries after immune stimulation of mussel Mytilus galloprovincialis. However, to date, the expression pattern, the antimicrobial activities and the immunomodulatory properties of the Myt C peptide have not been determined. In contrast, it is known that Myt C mRNA presents an unusual and high level of polymorphism of unidentified biological significance. Therefore, to provide a better understanding of the features of this interesting molecule, we have investigated its function using four different cloned and expressed variants of Myt C cDNA and polyclonal anti-Myt C sera. The in vivo results suggest that this AMP, mainly present in hemocytes, could be acting as an immune system modulator molecule because its overexpression was able to alter the expression of mussel immune-related genes (as the antimicrobial peptides Myticin B and Mytilin B, the C1q domain-containing protein MgC1q, and lysozyme). Moreover, the in vitro results indicate that Myt C peptides have antimicrobial and chemotactic properties. Their recombinant expression in a fish cell line conferred protection against two different fish viruses (enveloped and non-enveloped). Cell extracts from Myt C expressing fish cells were also able to attract hemocytes. All together, these results suggest that Myt C should be considered not only as an AMP but also as the first chemokine/cytokine-like molecule identified in bivalves and one of the few examples in all of the invertebrates.

  5. Mytilus galloprovincialis myticin C: a chemotactic molecule with antiviral activity and immunoregulatory properties.

    Directory of Open Access Journals (Sweden)

    Pablo Balseiro

    Full Text Available Previous research has shown that an antimicrobial peptide (AMP of the myticin class C (Myt C is the most abundantly expressed gene in cDNA and suppressive subtractive hybridization (SSH libraries after immune stimulation of mussel Mytilus galloprovincialis. However, to date, the expression pattern, the antimicrobial activities and the immunomodulatory properties of the Myt C peptide have not been determined. In contrast, it is known that Myt C mRNA presents an unusual and high level of polymorphism of unidentified biological significance. Therefore, to provide a better understanding of the features of this interesting molecule, we have investigated its function using four different cloned and expressed variants of Myt C cDNA and polyclonal anti-Myt C sera. The in vivo results suggest that this AMP, mainly present in hemocytes, could be acting as an immune system modulator molecule because its overexpression was able to alter the expression of mussel immune-related genes (as the antimicrobial peptides Myticin B and Mytilin B, the C1q domain-containing protein MgC1q, and lysozyme. Moreover, the in vitro results indicate that Myt C peptides have antimicrobial and chemotactic properties. Their recombinant expression in a fish cell line conferred protection against two different fish viruses (enveloped and non-enveloped. Cell extracts from Myt C expressing fish cells were also able to attract hemocytes. All together, these results suggest that Myt C should be considered not only as an AMP but also as the first chemokine/cytokine-like molecule identified in bivalves and one of the few examples in all of the invertebrates.

  6. Measurement of homonuclear three-bond J(HNH{alpha}) coupling constants in unlabeled peptides complexed with labeled proteins: Application to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of hepatitis C virus (HCV)

    Energy Technology Data Exchange (ETDEWEB)

    Cicero, Daniel O.; Barbato, Gaetano; Koch, Uwe; Ingallinella, Paolo; Bianchi, Elisabetta; Sambucini, Sonia; Neddermann, Petra; De Francesco, Raffaele; Pessi, Antonello; Bazzo, Renzo

    2001-05-15

    A new isotope-filtered experiment has been designed to measure homonuclear three-bond J(H{sup N}H{sup {alpha}}) coupling constants of unlabeled peptides complexed with labeled proteins. The new experiment is based on the 3D HNHA pulse scheme, and belongs to the 'quantitative J-correlation' type. It has been applied to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of human hepatitis C virus (HCV)

  7. Peptide arrays for screening cancer specific peptides.

    Science.gov (United States)

    Ahmed, Sahar; Mathews, Anu Stella; Byeon, Nara; Lavasanifar, Afsaneh; Kaur, Kamaljit

    2010-09-15

    In this paper, we describe a novel method to screen peptides for specific recognition by cancer cells. Seventy peptides were synthesized on a cellulose membrane in an array format, and a direct method to study the peptide-whole cell interaction was developed. The relative binding affinity of the cells for different peptides with respect to a lead 12-mer p160 peptide, identified by phage display, was evaluated using the CyQUANT fluorescence of the bound cells. Screening allowed identification of at least five new peptides that displayed higher affinity (up to 3-fold) for MDA-MB-435 and MCF-7 human cancer cells compared to the p160 peptide. These peptides showed very little binding to the control (noncancerous) human umbilical vein endothelial cells (HUVECs). Three of these peptides were synthesized separately and labeled with fluorescein isothiocyanate (FITC) to study their uptake and interaction with the cancer and control cells using confocal laser scanning microscopy and flow cytometry. The results confirmed the high and specific affinity of an 11-mer peptide 11 (RGDPAYQGRFL) and a 10-mer peptide 18 (WXEAAYQRFL) for the cancer cells versus HUVECs. Peptide 11 binds different receptors on target cancer cells as its sequence contains multiple recognition motifs, whereas peptide 18 binds mainly to the putative p160 receptor. The peptide array-whole cell binding assay reported here is a complementary method to phage display for further screening and optimization of cancer targeting peptides for cancer therapy and diagnosis.

  8. Effects of linker variation on the in vitro and in vivo characteristics of an {sup 111}In-labeled RGD peptide

    Energy Technology Data Exchange (ETDEWEB)

    Dijkgraaf, Ingrid [Department of Nuclear Medicine, Radboud University Nijmegen Medical Center, PO Box 9101, HB Nijmegen 6500 (Netherlands) and Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3508 TB Utrecht (Netherlands)]. E-mail: i.dijkgraaf@nucmed.umcn.nl; Liu, Shuang [School of Health Sciences, Purdue University, West Lafayette, Indiana (United States); Kruijtzer, John A.W. [Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3508 TB Utrecht (Netherlands); Soede, Annemieke C. [Department of Nuclear Medicine, Radboud University Nijmegen Medical Center, PO Box 9101, HB Nijmegen 6500 (Netherlands); Oyen, Wim J.G. [Department of Nuclear Medicine, Radboud University Nijmegen Medical Center, PO Box 9101, HB Nijmegen 6500 (Netherlands); Liskamp, Rob M.J. [Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3508 TB Utrecht (Netherlands); Corstens, Frans H.M. [Department of Nuclear Medicine, Radboud University Nijmegen Medical Center, PO Box 9101, HB Nijmegen 6500 (Netherlands); Boerman, Otto C. [Department of Nuclear Medicine, Radboud University Nijmegen Medical Center, PO Box 9101, HB Nijmegen 6500 (Netherlands)

    2007-01-15

    Introduction: Due to the selective expression of the {alpha}{sub v}{beta}{sub 3} integrin in tumors, radiolabeled arginine-glycine-aspartic acid (RGD) peptides are attractive candidates for tumor targeting. Minor modifications of these peptides could have a major impact on in vivo characteristics. In this study, we systematically investigated the effects of linker modification between two cyclic RGD sequences and DOTA (1,4,7,10-tetraazadodecane-N,N',N{sub '},N'''-tetraacetic acid) on the in vitro and in vivo characteristics of the tracer. Methods: A dimeric RGD peptide was synthesized and conjugated either directly with DOTA or via different linkers: PEG{sub 4} (polyethylene glycol), glutamic acid or lysine. The RGD peptides were radiolabeled with {sup 111}In, and their in vitro and in vivo {alpha}{sub v}{beta}{sub 3}-binding characteristics were determined. Results: LogP values varied between -2.82{+-}0.06 and -3.95{+-}0.33. The IC{sub 50} values for DOTA-E-[c(RGDfK)]{sub 2}, DOTA-PEG{sub 4}-E-[c(RGDfK)]{sub 2}, DOTA-E-E-[c(RGDfK)]{sub 2} and DOTA-K-E-[c(RGDfK)]{sub 2} were comparable. Two hours after injection, the tumor uptakes of the {sup 111}In-labeled compounds were not significantly different. The kidney accumulation of [{sup 111}In]-DOTA-K-E-[c(RGDfK)]{sub 2} [4.05{+-}0.20% of the injected dose per gram (ID/g)] was significantly higher as compared with that of [{sup 111}In]-DOTA-E-[c(RGDfK)]{sub 2} (2.63{+-}0.19% ID/g; P<.05) as well as that of [{sup 111}In]-DOTA-E-E-[c(RGDfK)]{sub 2} (2.16{+-}0.21% ID/g; P<.01). The liver uptake of [{sup 111}In]-DOTA-E-E-[c(RGDfK)]{sub 2} (2.12{+-}0.09% ID/g) was significantly higher as compared with that of [{sup 111}In]-DOTA-E-[c(RGDfK)]{sub 2} (1.64{+-}0.1% ID/g; P<.05) as well as that of [{sup 111}In]-DOTA-K-E-[c(RGDfK)]{sub 2} (1.52{+-}0.04% ID/g; P<.01). Conclusions: Linker variation did not affect affinity for {alpha}{sub v}{beta}{sub 3} and tumor uptake. Insertion of lysine caused enhanced

  9. Radioiodinated Exendin-4 Is Superior to the Radiometal-Labelled Glucagon-Like Peptide-1 Receptor Probes Overcoming Their High Kidney Uptake

    Science.gov (United States)

    Läppchen, Tilman; Tönnesmann, Roswitha; Eersels, Jos; Meyer, Philipp T.; Maecke, Helmut R.; Rylova, Svetlana N.

    2017-01-01

    GLP-1 receptors are ideal targets for preoperative imaging of benign insulinoma and for quantifying the beta cell mass. The existing clinical tracers targeting GLP-1R are all agonists with low specific activity and very high kidney uptake. In order to solve those issues we evaluated GLP-1R agonist Ex-4 and antagonist Ex(9–39) radioiodinated at Tyr40 side by side with [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4 (68Ga-Ex-4) used in the clinic. The Kd, Bmax, internalization and binding kinetics of [Nle14,125I-Tyr40-NH2]Ex-4 and [Nle14,125I-Tyr40-NH2]Ex(9–39) were studied in vitro using Ins-1E cells. Biodistribution and imaging studies were performed in nude mice bearing Ins-1E xenografts. In vitro evaluation demonstrated high affinity binding of the [Nle14,125I-Tyr40-NH2]Ex-4 agonist to the Ins-1E cells with fast internalization kinetics reaching a plateau after 30 min. The antagonist [Nle14,125I-Tyr40-NH2]Ex(9–39) did not internalize and had a 4–fold higher Kd value compared to the agonist. In contrast to [Nle14,125I-Tyr40-NH2]Ex(9–39), which showed low and transient tumor uptake, [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated excellent in vivo binding properties with tumor uptake identical to that of 68Ga-Ex-4, but substantially lower kidney uptake resulting in a tumor-to-kidney ratio of 9.7 at 1 h compared to 0.3 with 68Ga-Ex-4. Accumulation of activity in thyroid and stomach for both peptides, which was effectively blocked by irenat, confirms that in vivo deiodination is the mechanism behind the low kidney retention of iodinated peptides. The 124I congener of [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated a similar favourable biodistribution profile in the PET imaging studies in contrast to the typical biodistribution pattern of [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4. Our results demonstrate that iodinated Ex-4 is a very promising tracer for imaging of benign insulinomas. It solves the problem of high kidney uptake of the radiometal-labelled tracers by improving the tumor

  10. Biodistribution and SPECT Imaging Study of 99mTc Labeling NGR Peptide in Nude Mice Bearing Human HepG2 Hepatoma

    Directory of Open Access Journals (Sweden)

    Wenhui Ma

    2014-01-01

    Full Text Available A peptide containing Asn-Gly-Arg(NGR sequence was synthesized and directly labeled with Tc. Its radiochemical characteristics, biodistribution, and SPECT imaging were evaluated in nude mice bearing human HepG2 hepatoma. Nude mice bearing HepG2 were randomly divided into 5 groups with 5 mice in each group and injected with ~7.4 MBq Tc-NGR. The SPECT images were acquired in 1, 4, 8, and 12 h postinjection via caudal vein. The metabolism of tracers was determined in major organs at different time points, which demonstrated rapid, significant tumor uptake and slow tumor washout. The control group mice were blocked by coinjecting unlabelled NGR (20 mg/kg. Tumor uptake was (2.52±0.83% ID/g at 1 h, with the highest uptake of (3.26±0.63% ID/g at 8 h. In comparison, the uptake of the blocked control group was (1.65±0.61% ID/g at 1 h after injection. The SPECT static images and the tumor/muscle (T/NT value were obtained. The highest T/NT value was 7.58±1.92 at 8 h. The xenografted tumor became visible at 1 h and the clearest image of the tumor was observed at 8 h. In conclusion, Tc-NGR can be efficiently prepared and it exhibited good properties for the potential SPECT imaging agent of tumor.

  11. Post-translational heterocyclic backbone modifications in the 43-peptide antibiotic microcin B17. Structure elucidation and NMR study of a 13C,15N-labelled gyrase inhibitor.

    Science.gov (United States)

    Bayer, A; Freund, S; Jung, G

    1995-12-01

    Microcin B17 (McB17), the first known gyrase inhibitor of peptidic nature, is produced by ribosomal synthesis and post-translational modification of the 69-residue precursor protein by an Escherichia coli strain. To elucidate the chemical structure of the mature 43-residue peptide antibiotic, fermentation and purification protocols were established and optimized which allowed the isolation and purification of substantial amounts of highly pure McB17 (non-labelled, 15N-labelled and 13C/15N-labelled peptide. By ultraviolet-absorption spectroscopy. HPLC-electrospray mass spectrometry and GC-mass spectrometry, amino acid analysis, protein sequencing, and, in particular, multidimensional NMR, we could demonstrate and unequivocally prove that the enzymic modification of the precursor backbone at Gly-Cys and Gly-Ser segments leads to the formation of 2-aminomethylthiazole-4-carboxylic acid and 2-aminomethyloxazole-4-carboxylic acid, respectively. In addition, two bicyclic modifications 2-(2-aminomethyloxazolyl)thiazole-4-carboxylic acid and 2-(2-aminomethylthiazolyl)oxazole-4-carboxylic acid were found that consist of directly linked thiazole and oxazole rings derived from one Gly-Ser-Cys and one Gly-Cys-Ser segment. Analogous to the thiazole and oxazole rings found in antitumor peptides of microbial and marine origin, these heteroaromatic ring systems of McB17 presumably play an important role in its gyrase-inhibiting activity, e.g. interacting with the DNA to trap the covalent protein-DNA intermediate of the breakage-reunion reaction of the gyrase.

  12. Jeans type instability for a chemotactic model of cellular aggregation

    CERN Document Server

    Chavanis, Pierre-Henri

    2008-01-01

    We consider an inertial model of chemotactic aggregation generalizing the Keller-Segel model and we study the linear dynamical stability of an infinite and homogeneous distribution of cells (bacteria, amoebae, endothelial cells,...) when inertial effects are accounted for. These inertial terms model cells directional persistance. We determine the condition of instability and the growth rate of the perturbation as a function of the cell density and the wavelength of the perturbation. We discuss the differences between overdamped (Keller-Segel) and inertial models. Finally, we show the analogy between the instability criterion for biological populations and the Jeans instability criterion in astrophysics.

  13. Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules.

    Science.gov (United States)

    Altai, Mohamed; Wållberg, Helena; Orlova, Anna; Rosestedt, Maria; Hosseinimehr, Seyed Jalal; Tolmachev, Vladimir; Ståhl, Stefan

    2012-05-01

    Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide 99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of 99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied 99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of 99mTc. The biodistribution of all 99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of 99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

  14. The Effect of Superparamagnetic Iron Oxide with iRGD Peptide on the Labeling of Pancreatic Cancer Cells In Vitro: A Preliminary Study

    OpenAIRE

    Hou Dong Zuo; Wei Wu Yao; Tian Wu Chen; Jiang Zhu; Juan Juan Zhang; Yu Pu; Gang Liu; Xiao Ming Zhang

    2014-01-01

    The iRGD peptide loaded with iron oxide nanoparticles for tumor targeting and tissue penetration was developed for targeted tumor therapy and ultrasensitive MR imaging. Binding of iRGD, a tumor homing peptide, is mediated by integrins, which are widely expressed on the surface of cells. Several types of small molecular drugs and nanoparticles can be transfected into cells with the help of iRGD peptide. Thus, we postulate that SPIO nanoparticles, which have good biocompatibility, can also be t...

  15. Corneal organ cultures in tyrosinemia release chemotactic factors.

    Science.gov (United States)

    Lohr, K M; Hyndiuk, R A; Hatchell, D L; Kurth, C E

    1985-05-01

    Corneal inflammation with subsequent scarring and blindness occurs in the inherited human metabolic disease tyrosinemia type II, yet putative inflammatory mediators in this disorder and in the avascular cornea in general are poorly defined. In a Tyr-fed rat model of tyrosinemia type II, intracellular crystals, presumably Tyr, are hypothesized to be responsible for the increased lysosomal activity observed in corneal epithelial lesions. Because polymorphonuclear leukocytes (PMNs) are seen only at the site of these lesions, we used this model to study humoral mediators released from Tyr-fed rat corneal organ cultures. Only Tyr-fed rats developed stromal edema and linear granular opacities in gray edematous corneal epithelium, compatible with a noninfectious keratitis. Electron micrographs confirmed epithelial edema and showed focal epithelial necrosis with PMN invasion of the stroma. Only Tyr-fed rat corneal culture supernatants contained chemotactic activity that was heat labile and moderately trypsin sensitive. Four peaks with varying amounts of chemotactic activity were found on Sephadex G-75 chromatography. Although the identity of these peaks of activity has not yet been established, we suggest that they may be responsible for the PMN infiltration observed in this model of corneal inflammation.

  16. DETECTION OF A NEUTROPHIL CHEMOTACTIC FACTOR IN JAPANESE ENCEPHALITIS PATIENTS

    Directory of Open Access Journals (Sweden)

    Aditi Singh

    2012-12-01

    Full Text Available Japanese encephalitis (JE one of the most common cause of acute encephalitis in tropical regions, has generated much public anxiety in India. An early influx of macrophages followed by neutrophils at the site of injury in different organs in humans and mice has previously been reported. It correlated with production of a neutrophil chemotactic protein derived from macrophages. In the present study out of a total of 324 acute encephalitic patients, admitted in Gandhi memorial and associated hospitals, Lucknow, 121 patients with one or more indicators of JE virus infection were included. Significant pleocytosis (mean TLC value of 126+52 cells / mm3 in CSF and leucocytosis (>11,000 cells/mm3 in peripheral blood was observed at the time of admission. The leucocytosis increased significantly during second week in 67% of patients. The peripheral blood mononuclear cells culture done on alternate days was tested for chemotactic activity (hMDF, which was observed to be highest in second week of illness. The direct detection of hMDF in circulation by dot blot was positive in 92% of acute serum samples, with negligible (12.5% reactivity for convalescent sera. A correlation between the hMDF levels and severity of illness has also been observed.

  17. Collective cell motility promotes chemotactic prowess and resistance to chemorepulsion.

    Science.gov (United States)

    Malet-Engra, Gema; Yu, Weimiao; Oldani, Amanda; Rey-Barroso, Javier; Gov, Nir S; Scita, Giorgio; Dupré, Loïc

    2015-01-19

    Collective cell migration is a widespread biological phenomenon, whereby groups of highly coordinated, adherent cells move in a polarized fashion. This migration mode is a hallmark of tissue morphogenesis during development and repair and of solid tumor dissemination. In addition to circulating as solitary cells, lymphoid malignancies can assemble into tissues as multicellular aggregates. Whether malignant lymphocytes are capable of coordinating their motility in the context of chemokine gradients is, however, unknown. Here, we show that, upon exposure to CCL19 or CXCL12 gradients, malignant B and T lymphocytes assemble into clusters that migrate directionally and display a wider chemotactic sensitivity than individual cells. Physical modeling recapitulates cluster motility statistics and shows that intracluster cell cohesion results in noise reduction and enhanced directionality. Quantitative image analysis reveals that cluster migration runs are periodically interrupted by transitory rotation and random phases that favor leader cell turnover. Additionally, internalization of CCR7 in leader cells is accompanied by protrusion retraction, loss of polarity, and the ensuing replacement by new leader cells. These mechanisms ensure sustained forward migration and resistance to chemorepulsion, a behavior of individual cells exposed to steep CCL19 gradients that depends on CCR7 endocytosis. Thus, coordinated cluster dynamics confer distinct chemotactic properties, highlighting unexpected features of lymphoid cell migration.

  18. Radioassay of granulocyte chemotaxis. Studies of human granulocytes and chemotactic factors. [/sup 51/Cr tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Gallin, J.I.

    1974-01-01

    The above studies demonstrate that the /sup 51/Cr radiolabel chemotactic assay is a relatively simple and objective means for studying leukocyte chemotaxis in both normal and pathological conditions. Application of this method to studies of normal human chemotaxis revealed a relatively narrow range of normal and little day-to-day variability. Analysis of this variability revealed that there is more variability among the response of different granulocytes to a constant chemotactic stimulus than among the chemotactic activity of different sera to a single cell source. Utilizing the /sup 51/Cr radioassay, the abnormal granulocyte chemotactic behavior reported in Chediak-Higashi syndrome and a patient with recurrent pyogenic infections and mucocutaneous candidiasis has been confirmed. The /sup 51/Cr chemotactic assay has also been used to assess the generation of chemotactic activity from human serum and plasma. The in vitro generation of two distinct chemotactic factors were examined; the complement product (C5a) and kallikrein, an enzyme of the kinin-generating pathway. Kinetic analysis of complement-related chemotactic factor formation, utilizing immune complexes or endotoxin to activate normal sera in the presence or absence of EGTA as well as kinetic analysis of activation of C2-deficient human serum, provided an easy means of distinguishing the classical (antibody-mediated) complement pathway from the alternate pathway. Such kinetic analysis is necessary to detect clinically important abnormalities since, after 60 min of generation time, normal chemotactic activity may be present despite complete absence or inhibition of one complement pathway. The chemotactic factor generated by either pathway of complement activation appears to be predominately attributable to C5a.

  19. Controlling neural activity in Caenorhabditis elegans to evoke chemotactic behavior

    Science.gov (United States)

    Kocabas, Askin; Shen, Ching-Han; Guo, Zengcai V.; Ramanathan, Sharad

    2013-03-01

    Animals locate and track chemoattractive gradients in the environment to find food. With its simple nervous system, Caenorhabditis elegans is a good model system in which to understand how the dynamics of neural activity control this search behavior. To understand how the activity in its interneurons coordinate different motor programs to lead the animal to food, here we used optogenetics and new optical tools to manipulate neural activity directly in freely moving animals to evoke chemotactic behavior. By deducing the classes of activity patterns triggered during chemotaxis and exciting individual neurons with these patterns, we identified interneurons that control the essential locomotory programs for this behavior. Notably, we discovered that controlling the dynamics of activity in just one interneuron pair was sufficient to force the animal to locate, turn towards and track virtual light gradients.

  20. Phenomenological understanding of aggregation and dispersion of chemotactic cells

    CERN Document Server

    Iwasa, Masatomo

    2011-01-01

    We present a simple model that describes the motion of a single chemotactic cell exposed to a traveling wave of the chemoattractant. The model incorporates two types of responses to stimulation by the chemoattractant, i.e., change in polarity and change in motility of the cell. The periodic change in motility is assumed to be induced by the periodic stimulation by the chemoattractant on the basis of previous observations. Consequently, net migration of the cell occurs in a particular direction with respect to wave propagation, which explains the migration of Dictyostelium cells in aggregation processes. The difference between two time delays from the stimulation to the two responses and the wave frequency determined by the frequency of the secretion of the chemoattractant are important parameters that determine the direction of migration and the effective interaction between cells in a population. This result explains the dispersed state of a population of vegetative cells and cells in preaggregation without ...

  1. Utilization of a novel electrochemical {sup 90}Sr/{sup 90}Y generator for the preparation of {sup 90}Y-labeled RGD peptide dimer in clinically relevant dose

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Sudipta; Chakravarty, Rubel; Pillai, Maroor Raghavan Ambikalmajan; Dash, Ashutosh [Bhabha Atomic Research Centre, Mumbai (India). Radiopharmaceuticals Div.; Sarma, Haladhar Dev [Bhabha Atomic Research Centre, Mumbai (India). Radiation Biology and Health Sciences Div.

    2014-09-01

    The work reported in this paper provides a systematic study towards the development of an optimized strategy for preparation of a clinically relevant dose of {sup 90}Y-labeled dimeric RGD peptide derivative, DOTA-E[c(RGDfK)]{sub 2} [DOTA-(RGD){sub 2}] for in vivo targeted therapy utilizing {sup 90}Y obtained from a novel electrochemical {sup 90}Sr/{sup 90}Y generator. The performance of the generator was evaluated to ensure its suitability for providing {sup 90}Y in adequate quantity and purity required for formulation of clinically relevant dose for PRRT. {sup 90}Y-DOTA-(RGD){sub 2} was synthesized in high yield (86.2 ± 2.5%) and radiochemical purity (98.4 ± 0.5%) using clinically relevant dose (∝ 3.8 GBq) of {sup 90}Y. In vitro stability studies revealed that the radiolabeled conjugate retained its radiochemical purity in normal saline and human serum. Preliminary biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors showed that the preparation exhibited significant tumor uptake (5.30 ± 0.78% of injected activity at 30 min post-injection) with good tumor to background ratio. The optimized radiolabeling protocol seems to be an attractive strategy which is largely viewed as a springboard to realize scope of developing {sup 90}Y labeled cyclic RGD peptides for targeted therapy of tumors over-expressing integrin-α{sub ν}β{sub 3} receptors. (orig.)

  2. Molecular imaging of neuroendocrine tumors using {sup 68}Ga-labeled peptides (Somatostatin receptor PET/CT); Molekulare Bildgebung neuroendokriner Tumoren mit {sup 68}Ga-markierten Peptiden (Somatostatinrezeptor-PET/CT)

    Energy Technology Data Exchange (ETDEWEB)

    Baum, R.P.; Prasad, V. [Zentralklinik Bad Berka GmbH (Germany). Klinik fuer Nuklearmedizin/PET-Zentrum; Hoersch, D. [Zentralklinik Bad Berka GmbH (Germany). Klinik fuer Innere Medizin, Gastroenterologie, Onkologie, Endokrionologie

    2009-06-15

    Receptor PET/CT using {sup 68}Ga-labeled somatostatin analogues (DOTA-NOC, DOTA-TOC or DOTA-TATE) enables the highly sensitive molecular imaging of neuroendocrine tumors (NETs) based on the expression of somatostatin receptors and even the detection of receptor subtypes. Our experience after more than 3000 studies shows that receptor PET/CT has a significantly higher tumor detection rate than conventional scintigraphy (even in SPECT/CT technique), and that tumor lesions can be very accurately localized. By calculating standardized uptake values (SUV) - which are reproducible and investigator-independent - patients can be selected for peptide receptor radiotherapy and also the course after therapy can be controlled. Receptor-PET/CT is the most sensitive imaging modality for the detection of unknown primary tumors (CUP syndrome), which is especially true for the detection of neuroendocrine tumors of the pancreas and small bowel; whole-body staging (''one stop shop'') as well as restaging and selection of patients for peptide receptor radiotherapy can be performed using a patient-friendly procedure (examination finished within one hour) exposing the patient to less radiation than whole-body CT scanning. The {sup 68}Ge/{sup 68}Ga generator has proved very reliable over the years - even in a hospital environment. The effective costs for {sup 68}Ga labeled somatostatin analogues might be less than for scintigraphic agents, provided a certain number of studies per year are performed. The development of new tumor-specific peptides as well as of other DOTA- or NOTA-coupled radiopharmaceuticals opens a new avenue into the future: finally, the {sup 68}Ga generator could play a similar important role for PET/CT as did the {sup 99m}Tc-Generator for conventional gamma camera imaging over the last decades. (orig.)

  3. Data on biodistribution and radiation absorbed dose profile of a novel 64Cu-labeled high affinity cell-specific peptide for positron emission tomography imaging of tumor vasculature

    Directory of Open Access Journals (Sweden)

    Joseph R. Merrill

    2016-06-01

    Full Text Available New peptide-based diagnostic and therapeutic approaches hold promise for highly selective targeting of cancer leading to more precise and effective diagnostic and therapeutic modalities. An important feature of these approaches is to reach the tumor tissue while limiting or minimizing the dose to normal organs. In this context, efforts to design and engineer materials with optimal in vivo targeting and clearance properties are important. This Data In Brief article reports on biodistribution and radiation absorbed dose profile of a novel high affinity radiopeptide specific for bone marrow-derived tumor vasculature. Background information on the design, preparation, and in vivo characterization of this peptide-based targeted radiodiagnostic is described in the article “Synthesis and comparative evaluation of novel 64Cu-labeled high affinity cell-specific peptides for positron emission tomography of tumor vasculature” (Merrill et al., 2016 [1]. Here we report biodistribution measurements in mice and calculate the radiation absorbed doses to normal organs using a modified Medical Internal Radiation Dosimetry (MIRD methodology that accounts for physical and geometric factors and cross-organ beta doses.

  4. Absolute Quantification of Prion Protein (90-231) Using Stable Isotope-Labeled Chymotryptic Peptide Standards in a LC-MRM AQUA Workflow

    Science.gov (United States)

    Sturm, Robert; Sheynkman, Gloria; Booth, Clarissa; Smith, Lloyd M.; Pedersen, Joel A.; Li, Lingjun

    2012-09-01

    Substantial evidence indicates that the disease-associated conformer of the prion protein (PrPTSE) constitutes the etiologic agent in prion diseases. These diseases affect multiple mammalian species. PrPTSE has the ability to convert the conformation of the normal prion protein (PrPC) into a β-sheet rich form resistant to proteinase K digestion. Common immunological techniques lack the sensitivity to detect PrPTSE at subfemtomole levels, whereas animal bioassays, cell culture, and in vitro conversion assays offer higher sensitivity but lack the high-throughput the immunological assays offer. Mass spectrometry is an attractive alternative to the above assays as it offers high-throughput, direct measurement of a protein's signature peptide, often with subfemtomole sensitivities. Although a liquid chromatography-multiple reaction monitoring (LC-MRM) method has been reported for PrPTSE, the chemical composition and lack of amino acid sequence conservation of the signature peptide may compromise its accuracy and make it difficult to apply to multiple species. Here, we demonstrate that an alternative protease (chymotrypsin) can produce signature peptides suitable for a LC-MRM absolute quantification (AQUA) experiment. The new method offers several advantages, including: (1) a chymotryptic signature peptide lacking chemically active residues (Cys, Met) that can confound assay accuracy; (2) low attomole limits of detection and quantitation (LOD and LOQ); and (3) a signature peptide retaining the same amino acid sequence across most mammals naturally susceptible to prion infection as well as important laboratory models. To the authors' knowledge, this is the first report on the use of a non-tryptic peptide in a LC-MRM AQUA workflow.

  5. Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment.

    Science.gov (United States)

    Clark, R A; Wikner, N E; Doherty, D E; Norris, D A

    1988-08-25

    Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell

  6. Urease-independent chemotactic responses of Helicobacter pylori to urea, urease inhibitors, and sodium bicarbonate.

    OpenAIRE

    Mizote, T; Yoshiyama, H; T. Nakazawa

    1997-01-01

    Helicobacter pylori CPY3401 and an isogenic urease-negative mutant, HPT73, showed chemotactic responses to urea, flurofamide (a potent urease inhibitor), and sodium bicarbonate. Since urea and sodium bicarbonate are secreted through the gastric epithelial surface and hydrolysis of urea by urease on the bacterial surface is essential for colonization, the chemotactic response of H. pylori may be crucial for its colonization and persistence in the stomach.

  7. Chemotactic Activity on Human Neutrophils to Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Tetiana Haniastuti

    2013-07-01

    Full Text Available Objective: The aim of this study was to evaluate chemotactic activity o neutrophil to S. mutans. Chemotaxis assay was performed in blind well chambers. Materials and Methods: Hanks balanced salt solution (HBSS containing 106 S. mutans,  108 S. mutans, 10-8 M fMLP, or HBSS alone were placed in the lower wells of the chamber and covered with polycorbonate membrane filter. Neutrophils suspension (2x105 cells was then placed in the upper compartment. After incubation for 60 mins at 37ºC in a humidified atmosphere with 5% CO2, the filters were removed and stained with Giemsa. Result: ANOVA revealed statistically significant differences among groups (p<0.05, indicating that S. mutans induced neutrophils chemotaxis. The number of neutrophils migration in response to 108 S. mutans and 106 S. mutans were signifiantly greater compared to fMLP (p<0.05. Conclusion: S. mutans may activate human neutrophils, resulting in the chemotaxis of the neutrophils.DOI: 10.14693/jdi.v16i2.99

  8. Human sperm pattern of movement during chemotactic re-orientation towards a progesterone source

    Institute of Scientific and Technical Information of China (English)

    Cecilia Soledad Blengini; Maria Eugenia Teves; Diego Rafael Unates; Hector Alejandro Guidobaldi; Laura Virginia Gatica; Laura Cecilia Giojalas

    2011-01-01

    @@ Human spermatozoa may chemotactically find out the egg by following an increasing gradient of attractant molecules.Although human spermatozoa have been observed to show several of the physiological characteristics of chemotaxis,the chemotactic pattern of movement has not been easy to describe.However,it is apparent that chemotactic cells may be identified while returning to the attractant source.This study characterizes the pattern of movement of human spermatozoa during chemotactic re-orientation towards a progesterone source,which is a physiological attractant candidate.By means of videomicroscopy and image analysis,a chemotactic pattern of movement was identified as the spermatozoon returned towards the source of a chemotactic concentration of progesterone (10 pmol l-1).First,as a continuation of its original path,the spermatozoon swims away from the progesterone source with linear movement and then turns back with a transitional movement that can be characterized by an increased velocity and decreased linearity.This sperm behaviour may help the spermatozoon to re-orient itself towards a progesterone source and may be used to identify the few cells that are undergoing chemotaxis at a given time.

  9. Effect of monocyte chemoattractant protein-1 on chemotactic gene expression by macrophage cell line U937

    Institute of Scientific and Technical Information of China (English)

    BIAN Guang-xing; GUO Bao-yu; MIAO Hong; QIU Lei; CAO Dong-mei; DAO Shu-yan; ZHANG Ran

    2004-01-01

    Objective: To study the chemotactic superfamily genes expression profiling of macrophage line U937 treated with monocyte chemoattractant protein-1 (MCP-1) using gene chip technique. Methods: Total RNA from macrophage line U937 (as control) and U937 with MCP-1 was extracted, made reverse transcript to cDNA and tested with gene expression chip HO2 human. Results: Some chemotactic-related gene expressions were changed in all analyzed genes. Regulated upon activation, normal T cell expressed and secreted (RANTES) was up-regulated over 2-fold and 7 chemotactic-related genes (CCR2, CCR5, CCL16, GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2) were down-regulated over 2-fold inMCP-1 treated U937 cells at mRNA level. Conclusion: MCP-1 can influence some chemokines and receptors expression in macrophage in vitro, in which MCP-1 mainly down-regulates the chemotactic genes expression of those influencing neutrophilic granulocyte (GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2). Another novel finding is that it can also down-regulate the mRNA level of CCR5, which plays a critical role in many disorders and illnesses.

  10. Animal experiment study of silica nanoparticles labeled peptides with 125 I%包裹125 I标记多肽的二氧化硅纳米颗粒的动物实验研究

    Institute of Scientific and Technical Information of China (English)

    王风秀; 王加芬; 陆小伟

    2014-01-01

    目的:探讨纳米二氧化硅是否可作为生物大分子药物口服载体。方法研究钙离子的量对二氧化硅纳米颗粒形态的影响、不同钙离子存在下二氧化硅纳米颗粒对蛋白或多肽的包裹率、蛋白或多肽从二氧化硅纳米颗粒中释放情况、二氧化硅包裹蛋白酶解情况、以及二氧化硅纳米颗粒包裹125 I标记多肽在小鼠体内释放情况。结果实验表明合成的二氧化硅纳米颗粒包裹多肽在小白鼠体内可以顺利通过胃酸到达小肠,有作为生物活性大分子口服药物载体的潜力。结论蛋白或多肽比较容易在碱性条件下释放,在pH 2的酸性条件下释放极少(胃酸的pH值),制备的二氧化硅纳米颗粒有作为口服药物载体的潜力。%Objective To study whether the silica nanoparticles can be used as a biomacromolecular carrier of oral drugs.Methods Studyed the influence of the amount of calcium ions on nano silica nanoparticles morphology,the encapsulation rate of silica nanoparticles on proteins or peptides in the presence of different calcium ions,the situation of proteins or peptides released from silica nanoparticles,enzymolysis of protein packaged by silica, silica nanoparticles labeled peptides with 125 I released in mice.Results Experiments showed that the synthetic silica nanoparticles packaged with peptides could smoothly reach the small intestine through the gastric acid in mice, it had potential to be biomacromolecular carrier of oral drug. Conclusion Proteins or peptides is easy to release in alkaline condition,but release few in acidic condition of pH2 (PH of gastric acid).The silica nanoparticles prepared have the potential to be oral drug carrier.

  11. Development of a formulation lyophilized for the obtention of a antimicrobial peptide Ubiquicidine labelled with {sup 99m} Tc; Desarrollo de una formulacion liofilizada para la obtencion del peptido antimicrobiano Ubiquicidina marcado con Tc-99m

    Energy Technology Data Exchange (ETDEWEB)

    Palomares R, P.; Hernandez B, C.A.; Contreras N, G.; Garcia P, M.L.; Pantoja H, I.E. [UAEM, Toluca (Mexico); Ferro F, G. [ININ, A.P. 18-1027, 11801 Mexico D.F. (Mexico)]. E-mail: patodish@hotmail.com

    2004-07-01

    The {sup 99m} Tc-UBI 29-41 are a labelled fragment of the antimicrobial human peptide Ubiquicidine proposed as a new radiopharmaceutical able to differentiate an infectious process of an inflammatory one through the gamma graphic image. It has been demonstrated that the {sup 99m} Tc-UBI 29-41 unite to bacteria in vitro and that accumulates in infection sites in human with minimum captivation in inflammation sites. In this work the development of a pharmaceutical lyophilized formulation is presented for the instantaneous marked one of the UBI 29-41 with {sup 99m} Tc. The selection of the components of the formulation settled down by means of the employment of an experimental design of 3 factors with mixed levels, evaluating the effect of the diluent type, concentration of tinny chloride and the reaction volume. The obtained formulations showed to be stable until for 6 months, being obtained complexes of the radiolabelled peptide with radiochemical purity > 95 % in sterile form and apirogen. The developed pharmaceutical form, will facilitate the routinary use of this new radiopharmaceutical in the diverse hospital departments of nuclear medicine. (Author)

  12. Food Labels

    Science.gov (United States)

    ... Loss Surgery? A Week of Healthy Breakfasts Shyness Food Labels KidsHealth > For Teens > Food Labels Print A ... have at least 95% organic ingredients. continue Making Food Labels Work for You The first step in ...

  13. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells.

    OpenAIRE

    Cushing, S D; Berliner, J A; Valente, A. J.; Territo, M C; Navab, M; Parhami, F; Gerrity, R; Schwartz, C J; Fogelman, A M

    1990-01-01

    After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2- to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human ...

  14. Rabbit neutrophil chemotactic protein (NCP) activates both CXCR1 and CXCR2 and is the functional homologue for human CXCL6.

    Science.gov (United States)

    Catusse, Julie; Struyf, Sofie; Wuyts, Anja; Weyler, Myke; Loos, Tamara; Gijsbers, Klara; Gouwy, Mieke; Proost, Paul; Van Damme, Jo

    2004-11-15

    Neutrophil chemotactic protein (NCP) is a rabbit CXC chemokine with activating and chemotactic properties on neutrophilic granulocytes. Although its selective activity on neutrophils is demonstrated, its interactions with specific chemokine receptors are not defined. For further functional characterization, NCP was chemically synthesized and was found to be equipotent as natural NCP in neutrophil chemotaxis. To identify its human homologue, we separately expressed two potential rabbit NCP receptors (CXCR1 and CXCR2) in Jurkat cells. Pure synthetic NCP was equally efficient to promote chemotaxis through either rabbit CXCR1 or CXCR2. Moreover, chemotaxis assays on rabbit CXCR1 and CXCR2 transfectants showed that NCP uses the same receptors as interleukin-8 (IL-8), a major rabbit CXC chemokine, but not rabbit GROalpha, which only recognized CXCR2. In addition, specific inhibitors for CXCR1 or CXCR2 reduced rabbit neutrophil chemotaxis induced by NCP and rabbit IL-8. Furthermore, NCP and the structurally related human CXCR1/CXCR2 agonist CXCL6/GCP-2 (granulocyte chemotactic protein-2) cross-desensitized each other in intracellular calcium release assays on human neutrophils, further indicating that both chemokines share the same receptors. The inflammatory role of NCP was also evidenced by its potent granulocytosis inducing capacity in rabbits upon systemic administration. This study provides in vitro and in vivo evidences that NCP is the functional rabbit homologue for human CXCL6/GCP-2 rather than the most related CXCR2 agonist CXCL5/ENA-78 (epithelial cell-derived neutrophil activating peptide-78). It is concluded that the rabbit is a better model to study human neutrophil activation compared to mice, which lack CXCL8/IL-8.

  15. Enhanced Retention of Chemotactic Bacteria in a Pore Network with Residual NAPL Contamination.

    Science.gov (United States)

    Wang, Xiaopu; Lanning, Larry M; Ford, Roseanne M

    2016-01-01

    Nonaqueous-phase liquid (NAPL) contaminants are difficult to eliminate from natural aquifers due, in part, to the heterogeneous structure of the soil. Chemotaxis enhances the mixing of bacteria with contaminant sources in low-permeability regions, which may not be readily accessible by advection and dispersion alone. A microfluidic device was designed to mimic heterogeneous features of a contaminated groundwater aquifer. NAPL droplets (toluene) were trapped within a fine pore network, and bacteria were injected through a highly conductive adjacent macrochannel. Chemotactic bacteria (Pseudomonas putida F1) exhibited greater accumulation near the pore network at 0.5 m/day than both the nonchemotactic control and the chemotactic bacteria at a higher groundwater velocity of 5 m/day. Chemotactic bacteria accumulated in the vicinity of NAPL droplets, and the accumulation was 15% greater than a nonchemotactic mutant. Indirect evidence showed that chemotactic bacteria were retained within the contaminated low-permeability region longer than nonchemotactic bacteria at 0.25 m/day. This retention was diminished at 5 m/day. Numerical solutions of the bacterial-transport equations were consistent with the experimental results. Because toluene is degraded by P. putida F1, the accumulation of chemotactic bacteria around NAPL sources is expected to increase contaminant consumption and improve the efficiency of bioremediation.

  16. Collective Chemotactic Dynamics in the Presence of Self-Generated Fluid Flows

    CERN Document Server

    Lushi, Enkeleida; Shelley, Michael J

    2012-01-01

    In micro-swimmer suspensions locomotion necessarily generates fluid motion, and it is known that such flows can lead to collective behavior from unbiased swimming. We examine the complementary problem of how chemotaxis is affected by self-generated flows. A kinetic theory coupling run-and-tumble chemotaxis to the flows of collective swimming shows separate branches of chemotactic and hydrodynamic instabilities for isotropic suspensions, the first driving aggregation, the second producing increased orientational order in suspensions of "pushers" and maximal disorder in suspensions of "pullers". Nonlinear simulations show that hydrodynamic interactions can limit and modify chemotactically-driven aggregation dynamics. In puller suspensions the dynamics form aggregates that are mutually-repelling due to the non-trivial flows. In pusher suspensions chemotactic aggregation can lead to destabilizing flows that fragment the regions of aggregation.

  17. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery...

  18. Colloidal silver nanoparticles/rhamnolipid (SNPRL) composite as novel chemotactic antibacterial agent.

    Science.gov (United States)

    Bharali, P; Saikia, J P; Paul, S; Konwar, B K

    2013-10-01

    The antibacterial activity of silver nanoparticles and rhamnolipid are well known individually. In the present research, antibacterial and chemotactic activity due to colloidal silver nanoparticles (SNP), rhamnolipid (RL) and silver nanoparticles/rhamnolipid composite (SNPRL) were evaluated using Staphylococcus aureus (MTCC3160), Escherichia coli (MTCC40), Pseudomonas aeruginosa (MTCC8163) and Bacillus subtilis (MTCC441) as test strains. Further, the SNPRL nanoparticles were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). The observation clearly indicates that SNPRL shows prominent antibacterial and chemotactic activity in comparison to all of its individual precursor components.

  19. Monocyte chemotactic protein-1 expression in coronary atherosclerosis plaque of sudden coronary death patients

    Institute of Scientific and Technical Information of China (English)

    冯相平

    2006-01-01

    Objective To investigate the expression of monocyte chemotactic protein 1 (MCP-1) in coronary atherosclerosis plaque of sudden coronary death (SCD) patients and the relationship between MCP-1 expression and SCD. Methods Autopsy heart samples (n=90) collected during 2001 - 2003 were divided to SCD group (n=

  20. Radiolabelled peptides for oncological diagnosis

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-15

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

  1. Ultrastructure of the surface structures and electron immunogold labeling of peptide immunoreactivity in the nervous system of Pseudothoracocotyla indica (Polyopisthocotylea: Monogenea).

    Science.gov (United States)

    Brennan, G P; Ramasamy, P

    1996-01-01

    Transmission electron microscope studies of the tegument of the tropical marine fish monogenean parasite Pseudothoracocotyla indica describe surface specialisations and detail the ultrastructure of the tegument and the haptor. The tegument consists of a syncytium, numerous electron-dense granules, electron-lucent vesicles and large multivesicular bodies. The posterior tegumental syncytium is infolded to form tegumental ridges that are present on both the ventral and dorsal surfaces. A thin coat of glycocalyx is present on the tegument surface. In contrast, the tegumental syncytium of the haptor is relatively thin, containing electron-dense granules and various-sized electron-lucent vesicles. Exocytosis of the electron-dense and electron-lucent vesicles apparently occurs in the syncytium of the haptor and general body surface. Tegumental damage was observed on the dorsal surface in the mid-body region and may possibly have been due to natural mechanical forces. The haptor consists of electron-dense clamp sclerites embedded within a matrix covered by the tegumental syncytium. The sclerites are connected to each other and to the basal lamina by radially oriented muscle fibres. The haptor is richly supplied with non-myelinated nerve axons. Both uniciliated and non-ciliated presumed sensory structures are present on the body surface and haptor. Uniciliated sensory structures were found mainly around the oral sucker. Groups of neurons and nerve processes containing neurosecretory vesicles were frequently observed in the vicinity of the clamps. Electron immunogold labelling studies demonstrated that neuropeptide F [NPF (Moniezia expansa)] immunoreactivity was confined to electron-dense-cored neurosecretory vesicles in nerve fibres from the posterior and haptor regions of the fluke.

  2. Enhanced Retention of Chemotactic Bacteria in a Pore Network with Residual NAPL Contamination

    Science.gov (United States)

    Ford, R.; Wang, X.

    2013-12-01

    Nonaqueous phase liquid (NAPL) contaminants are difficult to eliminate from natural aquifers due, in part, to the heterogeneous structure of the soil matrix. Residual NAPL ganglia remain trapped in regions where the hydraulic conductivity is relatively low. Bioremediation processes depend on adequate mixing of microbial populations and the groundwater contaminants that they degrade. The ability of bacteria to sense a chemical gradient and swim preferentially toward locations of higher concentration, known as chemotaxis, can enhance the mixing of bacteria with contaminant sources that may not be readily accessible by advection and dispersion alone. The impact of chemotaxis on bacterial abundance within a low conductivity NAPL-contaminated region of a well-characterized porous matrix was investigated. A microfluidic device was designed to mimic heterogeneous features of a contaminated groundwater system. NAPL ganglia (toluene) were trapped within a fine pore network, and bacteria were injected into the system through a highly conductive adjacent channel. Chemotactic bacteria (P. putida F1) migrated preferentially towards and accumulated in the vicinity of NAPL contaminant sources. The accumulation of chemotactic bacteria was 15% greater in comparison to a nonchemotactic mutant (P. putida F1 CheA). Bacteria in the microfluidic device were subjected to different flow velocities from 0.25 to 5 m/d encompassing the range of typical groundwater flow rates. Chemotactic bacteria exhibited greater accumulation near the intersection between the macrochannel and the porous network at a flow velocity of 0.5 m/d than both the nonchemotactic mutant control and the chemotactic bacteria at a higher flow velocity of 5 m/d. Breakthrough curves observed at the outlet provided indirect evidence that chemotactic bacteria were retained within the contaminated low permeable region for a longer time than the nonchemotactic bacteria at a flow velocity of 0.25 m/d. This retention was

  3. Radiopharmaceutical development of radiolabelled peptides

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-15

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

  4. Preliminary radioimmunoimaging and biodistribution of 131iodine-labeled single-chain antibody fragment against progastrin-releasing peptide(31-98) in small cell lung cancer xenografts

    Institute of Scientific and Technical Information of China (English)

    Hong Zhihui; Shi Yizhen; Liu Zengli; Zhou Xiaolin; Yang Yi; Tang Jun

    2014-01-01

    Background Monoclonal antibodies (mAbs) such as DD3,raised against progastrin-releasing peptide(31-98) (ProGRP(31-98)) antigen,have been used to target small cell lung cancer (SCLC).However,as an intact mAb,DD3 is cleared slowly from the body,with an optimal radioimmunoimaging time of 72 hours.More recently,a singlechain antibody fragment has demonstrated reduced excretion time in blood and normal tissues and is increasingly used in diagnostic cancer research.Thereby,it potentially increases the radioimmunoimaging efficacy.However,there have been few studies with this antibody fragment.The aim of this study was to characterize the preliminary radioimmunoimaging and biodistribution of 1311I-anti-ProGRP(31-98)scFv in nude mice bearing SCLC xenografts.Methods Anti-ProGRP(31-98) scFv was used to detect ProGRP expression by flow cytometry analysis and immunohistochemistry.131I-anti-ProGRP(31-98) scFv was injected intravenously into healthy Kunming mice and the percentage injected dose per gram (%ID/g) in various organs was calculated.Similarly,the %ID/g and tumor/non-tumor ratio in xenograft-bearing mice was calculated.After injection of 131I-anti-ProGRP(31-98) scFv,treated mice were imaged at 1,24,and 30 hours.Then the tumor/base ratios were calculated.Results ProGRP was highly expressed in NCI-H446 cells and xenograft tissue.The metabolism of 131I-anti-ProGRP(31-98) scFv in healthy mice was consistent with a first-order and two-compartment model; T1/2α and T1/2β were 10.2 minutes and 5 hours 18 minutes,respectively.The %ID/g of 131I-anti-ProGRP(31-98) scFv in xenografts was much higher than in healthy tissues at 12 hours after injection,reaching a maximum of (5.38±0.92) %ID/g at 24 hours.Successful imaging of xenograft tissue was achieved as early as 1 hour post-injection and persisted until 30 hours,with 24 hours proving optimal.Conclusion 131I-anti-ProGRP(31-98)scFv shows highly selective tumor uptake with low accumulation in normal tissues and rapid

  5. 99mTc-labeled interleukin 8 for the scintigraphic detection of infection and inflammation: first clinical evaluation.

    NARCIS (Netherlands)

    Bleeker-Rovers, C.P.; Rennen, H.J.J.M.; Boerman, O.C.; Wymenga, A.B.; Visser, E.P.; Bakker, J.H.; Meer, J.W.M. van der; Corstens, F.H.M.; Oyen, W.J.G.

    2007-01-01

    Interleukin 8 (IL-8) is a chemotactic cytokine that binds with a high affinity to receptors expressed on neutrophils. Previous studies with various animal models showed that (99m)Tc-labeled IL-8 accumulates specifically and rapidly in infectious and inflammatory foci. The aims of the present study w

  6. Chemotactic effect of urokinase-type plasminogen activator on mouse spermatozoa in vitro

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The aim of this study is to investigate the chemotactic effect of urokinase-type plasminogen activator (uPA)on mouse spermatozoa.Capillary assays were applied to study the chemotactic activity of ascending and descending gradients of uPA.Firstly,the chemotactic effect of an ascending gradient of uPA on mouse spermatozoa was observed by counting the number of spermatozoa that migrated into the capillary after incubation with uPA for 5,10,20,and 30 min,respectively,compared with that after incubation with F10.Twenty minutes was a suitable incubation time to obtain a plateau of sperm accumulation.Meanwhile,to confirm the specific effect of uPA on mouse sperm chemotaxis,uPA inhibitor (PAI-1)and anti-uPAR rabbit IgG were added to the test solution containing 20 U/mL uPA,respectively.To exclude the possibility that PAI-1 and anti-uPAR rabbit IgG may affect sperm accumulation nonspecifically,PAIl and anti-uPAR rabbit IgG were added to F10,respectively.It was found that the chemotactic effect of uPA was neutralized completely by PAI-1 and anti-uPAR rabbit IgG.PAI-1 and anti-uPAR rabbit IgG had no neutralizing effect on the sperm chemotactic effect.Lastly,the sperm chemotaxis response to a descending gradient of uPA was also observed.Taken together,the results suggest that uPA can induce sperm chemotaxis in vitro by binding to its receptor on the sperm membrane and may act as a chemoattractant in precontacting sperm-egg communication thereby increasing the chance encounter of spermatozoa and eggs.

  7. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Cushing, S.D.; Berliner, J.A.; Valente, A.J.; Territo, M.C.; Navab, M.; Parhami, F.; Gerrity, R.; Schwartz, C.J.; Fogelman, A.M.

    1990-07-01

    After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2- to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human glioma U-105MG cell line. Antibody that had been prepared against cultured baboon smooth muscle cell chemotactic factor (anti-SMCF) did not inhibit monocyte migration induced by the potent bacterial chemotactic factor f-Met-Leu-Phe. However, anti-SMCF completely inhibited the monocyte chemotactic activity found in the media of U-105MG cells, EC, and SMC before and after exposure to MM-LDL. Moreover, monocyte migration into the subendothelial space of a coculture of EC and SMC that had been exposed to MM-LDL was completely inhibited by anti-SMCF. Anti-SMCF specifically immunoprecipitated 10-kDa and 12.5-kDa proteins from EC. Incorporation of (35S)methionine into the immunoprecipitated proteins paralleled the monocyte chemotactic activity found in the medium of MM-LDL stimulated EC and the levels of MCP-1 mRNA found in the EC. We conclude that SMCF is in fact MCP-1 and MCP-1 is induced by MM-LDL.

  8. Peptides and proteins

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-12-01

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

  9. Neuropeptides labelled with [sup 35]S

    Energy Technology Data Exchange (ETDEWEB)

    Kaspersen, F.M.; Nispen, J.W. van; Sperling, E.M.G.; Vader, J.F. (Organon Int BV, Oss (Netherlands))

    1993-01-01

    Methionine and cysteine containing peptides can be labelled with [sup 35]S by coupling of [sup 35]S-cysteine or [sup 35]S-methionine with a large excess of suitability protected peptide precursors. This is illustrated for [pGlu[sup 4], Cyt[sup 6

  10. 13 C solid-state NMR study of the 13 C-labeled peptide, (E)8 GGLGGQGAG(A)6 GGAGQGGYGG as a model for the local structure of Nephila clavipes dragline silk (MaSp1) before and after spinning.

    Science.gov (United States)

    Yazawa, Koji; Yamaguchi, Erika; Knight, David; Asakura, Tetsuo

    2012-06-01

    We prepared the water soluble model peptide, (E)(8) GGLGGQGAG(A)(6) GGAGQGGYGG, to throw light on the local structure of spidroin 1 (MaSpl) protein in spider dragline silk of Nephila clavipes before and after spinning. Solution (13) C NMR showed that the conformation of the peptide in aqueous solution was essentially random coil. Solid-state NMR was used to follow conformation-dependent (13) C chemical shifts in (13) C selectively labeled versions of the peptide. The peptide lyophilized from an aqueous solution at neutral pH (hereafter referred to as "without acid treatment)"was used to mimic the state of the spidroin stored in the spider's silk gland while the peptide precipitated from the acidic solution ("with acid treatment") was used to simulate the role of acid treatment in inducing conformation change in the natural spinning process. In without acid treatment, the fraction of random coil conformation was lowest in the N-terminal region (residues 15-18) when compared with the C-terminus. The conformational change produced by the acid treatment occurred in the sequence, G(15) AG(A)(6) GGAG(27), interposed between pairs of Gly residues pairs, Gly(12,13), and Gly(29,30). The acid treated peptide showed a remarkable decrease in the fraction of random coil conformation from A(20) to A(23) in the poly-Ala region when compared with the peptide without acid treatment. These observations taken together suggest that the peptide can be used as a model for studying the localization of the conformation change in spider silk fibroin in the natural spinning and the role of acid treatment in this process.

  11. Nutrition Labeling

    DEFF Research Database (Denmark)

    Grunert, Klaus G

    2013-01-01

    Nutrition labeling refers to the provision of information on a food product’s nutritional content on the package label. It can serve both public health and commercial purposes. From a public health perspective, the aim of nutrition labeling is to provide information that can enable consumers...... choices, but the nutritional content of food products may not always be clear to consumers, nutrition labeling can contribute to making the nutritional content more transparent, thus reducing the frequency of unhealthy choices. Nutrition labeling is sometimes also motivated by consumers’ right to know......, implying that the availability of information on the nutritional content on food products is of value in itself, no matter how this impacts consumer choices. Another argument for nutrition labeling is that making information about nutritional content transparent will lead to healthier products, partly...

  12. An enzyme immunoassay for detection of Japanese encephalitis virus-induced chemotactic cytokine

    Indian Academy of Sciences (India)

    Aditi Singh; Rajesh Kulshreshtha; Asha Mathur

    2000-03-01

    Japanese encephalitis virus (JEV) induces human peripheral blood monocytes to secrete a chemotactic cytokine [human macrophage-derived factor (hMDF)] which causes chemotaxis of neutrophils. The only known assay for hMDF cannot quantify its level in samples, so an enzyme immunoassay has been standardized for detection of hMDF and hMDF-specific antibodies in test samples. The reported enzyme linked immunosorbent assay (ELISA) was found to be sensitive (89%), specific (91%), accurate (92·2%) and reproducible and was able to detect a minimum concentration of 23 ng hMDF/ml in test samples. The chemotactic factor could be detected in JEV inoculated mouse sera and JEV infected culture fluids. Significant finding of the test was the detection of hMDF in sera of human cases of JE.

  13. Aberrant cGMP-binding activity in non-chemotactic Dictyostelium discoideum mutants

    NARCIS (Netherlands)

    Kuwayama, Hidekazu; Viel, Gerhard T.; Ishida, Shuji; Haastert, Peter J.M. van

    1995-01-01

    The kinetics of cGMP-binding to the major cGMP-binding activity in Dictyostelium, were investigated in 10 non-chemotactic mutants (KI mutants; KI-1 similar to 10). A wild-type cell contains about 3000 binding sites with a K-d of 1.5 nM. cGMP may dissociate from these binding sites with fast (F-type)

  14. Chemotactic model for interaction of antagonistic microflora colonies and numerical simulations

    CERN Document Server

    Malaga, Carlos; Plaza, Ramon G; Simeoni, Chiara

    2011-01-01

    This paper studies a two-dimensional chemotactic model for two species in which one of them produces a chemo-repellent for the other. Under these circumstances, the chemical inhibits the invasion of a moving front for the second species. It is shown asymptotically and numerically how stable steady states, which depend on the chemical concentration, can be reached. The results qualitatively explain experimental observations by Swain and Ray, where colonies of bacteria produce metabolite agents which prevent the invasion of fungi.

  15. Rapid chemotactic response enables marine bacteria to exploit ephemeral microscale nutrient patches

    OpenAIRE

    Stocker, Roman; Seymour, Justin R.; Samadani, Azadeh; Dana E Hunt; Polz, Martin F.

    2008-01-01

    Because ocean water is typically resource-poor, bacteria may gain significant growth advantages if they can exploit the ephemeral nutrient patches originating from numerous, small sources. Although this interaction has been proposed to enhance biogeochemical transformation rates in the ocean, it remains questionable whether bacteria are able to efficiently use patches before physical mechanisms dissipate them. Here we show that the rapid chemotactic response of the marine bacterium Pseudoalte...

  16. LEGO bricks used as chemotactic chambers: evaluation by a computer-assisted image analysis technique.

    Science.gov (United States)

    Azzarà, A; Chimenti, M

    2004-01-01

    One of the main techniques used to explore neutrophil motility, employs micropore filters in chemotactic chambers. Many new models have been proposed, in order to perform multiple microassays in a rapid, inexpensive and reproducible way. In this work, LEGO bricks have been used as chemotactic chambers in the evaluation of neutrophil random motility and chemotaxis and compared with conventional Boyden chambers in a "time-response" experiment. Neutrophil motility throughout the filters was evaluated by means of an image-processing workstation, in which a dedicated algorithm recognizes and counts the cells in several fields and focal planes throughout the whole filter; correlates counts and depth values; performs a statistical analysis of data; calculates the true value of neutrophil migration; determines the distribution of cells; and displays the migration pattern. By this method, we found that the distances travelled by the cells in conventional chambers and in LEGO bricks were perfectly identical, both in random migration and under chemotactic conditions. Moreover, no interference with the physiological behaviour of neutrophils was detectable. In fact, the kinetics of migration was identical both in random migration (characterized by a gaussian pattern) and in chemotaxis (characterized by a typical stimulation peak, previously identified by our workstation). In conclusion, LEGO bricks are extremely precise devices. They are simple to use and allow the use of small amounts of chemoattractant solution and cell suspension, supplying by itself a triplicate test. LEGO bricks are inexpensive, fast and suitable for current diagnostic activity or for research investigations in every laboratory.

  17. Polymorphonuclear neutrophil granulocyte chemotactic hyperresponsiveness in a case of canine acromegaly.

    Science.gov (United States)

    Jensen, A L; Thomsen, M K; Aaes, H; Andreasen, M; Søndergaard, J

    1993-08-01

    Growth hormone (GH) has recently been shown to affect polymorphonuclear neutrophil granulocyte (PMN) function and to be secreted by mononuclear cells, indicating that the hormone may be active in an immunophysiologic network, acting as an endo- or paracrine priming agent. The purpose of the present study was to evaluate the chemotactic responsiveness of canine peripheral PMN in a dog with acromegaly, caused by spontaneous, progesterone-induced hypersecretion of GH and, secondary to this, a seven-fold increase in insulin-like growth factor I (IGF-I). The chemotactic responsiveness towards zymosan-activated serum (ZAS) and leukotriene B4 (LTB4) was evaluated at a time when the dog suffered from acromegaly and again 57 days after corrective surgery (ovariohysterectomy). The experiments showed that PMN from the patient exhibited enhanced chemotactic migration that appeared to be associated with the hypersomatotropic condition as judged from the reversibility of the phenomenon. The glucose intolerance and elevated serum alkaline phosphatase that were observed in the acromegalic dog were also shown to be reversible following surgery.

  18. Labeling of vasoactive intestinal peptide (VIP) and VIP 10-28 fragment with radioiodine by direct method. Comparative study of the kinetics biodistribution and affinity for neuroendocrine tumor cells; Marcacao do peptideo intestinal vasoativo (VIP) e do fragmento VIP10-28 com radioiodo por metodo direto. Estudo comparativo da cinetica de biodistribuicao e da afinidade por celulas de tumor neuroendocrino

    Energy Technology Data Exchange (ETDEWEB)

    Colturato, Maria Tereza

    2005-07-01

    In the progress of the Nuclear Medicine, many protein based radiopharmaceuticals have been developed in the last years using antibodies and, more recently, biologically active natural peptides or similar synthetic peptides. In the search for agents with specificity for the target tissue in tumors detection, it was verified that small sequences of amino acids may interact with selective sites, with homogenous distribution, fast accumulation in tissues and fast blood clearance when compared to the antibodies. Among the peptides used in the diagnosis of tumors, Vasoactive Intestinal Peptide (VIP) has been studied. VIP labeled with iodine-123 is applied in the images of intestinal adenocarcinoma and endocrine tumors. The molecule of VIP contains two tyrosine residues, in the positions 10 and 22 that are, theoretically, equally susceptible to radioiodination for direct method. The objective of this work was to produce VIP labeled with radioiodine (iodine-123), in order to introduce to the brazilian medical class this radiopharmaceutical of interest for the diagnosis and recurrence of tumors that express specific receptors. In an unpublished way, the work studied the labeling and the kinetic distribution of the VIP fragment (VIP 10-28) and verified its potential as radiopharmaceutical applied in the identification of tumors that express VIP receptors. After the choice of the appropriated technique for labeling VIP and VIP 10-28 with radioiodine, using Ceremonial T as oxidant agent and sodium metabisulfite as reducing agent, the quality control procedures were accomplished (electrophoresis and high performance liquid chromatography, HPLC) for radiochemical purity determination as well as the separation of the radiochemical species obtained. Labeling and quality control procedures applied were efficient and accurate. [{sup 131}I]VIP and [{sup 131}l]VIP 10-28 were obtained with high radiochemical purity (> 95%). The purification studies to remove free radioiodine in the

  19. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

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

    2013-01-01

    properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......Although peptide-oligonucleotide conjugates (POCs) are well-known for nucleic acids delivery and therapy, reports on internal attachment of peptides to oligonucleotides are limited in number. To develop a convenient route for preparation of internally labeled POCs with improved biomedical......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...

  20. Monocyte chemotactic protein 1 increases homing of mesenchymal stem cell to injured myocardium and neovascularization following myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:To investigate the effect of MCP-1 on mesenchymal stem cells(MSCs) homing to injured myocardium in a rat myocardial infarction(MI) model. Methods:Rat myocardial infarction model was established by permanent left anterior descending branch ligation. Mesenchymal stem cells from donor rats were cultured in IMDM and labeled with BrdU. The Rats were divided into two groups. Monocyte chemotactic protein 1(MCP-1) expression were measured by in situ hybridization and immunohistochemistry in the sham operated or infarcted hearts at 1,2, 4,7,14 and 28 days post operation in MCP-1 detection group. The rats were injected with MCP-1, anti-MCP-1 antibody or saline 4 days after myocardial infarction in intervention group. Then, a total of 5 × 106 cells in 2.5 ml of PBS were injected through the tail vein. The number of the labeled MSCs in the infarcted hearts was counted 3 days post injection. Cardiac function and blood vessel density were assessed 28 days post injection. Results:Self-generating MCP-1 expression was increased at the first day, peaked at the 7th day and decreased thereafter post MI and remained unchanged in sham operated hearts. The MSCs enrichment in the host hearts were more abundant in the MI groups than that in the non-Mi group(P = 0.000), the MSCs enrichment in the host hearts were more abundant in the MCP-1 injected group than that in the anti-MCP-1 antibody and saline injected groups (P = 0.000). Cardiac function was improved more in MCP-1 injected group than anti-MCP-1 antibody and saline injected groups(P= 0.000). Neovascularization in MCP-1 injected group significantly increased compared with that of other groups(P = 0.000). Conclusion: Myocardial MCP-1 expression was increased only in the early phase post MI. MCP-1 may enhance MSCs homing to the injured heart and improve cardiac function by promoting neovascularization.

  1. Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

    Science.gov (United States)

    Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

    2008-06-01

    Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

  2. Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands.

    Science.gov (United States)

    Lacal, Jesús; Alfonso, Carlos; Liu, Xianxian; Parales, Rebecca E; Morel, Bertrand; Conejero-Lara, Francisco; Rivas, Germán; Duque, Estrella; Ramos, Juan L; Krell, Tino

    2010-07-23

    We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (T(m)) and unfolding enthalpy (DeltaH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.

  3. Synthesis and evaluation of radiolabeled peptide multimers for tumor targeting

    NARCIS (Netherlands)

    Yim, C.B.

    2011-01-01

    Many cancer types express specific receptors on their cellular surface onto which regulatory peptides bind with high affinity. This mechanism can be exploited by labeling the peptide with a radionuclide and using the radiolabeled peptide as a vehicle to guide the radioactivity to receptor-rich cells

  4. Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

    Science.gov (United States)

    Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

    2016-03-10

    Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

  5. Improved methods for classification, prediction, and design of antimicrobial peptides.

    Science.gov (United States)

    Wang, Guangshun

    2015-01-01

    Peptides with diverse amino acid sequences, structures, and functions are essential players in biological systems. The construction of well-annotated databases not only facilitates effective information management, search, and mining but also lays the foundation for developing and testing new peptide algorithms and machines. The antimicrobial peptide database (APD) is an original construction in terms of both database design and peptide entries. The host defense antimicrobial peptides (AMPs) registered in the APD cover the five kingdoms (bacteria, protists, fungi, plants, and animals) or three domains of life (bacteria, archaea, and eukaryota). This comprehensive database ( http://aps.unmc.edu/AP ) provides useful information on peptide discovery timeline, nomenclature, classification, glossary, calculation tools, and statistics. The APD enables effective search, prediction, and design of peptides with antibacterial, antiviral, antifungal, antiparasitic, insecticidal, spermicidal, anticancer activities, chemotactic, immune modulation, or antioxidative properties. A universal classification scheme is proposed herein to unify innate immunity peptides from a variety of biological sources. As an improvement, the upgraded APD makes predictions based on the database-defined parameter space and provides a list of the sequences most similar to natural AMPs. In addition, the powerful pipeline design of the database search engine laid a solid basis for designing novel antimicrobials to combat resistant superbugs, viruses, fungi, or parasites. This comprehensive AMP database is a useful tool for both research and education.

  6. Effects of Garlic Oil on the Migration of Neutrophil-Like Cell Studied by Using a Chemotactic Gradient Labchip

    Directory of Open Access Journals (Sweden)

    Po-Chen Shih

    2010-01-01

    Full Text Available We have designed and fabricated a novel chemotactic gradient Labchip for studying cell migration quantitatively. Owing to the great potential of garlic and its preparations in developing antiinflammatory drugs, the aim of the present study is to investigate the effect of garlic oil on the locomotion of a neutrophil-like cell by measuring the dynamic features of cell migration including migration direction, average migration speed, chemotactic index (CI, and motility index (MI with the newly designed Labchip. We found that garlic oil treatment lowered the values of CI and MI and reduced the average speed of cell migration from 13 to 8 μm/min. The results indicate that garlic oil is a potential inhibitor for neutrophil-like cell migration and chemotactic responsiveness. By comparing with the effects of nocodazole and cytochalasin B, we also suggest that the antiinflammatory activity exhibited by garlic oil was mainly through inhibiting the assembly-disassembly processes of the cytoskeleton.

  7. Chemotactic behavior of deep subsurface bacteria toward carbohydrates, amino acids and a chlorinated alkene

    Energy Technology Data Exchange (ETDEWEB)

    Lopez de Victoria, G. (Puerto Rico Univ., Rio Piedras (Puerto Rico). Dept. of Biology)

    1989-02-01

    The chemotactic behavior of deep terrestrial subsurface bacteria toward amino acids, carbohydrates and trichloroethylene was assayed using a modification of the capillary method and bacterial enumeration by acridine orange direct counts. Eleven isolates of bacteria isolated from six different geological formations were investigated. A bimodal response rather than an absolute positive or negative response was observed in most assays. Most of the isolates were positively chemotactic to low concentrations of substrates and were repelled by high concentrations of the same substrate. However, this was not the case for trichloroethylene (TCE) which was mostly an attractant and elicited the highest responses in all the isolates when compared with amino acids and carbohydrates. The movement rates of these isolates in aseptic subsurface sediments in the absence and presence of TCE were also determined using a laboratory model. All of the isolates showed distinct response range, peak, and threshold concentrations when exposed to the same substrates suggesting that they are possibly different species as has been inferred from DNA homology studies. 101 refs., 4 figs., 57 tabs.

  8. Effect of Monocyte Chemotactic Protein-1 on the Intraperitoneal Adhesion Formation

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In order to study the role of monocyte chemotactic protein-1 (MCP-1) in the intra-peritoneal adhesion formation, 23 infertile patients undergoing laparoscopic operation were divided into two groups: experimental group including 12 patients with intra-peritoneal adhesion and control group including 11 patients without intra-peritoneal adhesion. Peritoneal fluid (PF) and peritoneum were collected from these patients during laparoscopic examination. The expression levels of MCP-l protein and MCP-1 mRNA were detected by using enzyme-linked immunosorbent assay (ELISA) and dot blot analysis method respectively. It was found that the levels of MCP-1 protein in PF of the patients with peritoneal adhesion were significantly higher than in the control group (0. 44±0.11 ng/ml vs 0. 19+0. 09 ng/ml respectively, P<0. 01 ). The level of MCP-1 mRNA in the peritoneum of the patients with peritoneal adhesion was significantly higher than in the control group (48.61±3.72 vs 19. 87±2.54 respectively, P<0. 01). It was suggested that MCP-1 might play a role in the adhesion formation, and chemotactic cytokines expressing in the peritoneal mesothelial cells might be take part in the process.

  9. Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration.

    Science.gov (United States)

    Howe, Alan K; Baldor, Linda C; Hogan, Brian P

    2005-10-04

    Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures formed during chemotaxis. This enrichment correlates with increased phosphorylation of key cytoskeletal substrates for PKA, including the vasodilator-stimulated phosphoprotein (VASP) and the protein tyrosine phosphatase containing a PEST motif. Importantly, inhibition of PKA activity or its ability to interact with A kinase anchoring proteins inhibited the activity of the Rac GTPase within pseudopodia. This effect correlated with both decreased guanine nucleotide exchange factor activity and increased GTPase activating protein activity. Finally, inhibition of PKA anchoring, like inhibition of total PKA activity, inhibited pseudopod formation and chemotactic cell migration. These data demonstrate that spatial regulation of PKA via anchoring is an important facet of normal chemotactic cell movement.

  10. Association of calprotectin with leukocyte chemotactic and inflammatory mediators following acute aerobic exercise.

    Science.gov (United States)

    Maharaj, Arun; Slusher, Aaron L; Zourdos, Michael C; Whitehurst, Michael; Fico, Brandon G; Huang, Chun-Jung

    2016-01-01

    The objective of this study was to examine whether acute aerobic exercise-mediated calprotectin in plasma would be associated with monocyte chemotactic protein-1 (MCP-1), myeloperoxidase (MPO), and interleukin-6 (IL-6) in healthy individuals. Eleven healthy participants, aged 18 to 30 years, were recruited to perform a 30-min bout of aerobic exercise at 75% maximal oxygen uptake. Acute aerobic exercise elicited a significant elevation across time in plasma calprotectin, MCP-1, MPO, and IL-6. Body mass index (BMI) was positively correlated with calprotectin area-under-the-curve with "respect to increase" (AUCi) and IL-6 AUCi. Furthermore, calprotectin AUCi was positively correlated with IL-6 AUCi and MPO AUCi, even after controlling for BMI. Although MPO AUCi was positively correlated with IL-6 AUCi, this relationship no longer existed after controlling for BMI. These results suggest that acute aerobic exercise could mediate innate immune response associated with calprotectin and its related leukocyte chemotactic and inflammatory mediators, especially in individuals with elevated BMI.

  11. Computational modeling of chemotactic signaling and aggregation of microglia around implantation site during deep brain stimulation

    Science.gov (United States)

    Silchenko, A. N.; Tass, P. A.

    2013-10-01

    It is well established that prolonged electrical stimulation of brain tissue causes massive release of ATP in the extracellular space. The released ATP and the products of its hydrolysis, such as ADP and adenosine, become the main elements mediating chemotactic sensitivity and motility of microglial cells via subsequent activation of P2Y2,12 as well as A3A and A2A adenosine receptors. The size of the sheath around the electrode formed by the microglial cells is an important criterion for the optimization of the parameters of electrical current delivered to brain tissue. Here, we study a purinergic signaling pathway underlying the chemotactic motion of microglia towards the implanted electrode during deep brain stimulation. We present a computational model describing formation of a stable aggregate around the implantation site due to the joint chemo-attractive action of ATP and ADP together with a mixed influence of extracellular adenosine. The model was built in accordance with the classical Keller-Segel approach and includes an equation for the cells' density as well as equations describing the hydrolysis of extracellular ATP via successive reaction steps ATP →ADP →AMP →adenosine. The results of our modeling allowed us to reveal the dependence of the width of the encapsulating layer around the electrode on the amount of ATP released due to permanent electrical stimulation. The dependences of the aggregates' size on the parameter governing the nonlinearity of interaction between extracellular adenosine and adenosine receptors are also analyzed.

  12. Distinct CCR7 glycosylation pattern shapes receptor signaling and endocytosis to modulate chemotactic responses.

    Science.gov (United States)

    Hauser, Mark A; Kindinger, Ilona; Laufer, Julia M; Späte, Anne-Katrin; Bucher, Delia; Vanes, Sarah L; Krueger, Wolfgang A; Wittmann, Valentin; Legler, Daniel F

    2016-06-01

    The homeostatic chemokines CCL19 and CCL21 and their common cognate chemokine receptor CCR7 orchestrate immune cell trafficking by eliciting distinct signaling pathways. Here, we demonstrate that human CCR7 is N-glycosylated on 2 specific residues in the N terminus and the third extracellular loop. Conceptually, CCR7 glycosylation adds steric hindrance to the receptor N terminus and extracellular loop 3, acting as a "swinging door" to regulate receptor sensitivity and cell migration. We found that freshly isolated human B cells, as well as expanded T cells, but not naïve T cells, express highly sialylated CCR7. Moreover, we identified that human dendritic cells imprint T cell migration toward CCR7 ligands by secreting enzymes that deglycosylate CCR7, thereby boosting CCR7 signaling on T cells, permitting enhanced T cell locomotion, while simultaneously decreasing receptor endocytosis. In addition, dendritic cells proteolytically convert immobilized CCL21 to a soluble form that is more potent in triggering chemotactic movement and does not desensitize the receptor. Furthermore, we demonstrate that soluble CCL21 functionally resembles neither the CCL19 nor the CCL21 phenotype but acts as a chemokine with unique features. Thus, we advance the concept of dendritic cell-dependent generation of micromilieus and lymph node conditioning by demonstrating a novel layer of CCR7 regulation through CCR7 sialylation. In summary, we demonstrate that leukocyte subsets express distinct patterns of CCR7 sialylation that contribute to receptor signaling and fine-tuning chemotactic responses.

  13. 4-[18F]Fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([18F]F-SA): a versatile building block for labeling of peptides, proteins and oligonucleotides with fluorine-18 via Cu(I)-mediated click chemistry.

    Science.gov (United States)

    Ramenda, Theres; Steinbach, Jörg; Wuest, Frank

    2013-04-01

    Cu(I)-mediated [3+2]cycloaddition between azides and alkynes has evolved into a valuable bioconjugation tool in radiopharmaceutical chemistry. We have developed a simple, convenient and reliable radiosynthesis of 4-[18F]fluoro-N-methyl-N-(propyl-2-yn-1-yl)benzenesulfonamide ([18F]F-SA) as a novel aromatic sulfonamide-based click chemistry building block. [18F]F-SA could be prepared in a remotely controlled synthesis unit in 32 ± 5% decay-corrected radiochemical yield in a total synthesis time of 80 min. The determined lipophilicity of [18F]F-SA (logP = 1.7) allows handling of the radiotracer in aqueous solutions. The versatility of [18F]F-SA as click chemistry building block was demonstrated by the labeling of a model peptide (phosphopeptide), protein (HSA), and oligonucleotide (L-RNA). The obtained radiochemical yields were 77% (phosphopeptide), 55-60% (HSA), and 25% (L-RNA), respectively. Despite the recent emergence of a multitude of highly innovative novel bioconjugation methods for 18F labeling of biopolymers, Cu(I)-mediated click chemistry with [18F]F-SA represents a reliable, robust and efficient radiolabeling technique for peptides, proteins, and oligonucleotides with the short-lived positron emitter 18F.

  14. A model for cell type localization in the migrating slug of Dictyostelium discoideum based on differential chemotactic sensitivity to cAMP and differential sensitivity to suppression of chemotaxis by ammonia

    Indian Academy of Sciences (India)

    Ira N Feit; Jeffrey Pawlikowski; Caroline Zawilski

    2007-03-01

    The three basic cell types in the migrating slug of Dictyostelium discoideum show differential chemotactic response to cyclic AMP (cAMP) and differential sensitivity to suppression of the chemotaxis by ammonia. The values of these parameters indicate a progressive maturation of chemotactic properties during the transdifferentiation of slug cell types. We present a model that explains the localization of the three cell types within the slug based on these chemotactic differences and on the maturation of their chemotactic properties.

  15. Monocyte Chemotactic Protein-1 Promotes the Myocardial Homing of Mesenchymal Stem Cells in Dilated Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Yunzeng Zou

    2013-04-01

    Full Text Available Dilated cardiomyopathy (DCM is the most common form of non-ischemic cardiomyopathy that leads to heart failure. Mesenchymal stem cells (MSCs are under active investigation currently as a potential therapy for DCM. However, little information is available about the therapeutic potential of intravenous administration of MSCs for DCM. Moreover, how MSCs home to the myocardium in DCM is also unclear. DCM was induced by intraperitoneally administering Doxorubicin and MSCs or vehicles were infused through the internal jugular vein. Cardiac functions including the percentage of fractional shortening, left ventricular diastolic dimension, left ventricular end-diastolic pressure, and left ventricular maximum dp/dt were evaluated by echocardiographic and hemodynamic studies. Fibrosis was determined by Masson’s trichrome staining. The mRNA expression levels of monocyte chemotactic protein-1 (MCP-1, stromal cell-derived factor-1 (SDF-1, macrophage inflammatory protein-1α (MIP-1α, and monocyte chemotactic protein-3 (MCP-3 were determined using real time polymerase chain reactions and the protein expression level of MCP-1 was detected with Western blot. The MSCs expression of C-C chemokine receptor type 2 (CCR2, a MCP-1 receptor, was confirmed by Western blot and flow cytometry analysis. The chemotactic effects of MCP-1/CCR2 were checked by assessing the migration in vitro and in vivo. MSCs transplantation improved the cardiac function and decreased the myocardial fibrosis of mice with DCM. MCP-1 was up-regulated in dilated myocardial tissue both at the mRNA and protein level while SDF-1, MIP-1α and MCP-3 remain unchanged. CCR2 was present in MSCs. MCP-1 promoted MSCs migration in vitro while CCR2 inhibition decreased the migration of MCP-1 to the dilated heart. This study provides direct evidences that peripheral intravenous infusion of MSCs can support the functional recovery of DCM. In addition, novel insights into the myocardial homing factor of MSCs

  16. Monocyte chemotactic protein-1 deficiency reduces spontaneous metastasis of Lewis lung carcinoma in mice fed a high-fat diet

    Science.gov (United States)

    Obesity is a risk factor for cancer. Adipose tissue produces pro-inflammatory adipokines that contribute obesity-related malignant progression. This study investigated the effects of monocyte chemotactic protein-1 (MCP-1) deficiency on pulmonary metastasis of Lewis lung carcinoma (LLC) in male C57...

  17. Introduction to Pesticide Labels

    Science.gov (United States)

    Pesticide product labels provide critical information about how to safely and legally handle and use pesticide products. Unlike most other types of product labels, pesticide labels are legally enforceable. Learn about pesticide product labels.

  18. How many consumer levels can survive in a chemotactic food chain?

    Institute of Scientific and Technical Information of China (English)

    Jing LIU; Chunhua OU

    2009-01-01

    We investigate the effect and the impact of predator-prey interactions, diffusivity and chemotaxis on the ability of survival of multiple consumer levels in a predator-prey microbial food chain. We aim at answering the question of how many consumer levels can survive from a dynamical system point of view. To solve this standing issue on food-chain length, first we construct a chemotactic food chain model. A priori bounds of the steady state populations are obtained. Then under certain sufficient conditions combining the effect of conversion efficiency, diffusivity and chemotaxis parameters, we derive the co-survival of all consumer levels, thus obtaining the food chain length of our model. Numerical simulations not only confirm our theoretical results, but also demonstrate the impact of conversion efficiency, diffusivity and chemotaxis behavior on the survival and stability of various consumer levels.

  19. PTEN functions to 'prioritize' chemotactic cues and prevent 'distraction' in migrating neutrophils.

    Science.gov (United States)

    Heit, Bryan; Robbins, Stephen M; Downey, Charlene M; Guan, Zhiwen; Colarusso, Pina; Miller, B Joan; Jirik, Frank R; Kubes, Paul

    2008-07-01

    Neutrophils encounter and 'prioritize' many chemoattractants in their pursuit of bacteria. Here we tested the possibility that the phosphatase PTEN is responsible for the prioritization of chemoattractants. Neutrophils induced chemotaxis by two separate pathways, the phosphatidylinositol-3-OH kinase (PI(3)K) phosphatase and tensin homolog (PTEN) pathway, and the p38 mitogen-activated protein kinase pathway, with the p38 pathway dominating over the PI(3)K pathway. Pten(-/-) neutrophils could not prioritize chemoattractants and were 'distracted' by chemokines when moving toward bacterial chemoattractants. In opposing gradients, PTEN became distributed throughout the cell circumference, which inhibited all PI(3)K activity, thus permitting 'preferential' migration toward bacterial products via phospholipase A(2) and p38. Such prioritization was defective in Pten(-/-) neutrophils, which resulted in defective bacterial clearance in vivo. Our data identify a PTEN-dependent mechanism in neutrophils to prioritize, 'triage' and integrate responses to multiple chemotactic cues.

  20. Study of the Chemotactic Response of Multicellular Spheroids in a Microfluidic Device

    Science.gov (United States)

    Ayuso, Jose M.; Basheer, Haneen A.; Monge, Rosa; Sánchez-Álvarez, Pablo; Doblaré, Manuel; Shnyder, Steven D.; Vinader, Victoria; Afarinkia, Kamyar

    2015-01-01

    We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it. PMID:26444904

  1. Imaging and biodistribution of ~(99)Tc~m labeled peptide high-binding VEGF receptor 3 in nude mice with ovarian cancer%~(99)Tc~m标记VEGFR-3高亲和融合多肽在荷人卵巢癌裸鼠体内的分布和显像研究

    Institute of Scientific and Technical Information of China (English)

    王玲; 梁志清; 侍立峰; 杨明福; 刘广元

    2009-01-01

    目的 研究~(99)Tc~m标记VEGFR-3高亲和融合肽(phage-SHSWHWLPNLRHYAS)在荷人卵巢癌裸鼠体内分布和放射免疫定位显像.方法 用NHS-MAG3为双功能鳌合剂,固相法合成多肽.~(99)Tc~m预亚锡直接标记法进行标记,纸层析法测定标记率.经尾静脉注射于荷瘤鼠体内,取不同时相进行SPECT显像,测定标记物在体内的分布情况并进行分析.结果 融合多肽的~(99)Tc~m标记率为95.27%,放射化学纯度为96%,放射性浓度24.6 MBq/ml.经鼠尾静脉注射后1 h,植瘤部位开始出现放射性浓集,肾脏、肝脏及膀胱组织也可见显像;注射后3 h肿瘤显像最清晰,每克组织注射百分剂量率(%ID/g)为(30.20±6.89).其余大部分脏器的T/NT值均达最高,最高为肌肉(13.13);注射后4 h肿瘤部位显像逐渐消退.对照组裸鼠的肿瘤部位始终未见显像.结论 筛选获得的VEGFR-3高亲和融合肽能够靶向荷瘤鼠体内肿瘤组织,实现肿瘤的靶向受体显像.%Objective To investigate the imaging and biodistribution of ~(99)Tc~m labeled peptide (phage-SHSWHWLPNLRHYAS) high-binding VEGF receptor 3 in nude mice with ovarian cancer. Methods We used NHS-MAG3 as the bifunctional chelating agent and synthesized the peptide above. It was completed with pre-stannous direct labeling by ~(99)Tc~m and then the labeling efficiency was determined by paper chromatography. The tumor bearing mice were injected via tail veins with radiolabeled peptide. The tumors were imaged with single photon emission computed tomography (SPECT). We measured and calculated the biodistribution of the radiolabeled peptide. Results The labeled rate was 95. 27% and the radiochemical purity was 96%. Under a SPECT apparatus, we observed tumor location image at 1 h post injection and the tumor images was the clearest at 3 h post injection, with the injected dose per gram of tissue (%ID/g) of (30. 20±6. 89) at the tumor sites. The tumor/muscle ratio was 13.13. Conclusion Our peptide can

  2. The role of eicosanoids in the chemotactic response to Pasteurella haemolytica infection.

    Science.gov (United States)

    Clarke, C R; Lauer, A K; Barron, S J; Wyckoff, J H

    1994-10-01

    The chemotactic role of eicosanoids in the pathogenesis of Pasteurella haemolytica infection was studied, using a tissue chamber infection model and pharmacological inhibitors of eicosanoid synthesis. Tissue chambers were implanted subcutaneously in 12 calves allotted to three treatment groups of equal size. At 45 days after implantation, calves received saline, dexamethasone, or phenylbutazone treatments, and tissue chambers in all animals were then inoculated with P. haemolytica. Chamber fluid samples were collected before inoculation and at 2, 6, 18, 40, and 90 h after inoculation. Bacterial counts, total leukocyte counts, pH and albumin concentrations in chamber fluids were determined using standard bacteriological and clinical pathological methods. Concentrations of eicosanoids and activity of interleukin-1 (IL-1) were measured by radioimmunoassay and a helper T cell bioassay, respectively. Concentrations of leukotriene B4 (LTB4), thromboxane B2 (TXB2), 6-keto-prostaglandin F1 alpha (PGF1 alpha) and prostaglandin E2 (PGE2) increased markedly after inoculation. An inhibitory effect of dexamethasone on both LTB4 production and neutrophil influx, together with the temporal relationship between these two events, suggested that LTB4 served as a chemo-attractant. Activity-time profiles for IL-1 in chamber fluids were similar to those of the eicosanoids. Phenylbutazone and dexamethasone reduced the severity of the inflammatory responses as measured by lower concentrations of albumin and higher pH in treated versus control chamber fluids. The results of this study suggest that eicosanoid inflammatory mediators play an important chemotactic role in the pathogenesis of P. haemolytica infection.

  3. Stochastic coordination of multiple actuators reduces latency and improves chemotactic response in bacteria.

    Science.gov (United States)

    Sneddon, Michael W; Pontius, William; Emonet, Thierry

    2012-01-17

    Individual neuronal, signal transduction, and regulatory pathways often control multiple stochastic downstream actuators, which raises the question of how coordinated response to a single input can be achieved when individual actuators fluctuate independently. In Escherichia coli, the bacterial chemotaxis pathway controls the activity of multiple flagellar motors to generate the run-and-tumble motion of the cell. High-resolution microscopy experiments have identified the key conformational changes adopted by individual flagella during this process. By incorporating these observations into a stochastic model of the flagellar bundle, we demonstrate that the presence of multiple motors imposes a trade-off on chemotactic performance. Multiple motors reduce the latency of the response below the time scale of the stochastic switching of a single motor, which improves performance on steep gradients of attractants. However, the uncoordinated switching of multiple motors interrupts and shortens cell runs, which thereby reduces signal detection and performance on shallow gradients. Remarkably, when slow fluctuations generated by the adaptation mechanism of the chemotaxis system are incorporated in the model at levels measured in experiments, the chemotactic sensitivity and performance in shallow gradients is partially restored with marginal effects for steep gradients. The noise is beneficial because it simultaneously generates long events in the statistics of individual motors and coordinates the motors to generate a long tail in the run length distribution of the cell. Occasional long runs are known to enhance exploration of random walkers. Here we show that they have the additional benefit of enhancing the sensitivity of the bacterium to very shallow gradients.

  4. CKbeta-8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues.

    Science.gov (United States)

    Votta, B J; White, J R; Dodds, R A; James, I E; Connor, J R; Lee-Rykaczewski, E; Eichman, C F; Kumar, S; Lark, M W; Gowen, M

    2000-05-01

    We have previously demonstrated that a tartrate-resistant acid phosphatase (TRAP)-positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKbeta-8, RANTES, and MIP-1alpha elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP-1beta, MCP-1, MCP-2, MCP-3, MCP-4, HCC-1, eotaxin-2, PARC, SLC, ELC) and 3 CXC chemokines (IL-8, GROalpha, SDF-1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG-63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross-desensitization studies demonstrate that RANTES and MIP-1alpha can partially inhibit the chemotactic response elicited by CKbeta-8. CKbeta-8, the most potent of the active CC chemokines (EC(max) 0.1-0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKbeta-8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKbeta-8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKbeta-8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption.

  5. On Online Labeling with Polynomially Many Labels

    DEFF Research Database (Denmark)

    Babka, Martin; Bulánek, Jan; Cunat, Vladimír

    2012-01-01

    In the online labeling problem with parameters n and m we are presented with a sequence of nkeys from a totally ordered universe U and must assign each arriving key a label from the label set {1,2,…,m} so that the order of labels (strictly) respects the ordering on U. As new keys arrive it may be...

  6. HIV-1 envelope gp41 peptides promote migration of human Fc epsilon RI+ cells and inhibit IL-13 synthesis through interaction with formyl peptide receptors.

    Science.gov (United States)

    de Paulis, Amato; Florio, Giovanni; Prevete, Nella; Triggiani, Massimo; Fiorentino, Isabella; Genovese, Arturo; Marone, Gianni

    2002-10-15

    We evaluated the effects of synthetic peptides (2017, 2019, 2020, 2021, 2023, 2027, 2029, 2030, 2031, and 2035) encompassing the structure of HIV-1(MN) envelope gp41 on both chemotaxis of human basophils and the release of preformed mediators (histamine) and of cytokines (IL-13). Peptides 2019 and 2021 were potent basophil chemoattractants, whereas the other peptides examined were ineffective. Preincubation of basophils with FMLP or gp41 2019 resulted in complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with low concentration (5 x 10(-7) M) of FMLP, which binds with high affinity to N-formyl peptide receptor (FPR), but not to FPR-like 1, did not affect the chemotactic response to a heterologous stimulus (gp41 2019). In contrast, a high concentration (10(-4) M) of FMLP, which binds also to FPR-like 1, significantly reduced the chemotactic response to gp41 2019. The FPR antagonist cyclosporin H inhibited chemotaxis induced by FMLP, but not by gp41 2019. None of these peptides singly induced the release of histamine or cytokines (IL-4 and IL-13) from basophils. However, low concentrations of peptides 2019 and 2021 (10(-8)-10(-6) M) inhibited histamine release from basophils challenged with FMLP but not the secretion caused by anti-IgE and gp120. Preincubation of basophils with peptides 2019 and 2021 inhibited the expression of both IL-13 mRNA, and the FMLP-induced release of IL-13 from basophils. These data highlight the complexity of the interactions between viral and bacterial peptides with FPR subtypes on human basophils.

  7. Peptide identification

    Science.gov (United States)

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  8. Förster Resonance Energy Transfer Mediated Photoluminescence Quenching in Stoichiometrically Assembled CdSe/ZnS Quantum Dot-Peptide Labeled Black Hole Quencher Conjugates for Matrix Metalloproteinase-2 Sensing.

    Science.gov (United States)

    Pillai, Sreenadh Sasidharan; Yukawa, Hiroshi; Onoshima, Daisuke; Biju, Vasudevanpillai; Baba, Yoshinobu

    2017-01-01

    The steady state and time-resolved photoluminescence quenching of streptavidin modified CdSe/ZnS quantum dots (QDs) instigated by biotin-peptide-BHQ-1 (biotin-pep-BHQ-1) molecule was investigated. Here, we have achieved an efficient photoluminescence (PL) quenching of QDs with the conjugation of dark quencher (black hole quencher-BHQ) molecules intermediated with the GPLGVRGK peptide. The luminescence of streptavidin-QDs585 was decreased upon titration with a nano molar concentration of the biotin-GPLGVRGK-BHQ-1 molecule. It has been suggested that the decrease of QDs PL occurred through a Förster resonance energy transfer (FRET) mechanism from the analysis of steady state photoluminescence intensity measurements as well as time resolved lifetime measurements of streptavidin-QDs and QDs-(pep-BHQ-1)n conjugates. The sequence of intermediate peptide GPLG↓VRGK can act as a target material for matrix metalloproteinases-2 (MMP-2) produced by cancer cells at its Gly and Val region, shown by the down-headed arrow. Interestingly, here the reported self-assembled QDs-(pep-BHQ-1)n conjugates could detect the presence MMP-2 at a detection limit of 1 ng/mL with a clear luminescence recovery.

  9. Anionic phospholipids modulate peptide insertion into membranes.

    Science.gov (United States)

    Liu, L P; Deber, C M

    1997-05-06

    While the insertion of a hydrophobic peptide or membrane protein segment into the bilayer can be spontaneous and driven mainly by the hydrophobic effect, anionic lipids, which comprise ca. 20% of biological membranes, provide a source of electrostatic attractions for binding of proteins/peptides into membranes. To unravel the interplay of hydrophobicity and electrostatics in the binding of peptides into membranes, we designed peptides de novo which possess the typical sequence Lys-Lys-Ala-Ala-Ala-X-Ala-Ala-Ala-Ala-Ala-X-Ala-Ala-Trp-Ala-Ala-X-Ala-Al a-Ala-Lys-Lys-Lys-Lys-amide, where X residues correspond to "guest" residues which encompass a range of hydrophobicity (Leu, Ile, Gly, and Ser). Circular dichroism spectra demonstrated that peptides were partially (40-90%) random in aqueous buffer but were promoted to form 100% alpha-helical structures by anionic lipid micelles. In neutral lipid micelles, only the relatively hydrophobic peptides (X = L and I) spontaneously adopted the alpha-helical conformation, but when 25% of negatively charged lipids were mixed in to mimic the content of anionic lipids in biomembranes, the less hydrophobic (X = S and G) peptides then formed alpha-helical conformations. Consistent with these findings, fluorescence quenching by the aqueous-phase quencher iodide indicated that in anionic (dimyristoylphosphatidylglycerol) vesicles, the peptide Trp residue was buried in the lipid vesicle hydrophobic core, while in neutral (dimyristoylphosphatidylcholine) vesicles, only hydrophobic (X = L and I) peptides were shielded from the aqueous solution. Trp emission spectra of peptides in the presence of phospholipids doxyl-labeled at the 5-, 7-, 10-, 12-, and 16-fatty acid positions implied not only a transbilayer orientation for inserted peptides but also that mixed peptide populations (transbilayer + surface-associated) may arise. Overall results suggest that for hydrophobic peptides with segmental threshold hydrophobicity below that which

  10. Synthesizing and modifying peptides for chemoselective ligation and assembly into quantum dot-peptide bioconjugates.

    Science.gov (United States)

    Algar, W Russ; Blanco-Canosa, Juan B; Manthe, Rachel L; Susumu, Kimihiro; Stewart, Michael H; Dawson, Philip E; Medintz, Igor L

    2013-01-01

    Quantum dots (QDs) are well-established as photoluminescent nanoparticle probes for in vitro or in vivo imaging, sensing, and even drug delivery. A critical component of this research is the need to reliably conjugate peptides, proteins, oligonucleotides, and other biomolecules to QDs in a controlled manner. In this chapter, we describe the conjugation of peptides to CdSe/ZnS QDs using a combination of polyhistidine self-assembly and hydrazone ligation. The former is a high-affinity interaction with the inorganic surface of the QD; the latter is a highly efficient and chemoselective reaction that occurs between 4-formylbenzoyl (4FB) and 2-hydrazinonicotinoyl (HYNIC) moieties. Two methods are presented for modifying peptides with these functional groups: (1) solid phase peptide synthesis; and (2) solution phase modification of pre-synthesized, commercial peptides. We further describe the aniline-catalyzed ligation of 4FB- and HYNIC-modified peptides, in the presence of a fluorescent label on the latter peptide, as well as subsequent assembly of the ligated peptide to water-soluble QDs. Many technical elements of these protocols can be extended to labeling peptides with other small molecule reagents. Overall, the bioconjugate chemistry is robust, selective, and modular, thereby potentiating the controlled conjugation of QDs with a diverse array of biomolecules for various applications.

  11. New opioid affinity labels containing maleoyl moiety.

    Science.gov (United States)

    Szatmári, I; Orosz, G; Rónai, A Z; Makó, E; Medzihradszky, K; Borsodi, A

    1999-01-01

    Opioid receptor binding properties and pharmacological profiles of novel peptides containing maleoyl function were determined in order to develop new affinity labels. Based on the enkephalin structure peptide ligands were synthesized and tested. Both in in vitro receptor binding experiments and pharmacological studies, all ligands showed agonist character with relatively high affinity (Ki values in the nanomolar range) and good to moderate selectivity. Replacement of Gly2 in the enkephalin frame with D-Ala led to higher affinities with a small decrease in selectivity. The longer peptide chains resulted in compounds with high percentage (up to 86%) of irreversible binding. The selectivity pattern of the ligands is in good agreement with the data obtained from the pharmacological assays (guinea pig ileum and mouse vas deferens bioassays). The newly synthesized peptides could be used in further studies in order to determine more detailed characteristics of the ligand-receptor interaction.

  12. Comamonas testosteroni uses a chemoreceptor for tricarboxylic acid cycle intermediates to trigger chemotactic responses towards aromatic compounds.

    Science.gov (United States)

    Ni, Bin; Huang, Zhou; Fan, Zheng; Jiang, Cheng-Ying; Liu, Shuang-Jiang

    2013-11-01

    Bacterial chemotaxis towards aromatic compounds has been frequently observed; however, knowledge of how bacteria sense aromatic compounds is limited. Comamonas testosteroni CNB-1 is able to grow on a range of aromatic compounds. This study investigated the chemotactic responses of CNB-1 to 10 aromatic compounds. We constructed a chemoreceptor-free, non-chemotactic mutant, CNB-1Δ20, by disruption of all 19 putative methyl-accepting chemotaxis proteins (MCPs) and the atypical chemoreceptor in strain CNB-1. Individual complementation revealed that a putative MCP (tagged MCP2201) was involved in triggering chemotaxis towards all 10 aromatic compounds. The recombinant sensory domain of MCP2201 did not bind to 3- or 4-hydroxybenzoate, protocatechuate, catechol, benzoate, vanillate and gentisate, but bound oxaloacetate, citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate and malate. The mutant CNB-1ΔpmdF that lost the ability to metabolize 4-hydroxybenzoate and protocatechuate also lost its chemotactic response to these compounds, suggesting that taxis towards aromatic compounds is metabolism-dependent. Based on the ligand profile, we proposed that MCP2201 triggers taxis towards aromatic compounds by sensing TCA cycle intermediates. Our hypothesis was further supported by the finding that introduction of the previously characterized pseudomonad chemoreceptor (McpS) for TCA cycle intermediates into CNB-1Δ20 likewise triggered chemotaxis towards aromatic compounds.

  13. Anti-coreceptor therapy drives selective T cell egress by suppressing inflammation-dependent chemotactic cues

    Science.gov (United States)

    Martin, Aaron J.; Clark, Matthew; Gojanovich, Gregory; Manzoor, Fatima; Miller, Keith; Kline, Douglas E.; Morillon, Y. Maurice; Wang, Bo

    2016-01-01

    There continues to be a need for immunotherapies to treat type 1 diabetes in the clinic. We previously reported that nondepleting anti-CD4 and -CD8 Ab treatment effectively reverses diabetes in new-onset NOD mice. A key feature of the induction of remission is the egress of the majority of islet-resident T cells. How this occurs is undefined. Herein, the effects of coreceptor therapy on islet T cell retention were investigated. Bivalent Ab binding to CD4 and CD8 blocked TCR signaling and T cell cytokine production, while indirectly downregulating islet chemokine expression. These processes were required for T cell retention, as ectopic IFN-γ or CXCL10 inhibited Ab-mediated T cell purging. Importantly, treatment of humanized mice with nondepleting anti–human CD4 and CD8 Ab similarly reduced tissue-infiltrating human CD4+ and CD8+ T cells. These findings demonstrate that Ab binding of CD4 and CD8 interrupts a feed-forward circuit by suppressing T cell–produced cytokines needed for expression of chemotactic cues, leading to rapid T cell egress from the islets. Coreceptor therapy therefore offers a robust approach to suppress T cell–mediated pathology by purging T cells in an inflammation-dependent manner.

  14. Emerging morphologies in round bacterial colonies: comparing volumetric versus chemotactic expansion.

    Science.gov (United States)

    Giverso, Chiara; Verani, Marco; Ciarletta, Pasquale

    2016-06-01

    Biological experiments performed on living bacterial colonies have demonstrated the microbial capability to develop finger-like shapes and highly irregular contours, even starting from an homogeneous inoculum. In this work, we study from the continuum mechanics viewpoint the emergence of such branched morphologies in an initially circular colony expanding on the top of a Petri dish coated with agar. The bacterial colony expansion, based on either a source term, representing volumetric mitotic processes, or a nonconvective mass flux, describing chemotactic expansion, is modeled at the continuum scale. We demonstrate that the front of the colony is always linearly unstable, having similar dispersion curves to the ones characterizing branching instabilities. We also perform finite element simulations, which not only prove the emergence of branching, but also highlight dramatic differences between the two mechanisms of colony expansion in the nonlinear regime. Furthermore, the proposed combination of analytical and numerical analysis allowed studying the influence of different model parameters on the selection of specific patterns. A very good agreement has been found between the resulting simulations and the typical structures observed in biological assays. Finally, this work provides a new interpretation of the emergence of branched patterns in living aggregates, depicted as the results of a complex interplay among chemical, mechanical and size effects.

  15. Effect of selective phosphodiesterase inhibitors on the rat eosinophil chemotactic response in vitro

    Directory of Open Access Journals (Sweden)

    Alessandra C Alves

    1997-12-01

    Full Text Available In the present study, we have performed a comparative analysis of the effect of selective inhibitors of phosphodiesterase (PDE type III, IV and V on eosinophil chemotaxis triggered by platelet activating factor (PAF and leukotriene B4 (LTB4 in vitro. The effect of the analogues N6-2'-O-dibutyryladenosine 3':5' cyclic monophosphate (Bt2 cyclic AMP and N2-2'-O- dibutyrylguanosine 3':5' cyclic monophosphate (Bt2 cyclic GMP has also been determined. The eosinophils were obtained from the peritoneal cavity of naive Wistar rats and purified in discontinuous Percoll gradients to 85-95% purity. We observed that pre-incubation of eosinophils with the PDE type IV inhibitor rolipram suppressed the chemotactic response triggered by PAF and LTB4, in association with an increase in the intracellular levels of cyclic AMP. In contrast, neither zaprinast (type V inhibitor nor type III inhibitors milrinone and SK&F 94836 affected the eosinophil migration. Only at the highest concentration tested did the analogue Bt2 cyclic AMP suppress the eosinophil chemotaxis, under conditions where Bt2 cyclic GMP was ineffective. We have concluded that inhibition of PDE IV, but not PDE III or V, was able to block the eosinophil chemotaxis in vitro, suggesting that the suppressive activity of selective PDE IV inhibitors on tissue eosinophil accumulation may, at least, be partially dependent on their ability to directly inhibit the eosinophil migration.

  16. Wave Patterns in Cell Membrane and Actin Cortex Uncoupled from Chemotactic Signals.

    Science.gov (United States)

    Gerisch, Günther; Ecke, Mary

    2016-01-01

    When cells of Dictyostelium discoideum orientate in a gradient of chemoattractant, they are polarized into a protruding front pointing toward the source of attractant, and into a retracting tail. Under the control of chemotactic signal inputs, Ras is activated and PIP3 is synthesized at the front, while the PIP3-degrading phosphatase PTEN decorates the tail region. As a result of signal transduction, actin filaments assemble at the front into dendritic structures associated with the Arp2/3 complex, in contrast to the tail region where a loose actin meshwork is associated with myosin-II and cortexillin, an antiparallel actin-bundling protein. In axenically growing strains of D. discoideum, wave patterns built by the same components evolve in the absence of any external signal input. Since these autonomously generated patterns are constrained to the plane of the substrate-attached cell surface, they are optimally suited to the optical analysis of state transitions between front-like and tail-like states of the membrane and the actin cortex. Here, we describe imaging techniques using fluorescent proteins to probe for the state of the membrane, the reorganization of the actin network, and the dynamics of wave patterns.

  17. Hydrocarbon biodegradation and dynamic laser speckle for detecting chemotactic responses at low bacterial concentration.

    Science.gov (United States)

    Nisenbaum, Melina; Sendra, Gonzalo Hernán; Gilbert, Gastón Alfredo Cerdá; Scagliola, Marcelo; González, Jorge Froilán; Murialdo, Silvia Elena

    2013-03-01

    We report on the biodegradation of pure hydrocarbons and chemotaxis towards these compounds by an isolated chlorophenol degrader, Pseudomonas strain H. The biochemical and phylogenetic analysis of the 16S rDNA sequence identified Pseudomonas strain H as having 99.56% similarity with P. aeruginosa PA01. This strain was able to degrade n-hexadecane, 1-undecene, 1-nonene, 1-decene, 1-dodecene and kerosene. It grew in the presence of 1-octene, while this hydrocarbons is toxic to other hydrocarbons degraders. Pseudomonas strain H was also chemotactic towards n-hexadecane, kerosene, 1-undecene and 1-dodecene. These results show that this Pseudomonas strain H is an attractive candidate for hydrocarbon-containing wastewater bioremediation in controlled environments. Since the classical standard techniques for detecting chemotaxis are not efficient at low bacterial concentrations, we demonstrate the use of the dynamic speckle laser method, which is simple and inexpensive, to confirm bacterial chemotaxis at low cell concentrations (less than 10(5) colony-forming unit per millilitre (CFU/mL)) when hydrocarbons are the attractants.

  18. A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole.

    Science.gov (United States)

    Yamaichi, Yoshiharu; Bruckner, Raphael; Ringgaard, Simon; Möll, Andrea; Cameron, D Ewen; Briegel, Ariane; Jensen, Grant J; Davis, Brigid M; Waldor, Matthew K

    2012-10-15

    The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation, recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles, chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella. Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus, a single polar hub is instrumental for establishing polar identity and organization.

  19. Hydrocarbon biodegradation and dynamic laser speckle for detecting chemotactic responses at low bacterial concentration

    Institute of Scientific and Technical Information of China (English)

    Melina Nisenbaum; Gonzalo Hernán Sendra; Gastón Alfredo Cerdá Gilbert; Marcelo Scagliola; Jorge Froilán González; Silvia Elena Murialdo

    2013-01-01

    We report on the biodegradation of pure hydrocarbons and chemotaxis towards these compounds by an isolated chlorophenol degrader,Pseudomonas strain H.The biochemical and phylogenetic analysis of the 16S rDNA sequence identified Pseudomonas strain H as having 99.56% similarity with P.aeruginosa PA01.This strain was able to degrade n-hexadecane,1-undecene,1-nonene,1-decene,1-dodecene and kerosene.It grew in the presence of 1-octene,while this hydrocarbons is toxic to other hydrocarbons degraders.Pseudomonas strain H was also chemotactic towards n-hexadecane,kerosene,1-undecene and 1-dodecene.These results show that this Pseudomonas strain H is an attractive candidate for hydrocarbon-containing wastewater bioremediation in controlled environments.Since the classical standard techniques for detecting chemotaxis are not efficient at low bacterial concentrations,we demonstrate the use of the dynamic speckle laser method,which is simple and inexpensive,to confirm bacterial chemotaxis at low cell concentrations (less than 105 colony-forming unit per millilitre (CFU/mL)) when hydrocarbons are the attractants.

  20. A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils.

    Science.gov (United States)

    Massena, Sara; Christoffersson, Gustaf; Hjertström, Elina; Zcharia, Eyal; Vlodavsky, Israel; Ausmees, Nora; Rolny, Charlotte; Li, Jin-Ping; Phillipson, Mia

    2010-09-16

    During infection, chemokines sequestered on endothelium induce recruitment of circulating leukocytes into the tissue where they chemotax along chemokine gradients toward the afflicted site. The aim of this in vivo study was to determine whether a chemokine gradient was formed intravascularly and influenced intraluminal neutrophil crawling and transmigration. A chemokine gradient was induced by placing a macrophage inflammatory protein-2 (MIP-2)-containing (CXCL2) gel on the cremaster muscle of anesthetized wild-type mice or heparanase-overexpressing transgenic mice (hpa-tg) with truncated heparan sulfate (HS) side chains. Neutrophil-endothelial interactions were visualized by intravital microscopy and chemokine gradients detected by confocal microscopy. Localized extravascular chemokine release (MIP-2 gel) induced directed neutrophil crawling along a chemotactic gradient immobilized on the endothelium and accelerated their recruitment into the target tissue compared with homogeneous extravascular chemokine concentration (MIP-2 superfusion). Endothelial chemokine sequestration occurred exclusively in venules and was HS-dependent, and neutrophils in hpa-tg mice exhibited random crawling. Despite similar numbers of adherent neutrophils in hpa-tg and wild-type mice, the altered crawling in hpa-tg mice was translated into decreased number of emigrated neutrophils and ultimately decreased the ability to clear bacterial infections. In conclusion, an intravascular chemokine gradient sequestered by endothelial HS effectively directs crawling leukocytes toward transmigration loci close to the infection site.

  1. A Worldwide Competition to Compare the Speed and Chemotactic Accuracy of Neutrophil-Like Cells

    Science.gov (United States)

    Wong, Elisabeth; Hamza, Bashar; Bae, Albert; Martel, Joseph; Kataria, Rama; Keizer-Gunnink, Ineke; Kortholt, Arjan; Van Haastert, Peter J. M.; Charras, Guillaume; Janetopoulos, Christopher; Irimia, Daniel

    2016-01-01

    Chemotaxis is the ability to migrate towards the source of chemical gradients. It underlies the ability of neutrophils and other immune cells to hone in on their targets and defend against invading pathogens. Given the importance of neutrophil migration to health and disease, it is crucial to understand the basic mechanisms controlling chemotaxis so that strategies can be developed to modulate cell migration in clinical settings. Because of the complexity of human genetics, Dictyostelium and HL60 cells have long served as models system for studying chemotaxis. Since many of our current insights into chemotaxis have been gained from these two model systems, we decided to compare them side by side in a set of winner-take-all races, the Dicty World Races. These worldwide competitions challenge researchers to genetically engineer and pharmacologically enhance the model systems to compete in microfluidic racecourses. These races bring together technological innovations in genetic engineering and precision measurement of cell motility. Fourteen teams participated in the inaugural Dicty World Race 2014 and contributed cell lines, which they tuned for enhanced speed and chemotactic accuracy. The race enabled large-scale analyses of chemotaxis in complex environments and revealed an intriguing balance of speed and accuracy of the model cell lines. The successes of the first race validated the concept of using fun-spirited competition to gain insights into the complex mechanisms controlling chemotaxis, while the challenges of the first race will guide further technological development and planning of future events. PMID:27332963

  2. Bound attractant at the leading vs. the trailing edge determines chemotactic prowess.

    Science.gov (United States)

    Herzmark, Paul; Campbell, Kyle; Wang, Fei; Wong, Kit; El-Samad, Hana; Groisman, Alex; Bourne, Henry R

    2007-08-14

    We have analyzed chemotaxis of neutrophil-differentiated HL60 cells in microfluidic devices that create exponential gradients of the chemoattractant, f-Met-Leu-Phe (fMLP). Such gradients expose each cell to a difference in fMLP concentration (DeltaC) across its diameter that is directly proportional to the ambient concentration (C) at that cell's position in the gradient, so the ratio DeltaC/C is constant everywhere. Cells exposed to ambient fMLP concentrations near the constant of dissociation (K(d)) for fMLP binding to its receptor ( approximately 10 nM) crawl much less frequently when DeltaC/C is 0.05 than when it is 0.09 or 0.13. Hence, cells can detect the gradient across their diameter without moving and, thus, without experiencing temporal changes in attractant concentration. At all DeltaC/C ratios tested, the average chemotactic prowess of individual cells (indicated by the distance a cell traveled in the correct direction divided by the length of its migration path) is maximal for cells that start migrating at concentrations near the K(d) and progressively decreases at higher or lower starting concentrations.

  3. A Worldwide Competition to Compare the Speed and Chemotactic Accuracy of Neutrophil-Like Cells.

    Directory of Open Access Journals (Sweden)

    Monica Skoge

    Full Text Available Chemotaxis is the ability to migrate towards the source of chemical gradients. It underlies the ability of neutrophils and other immune cells to hone in on their targets and defend against invading pathogens. Given the importance of neutrophil migration to health and disease, it is crucial to understand the basic mechanisms controlling chemotaxis so that strategies can be developed to modulate cell migration in clinical settings. Because of the complexity of human genetics, Dictyostelium and HL60 cells have long served as models system for studying chemotaxis. Since many of our current insights into chemotaxis have been gained from these two model systems, we decided to compare them side by side in a set of winner-take-all races, the Dicty World Races. These worldwide competitions challenge researchers to genetically engineer and pharmacologically enhance the model systems to compete in microfluidic racecourses. These races bring together technological innovations in genetic engineering and precision measurement of cell motility. Fourteen teams participated in the inaugural Dicty World Race 2014 and contributed cell lines, which they tuned for enhanced speed and chemotactic accuracy. The race enabled large-scale analyses of chemotaxis in complex environments and revealed an intriguing balance of speed and accuracy of the model cell lines. The successes of the first race validated the concept of using fun-spirited competition to gain insights into the complex mechanisms controlling chemotaxis, while the challenges of the first race will guide further technological development and planning of future events.

  4. Chemotactic systems in the presence of conflicts: A new functional inequality

    Science.gov (United States)

    Wolansky, G.

    2016-11-01

    The evolution of a chemotactic system involving a population of cells attracted to self-produced chemicals is described by the Keller-Segel system. In dimension 2, this system demonstrates a balance between the spreading effect of diffusion and the concentration due to self-attraction. As a result, there exists a critical "mass" (i.e. total cell's population) above which the solution of this system collapses in a finite time, while below this critical mass there is global existence in time. In particular, subcritical mass leads under certain additional conditions to the existence of steady states, corresponding to the solution of an elliptic Liouville equation. The existence of this critical mass is related to a functional inequality known as the Moser-Trudinger inequality. An extension of the Keller-Segel model to several cells populations was considered before in the literature. Here we review some of these results and, in particular, consider the case of conflict between two populations, that is, when population one attracts population two, while, at the same time, population two repels population one. This assumption leads to a new functional inequality which generalizes the Moser-Trudinger inequality. As an application of this inequality we derive sufficient conditions for the existence of steady states corresponding to solutions of an elliptic Liouville system.

  5. Profiling of differentially expressed chemotactic-related genes in MCP-1 treated macrophage cell line using human cDNA arrays

    Institute of Scientific and Technical Information of China (English)

    Guang-Xing Bian; Hong Miao; Lei Qiu; Dong-Mei Cao; Bao-Yu Guo

    2005-01-01

    AIM: To study the global gene expression of chemotactic genes in macrophage line U937 treated with human monocyte chemoattractant protein-1 (MCP-1) through the use of ExpreeChipTMHO2 cDNA array.METHODS: Total RNA was extracted from MCP-1 treated macrophage line U937 and normal U937 cells, reversely transcribed to cDNA, and then screened in parallel with HO2 human cDNA array chip. The scanned result was additionally validated using RT-PCR.RESULTS: The result of cDNA array showed that one chemotactic-related gene was up-regulated more than two-fold (RANTES) and seven chemotactic-related genes were down-regulated more than two-fold (CCR1, CCR5,ccl16, GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2) in MCP-1 treated U937 cells at mRNA level.RT-PCR analysis of four of these differentially expressed genes gave results consistent with cDNA array findings.CONCLUSION: MCP-1 could influence some chemokine and receptor expressions in macrophages in vitro. MCP-1mainly down-regulates the expression of chemotactic genes influencing neutrophilic granulocyte expression (GROβ, GROγ, IL-8 and granulocyte chemotactic protein 2), and the mRNA level of CCR5, which plays a critical role in many disorders and illnesses.

  6. Development of a renal collecting duct homing peptide using phage display

    DEFF Research Database (Denmark)

    Svenningsen, Per; Peti-Peterdi, Janos

    Homing peptides are useful for in vivo labeling and nonviral gene transfer to selective tissues and cell types. The aim of this project was to develop a renal collecting duct homing peptide. Using phage display, we identified a phage expressing a cyclic 7 amino acid peptide, which was internalize...

  7. 99mTc: Labeling Chemistry and Labeled Compounds

    Science.gov (United States)

    Alberto, R.; Abram, U.

    This chapter reviews the radiopharmaceutical chemistry of technetium related to the synthesis of perfusion agents and to the labeling of receptor-binding biomolecules. To understand the limitations of technetium chemistry imposed by future application of the complexes in nuclear medicine, an introductory section analyzes the compulsory requirements to be considered when facing the incentive of introducing a novel radiopharmaceutical into the market. Requirements from chemistry, routine application, and market are discussed. In a subsequent section, commercially available 99mTc-based radiopharmaceuticals are treated. It covers the complexes in use for imaging the most important target organs such as heart, brain, or kidney. The commercially available radiopharmaceuticals fulfill the requirements outlined earlier and are discussed with this background. In a following section, the properties and perspectives of the different generations of radiopharmaceuticals are described in a general way, covering characteristics for perfusion agents and for receptor-specific molecules. Technetium chemistry for the synthesis of perfusion agents and the different labeling approaches for target-specific biomolecules are summarized. The review comprises a general introduction to the common approaches currently in use, employing the N x S4-x , [3+1] and 2-hydrazino-nicotinicacid (HYNIC) method as well as more recent strategies such as the carbonyl and the TcN approach. Direct labeling without the need of a bifunctional chelator is briefly reviewed as well. More particularly, recent developments in the labeling of concrete targeting molecules, the second generation of radiopharmaceuticals, is then discussed and prominent examples with antibodies/peptides, neuroreceptor targeting small molecules, myocardial imaging agents, vitamins, thymidine, and complexes relevant to multidrug resistance are given. In addition, a new approach toward peptide drug development is described. The section

  8. Chemotactic signal transduction and phosphate metabolism as adaptive strategies during citrus canker induction by Xanthomonas citri.

    Science.gov (United States)

    Moreira, Leandro Marcio; Facincani, Agda Paula; Ferreira, Cristiano Barbalho; Ferreira, Rafael Marine; Ferro, Maria Inês Tiraboshi; Gozzo, Fabio Cesar; de Oliveira, Julio Cezar Franco; Ferro, Jesus Aparecido; Soares, Márcia Regina

    2015-03-01

    The genome of Xanthomonas citri subsp. Citri strain 306 pathotype A (Xac) was completely sequenced more than 10 years; to date, few studies involving functional genomics Xac and its host compatible have been developed, specially related to adaptive events that allow the survival of Xac within the plant. Proteomic analysis of Xac showed that the processes of chemotactic signal transduction and phosphate metabolism are key adaptive strategies during the interaction of a pathogenic bacterium with its plant host. The results also indicate the importance of a group of proteins that may not be directly related to the classical virulence factors, but that are likely fundamental to the success of the initial stages of the infection, such as methyl-accepting chemotaxis protein (Mcp) and phosphate specific transport (Pst). Furthermore, the analysis of the mutant of the gene pstB which codifies to an ABC phosphate transporter subunit revealed a complete absence of citrus canker symptoms when inoculated in compatible hosts. We also conducted an in silico analysis which established the possible network of genes regulated by two-component systems PhoPQ and PhoBR (related to phosphate metabolism), and possible transcriptional factor binding site (TFBS) motifs of regulatory proteins PhoB and PhoP, detaching high degree of conservation of PhoB TFBS in 84 genes of Xac genome. This is the first time that chemotaxis signal transduction and phosphate metabolism were therefore indicated to be fundamental to the process of colonization of plant tissue during the induction of disease associated with Xanthomonas genus bacteria.

  9. Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

    Directory of Open Access Journals (Sweden)

    Andrew J Muinonen-Martin

    2014-10-01

    Full Text Available The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.

  10. Chemotactic and mitogenic stimuli of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy

    Science.gov (United States)

    Fiala, Milan; Avagyan, Hripsime; Merino, Jose Joaquin; Bernas, Michael; Valdivia, Juan; Espinosa-Jeffrey, Araceli; Witte, Marlys; Weinand, Martin

    2012-01-01

    To identify the upstream signals of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy (TLE), we evaluated by immunohistochemistry and confocal microscopy brain tissues of 13 TLE patients and 5 control patients regarding expression of chemokines and cell-cycle proteins. The chemokine RANTES (CCR5) and other CC-chemokines and apoptotic markers (caspase-3, -8, -9) were expressed in lateral temporal cortical and hippocampal neurons of TLE patients, but not in neurons of control cases. The chemokine RANTES is usually found in cytoplasmic and extracellular locations. However, in TLE neurons, RANTES was displayed in an unusual location, the neuronal nuclei. In addition, the cell-cycle regulatory transcription factor E2F1 was found in an abnormal location in neuronal cytoplasm. The pro-inflammatory enzyme cyclooxygenase-2 and cytokine interleukin-1β were expressed both in neurons of patients suffering from temporal lobe epilepsy and from cerebral trauma. The vessels showed fibrin leakage, perivascular macrophages and expression of IL-6 on endothelial cells. In conclusion, the cytoplasmic effects of E2F1 and nuclear effects of RANTES might have novel roles in neuronal apoptosis of TLE neurons and indicate a need to develop new medical and/or surgical neuroprotective strategies against apoptotic signaling by these molecules. Both RANTES and E2F1 signaling are upstream from caspase activation, thus the antagonists of RANTES and/or E2F1 blockade might be neuroprotective for patients with medically intractable temporal lobe epilepsy. The results have implications for the development of new medical and surgical therapies based on inhibition of chemotactic and mitogenic stimuli of neuronal apoptosis in patients with medically intractable temporal lobe epilepsy. PMID:22444245

  11. Gaucher disease: chemotactic factors and immunological cell invasion in a mouse model.

    Science.gov (United States)

    Pandey, Manoj Kumar; Jabre, Nicholas A; Xu, You-Hai; Zhang, Wujuan; Setchell, Kenneth D R; Grabowski, Gregory A

    2014-02-01

    Gaucher disease results from mutations in GBA1 that cause functional disruption of the encoded lysosomal enzyme, acid β-glucosidase. The consequent excess accumulation of glucosylceramide and glucosylsphingosine in lysosomes is central to the disease pathogenesis with classical involvement of macrophage (Mфs) lineage cells of visceral organs, bone, or brain. Several studies have implicated the increased secretion of chemokines and infiltration of a variety of immunological cells into tissues of Gaucher disease patients. Trafficking of immunological cells to the sites of inflammation requires the presence of chemokines. Although increases of different immunological cells and several chemokines are present in Gaucher disease, the specific chemoattractants that cause the increased influx of immunological cells are not fully defined. Here, increased levels of I-309, MCP-5, CXCL-2, CXCL-9, CXCL-10, CXCL-11, CXCL-13, and their corresponding leukocytes, i.e., MOs (monocytes), Mфs, dendritic cells (DCs), polymorphonuclear neutrophils (PMNs), and T, and B cells were identified in the circulation of mice with Gba1 mutations (D409V/null). Sera from D409V/null mice contained chemoattractants for a variety of immunological cells as shown by ex vivo chemotaxis studies and by flow cytometry. Enhanced chemotaxis towards 9V/null sera was found for 9V/null lung-, spleen-, liver-, and bone marrow-derived Mфs (CD11b(+) F480(+)), PMNs (Gr1(high) CD11b(+)), DCs (CD11c(+) CD11b(+)), T lymphocytes (CD3(+) TCRB(+)), and B lymphocytes (B220(+) CD19(+)). These data support these chemotactic factors as causative to increased tissue infiltration of leukocytes in Gaucher disease.

  12. Chemotactic migration of T cells towards dendritic cells promotes the detection of rare antigens.

    Directory of Open Access Journals (Sweden)

    Renske M A Vroomans

    Full Text Available In many immunological processes chemoattraction is thought to play a role in guiding cells to their sites of action. However, based on in vivo two-photon microscopy experiments in the absence of cognate antigen, T cell migration in lymph nodes (LNs has been roughly described as a random walk. Although it has been shown that dendritic cells (DCs carrying cognate antigen in some circumstances attract T cells chemotactically, it is currently still unclear whether chemoattraction of T cells towards DCs helps or hampers scanning. Chemoattraction towards DCs could on the one hand help T cells to rapidly find DCs. On the other hand, it could be deleterious if DCs become shielded by a multitude of attracted yet non-specific T cells. Results from a recent simulation study suggested that the deleterious effect dominates. We re-addressed the question whether T cell chemoattraction towards DCs is expected to promote or hamper the detection of rare antigens using the Cellular Potts Model, a formalism that allows for dynamic, flexible cellular shapes and cell migration. Our simulations show that chemoattraction of T cells enhances the DC scanning efficiency, leading to an increased probability that rare antigen-specific T cells find DCs carrying cognate antigen. Desensitization of T cells after contact with a DC further improves the scanning efficiency, yielding an almost threefold enhancement compared to random migration. Moreover, the chemotaxis-driven migration still roughly appears as a random walk, hence fine-tuned analysis of cell tracks will be required to detect chemotaxis within microscopy data.

  13. Chemotactic migration of T cells towards dendritic cells promotes the detection of rare antigens.

    Science.gov (United States)

    Vroomans, Renske M A; Marée, Athanasius F M; de Boer, Rob J; Beltman, Joost B

    2012-01-01

    In many immunological processes chemoattraction is thought to play a role in guiding cells to their sites of action. However, based on in vivo two-photon microscopy experiments in the absence of cognate antigen, T cell migration in lymph nodes (LNs) has been roughly described as a random walk. Although it has been shown that dendritic cells (DCs) carrying cognate antigen in some circumstances attract T cells chemotactically, it is currently still unclear whether chemoattraction of T cells towards DCs helps or hampers scanning. Chemoattraction towards DCs could on the one hand help T cells to rapidly find DCs. On the other hand, it could be deleterious if DCs become shielded by a multitude of attracted yet non-specific T cells. Results from a recent simulation study suggested that the deleterious effect dominates. We re-addressed the question whether T cell chemoattraction towards DCs is expected to promote or hamper the detection of rare antigens using the Cellular Potts Model, a formalism that allows for dynamic, flexible cellular shapes and cell migration. Our simulations show that chemoattraction of T cells enhances the DC scanning efficiency, leading to an increased probability that rare antigen-specific T cells find DCs carrying cognate antigen. Desensitization of T cells after contact with a DC further improves the scanning efficiency, yielding an almost threefold enhancement compared to random migration. Moreover, the chemotaxis-driven migration still roughly appears as a random walk, hence fine-tuned analysis of cell tracks will be required to detect chemotaxis within microscopy data.

  14. Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure.

    Science.gov (United States)

    Tolosa, Ezequiel J; Fernández-Zapico, Martín E; Battiato, Natalia L; Rovasio, Roberto A

    2016-01-01

    Our aim was to understand the involvement of Sonic hedgehog (Shh) morphogen in the oriented distribution of neural crest cells (NCCs) toward the optic vesicle and to look for potential disorders of this guiding mechanism after ethanol exposure. In vitro directional analysis showed the chemotactic response of NCCs up Shh gradients and to notochord co-cultures (Shh source) or to their conditioned medium, a response inhibited by anti-Shh antibody, receptor inhibitor cyclopamine and anti-Smo morpholino (MO). Expression of the Ptch-Smo receptor complex on in vitro NCCs was also shown. In whole embryos, the expression of Shh mRNA and protein was seen in the ocular region, and of Ptch, Smo and Gli/Sufu system on cephalic NCCs. Anti-Smo MO or Ptch-mutated plasmid (Ptch1(Δloop2)) impaired cephalic NCC migration/distribution, with fewer cells invading the optic region and with higher cell density at the homolateral mesencephalic level. Beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic signal) supported the role of Shh as an in vivo guide molecule for cephalic NCCs. Ethanol exposure perturbed in vitro and in vivo NCC migration. Early stage embryos treated with ethanol, in a model reproducing Fetal Alcohol Syndrome, showed later disruptions of craniofacial development associated with abnormal in situ expression of Shh morphogen. The results show the Shh/Ptch/Smo-dependent migration of NCCs toward the optic vesicle, with the support of specific inactivation with genetic and pharmacological tools. They also help to understand mechanisms of accurate distribution of embryonic cells and of their perturbation by a commonly consumed teratogen, and demonstrate, in addition to its other known developmental functions, a new biological activity of cellular guidance for Shh.

  15. Leukocyte chemotactic factor 2 amyloidosis cannot be reliably diagnosed by immunohistochemical staining.

    Science.gov (United States)

    Paueksakon, Paisit; Fogo, Agnes B; Sethi, Sanjeev

    2014-07-01

    We investigated the role of leukocyte chemotactic factor (LECT2) immunohistochemical staining in the diagnosis of type of renal amyloidosis. Fifty renal amyloidosis cases with available paraffin blocks in our 2002 to 2012 renal biopsy files were reviewed. Patients were designated as a defined amyloid, including amyloid light chain (AL) and amyloid-associated amyloid (AA), or a non-AL/non-AA amyloid group. LECT2-specific antibody immunohistochemistry was performed in all 50 cases. Laser microdissection and mass spectrometry (LMD/MS) were performed in 10 cases. Forty-five patients had amyloid classified as either AL (44) or AA (1), and 5 had undetermined amyloid. Three of the five non-AL/non-AA group patient biopsies showed positive LECT2 immunohistochemical staining, and of these, LECT2 was also identified by LMD/MS in 1 patient, fibrinogen-α was identified in 1 patient, and apolipoprotein IV was identified in 1 patient. Two of these non-AL/non-AA patients showed negative LECT2 staining, and LMD/MS showed apolipoprotein IV as a major protein component. Five of the 44 AL amyloid patients showed weakly positive LECT2 staining. However, LECT2 was not identified by LMD/MS in any of these 5 cases. The single patient with AA amyloid was negative for LECT2 by immunohistochemical staining. Among 5 non-AL and non-AA amyloidosis patients in our study, 1 had LECT2, 1 had fibrinogen-α, and 3 had apolipoprotein IV as a major protein component. The data from this study show that weak LECT2 staining should be regarded as indeterminate or a negative result and does not per se allow diagnosis of specific amyloid type. The diagnosis of LECT2 renal amyloidosis may require LMD/MS confirmation.

  16. Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils.

    Directory of Open Access Journals (Sweden)

    Cheng-Yuk Lee

    Full Text Available Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis as well as upon contact (by serum-dependent adhesion and phagocytosis. This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.

  17. Antimicrobial Peptides

    Directory of Open Access Journals (Sweden)

    Ali Adem Bahar

    2013-11-01

    Full Text Available The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs, a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics.

  18. Neutrophil antibacterial peptides, multifunctional effector molecules in the mammalian immune system.

    Science.gov (United States)

    Gudmundsson, G H; Agerberth, B

    1999-12-17

    The bactericidal machinery of mammalian neutrophils is built up of many components with different chemical properties, involving proteins, peptides and oxygen-dependent radicals. All these components work in synergy, leading to destruction and elimination of ingested microbes. During the eighties, it gradually became clear, that cationic peptides are a part of the oxygen-independent bactericidal effectors in phagocytic cells. In mammals, these antimicrobial peptides are represented by two families, the defensins and the cathelicidins. These potent broad spectra peptides are included as immediate effector molecules in innate immunity. The detailed killing mechanism for these effectors is partly known, but nearly all of them have membrane affinity, and permeate bacterial membranes, resulting in lysis of the bacteria. This peptide-membrane interaction includes also eukaryotic membranes, that implicates cytotoxic effects on host cells. Studies in vitro have established that the microenvironment is critical for their activities. In connection to cystic fibrosis, the effects of microenvironment changes are apparent, causing inactivation of peptide defences and leading to repeated serious bacterial infections. Thus, the importance of the microenvironment is also supported in vivo. Additional functions of these peptides such as chemotactic, mitogenic and stimulatory in the wound healing process suggest further important roles for these peptides.

  19. The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate.

    Science.gov (United States)

    Miller, J C; Waley, S G

    1971-06-01

    1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.

  20. Effects of Spin-Labels on Membrane Burial Depth of MARCKS-ED Residues.

    Science.gov (United States)

    Qi, Yifei; Klauda, Jeffery B; Im, Wonpil

    2016-10-18

    Site-directed spin-labeling electron paramagnetic resonance spectroscopy is a useful tool to obtain information about the environment of specific residues. One of its applications is to investigate membrane protein topology based on the accessibility of the spin label, with the assumption that the position of the spin label in the membrane is close to that of the native residue. This assumption is valid in proteins with well-ordered structures, but could be problematic in small peptides because the labeling may cause a perturbation that is large enough to change local interactions between the peptide and the membrane. To quantitatively characterize such effects, we have simulated the association of a 25-amino-acid peptide, MARCKS-ED, to membranes with and without spin labels. Our simulations show that the depths of spin labels are ∼6-17 Å deeper than the unlabeled charged and polar residues in the wild-type. When the hydrophobic residue Phe is labeled, however, the spin-label depth is close to that of the native residue as well as the experimental value. Our study suggests that one should be cautious in interpretation of spin label data when charged and polar residues in small peptides are labeled.

  1. Solid Phase Formylation of N-Terminus Peptides

    Directory of Open Access Journals (Sweden)

    Anna Lucia Tornesello

    2016-06-01

    Full Text Available Formylation of amino groups is a critical reaction involved in several biological processes including post-translational modification of histones. The addition of a formyl group (CHO to the N-terminal end of a peptide chain generates biologically active molecules. N-formyl-peptides can be produced by different methods. We performed the N-formylation of two chemotactic hexapetides, Met1-Leu2-Lys3-Leu4-Ile5-Val6 and Met1-Met2-Tyr3-Ala4-Leu5-Phe6, carrying out the reaction directly on peptidyl-resin following pre-activation of formic acid with N,N-dicyclohexylcarbodiimmide (DCC in liquid phase. The overnight incubation at 4 °C resulted in a significant increase in production yields of formylated peptides compared to the reaction performed at room temperature. The method is consistently effective, rapid, and inexpensive. Moreover, the synthetic strategy can be applied for the formylation of all primary amines at N-terminus of peptide chains or amino groups of lysine side-chains in solid phase.

  2. MiR-124 suppresses the chemotactic migration of rat mesenchymal stem cells toward HGF by downregulating Wnt/β-catenin signaling.

    Science.gov (United States)

    Yue, Qing; Zhang, Yu; Li, Xianyang; He, Lihong; Hu, Ya'nan; Wang, Xianyao; Xu, Xiaojing; Shen, Yixin; Zhang, Huanxiang

    2016-09-01

    Mesenchymal stem cells (MSCs) exhibit the potential to repair a wide variety of injured adult tissues. The migration capability of MSCs is an important determinant of the efficiency of MSC transplant therapy. MicroRNAs (miRNAs) are increasingly implicated in regulating the migration of MSCs. Herein, we show that the expression of miR-124 was downregulated in rat MSCs (rMSCs) treated with hepatocyte growth factor (HGF). Overexpression of miR-124 significantly reduced the chemotactic migration of rMSCs toward HGF, while inhibition of endogenous miR-124 promoted the chemotactic migration. A further study revealed that miR-124 directly targeted FZD4 and LRP6, which encode a receptor and co-receptor of the Wnt/β-catenin signaling pathway, respectively, thus reducing the activity of this signaling. Consistently, activation of the Wnt/β-catenin signaling pathway by LiCl and ΔN89β-catenin rescued the inhibitory effect of miR-124 on the chemotactic migration of rMSCs toward HGF, while inhibition of Wnt/β-catenin signaling by FH535 abrogated the enhanced chemotactic response achieved by the miR-124 inhibitor. Collectively, our study demonstrates that miR-124 downregulates Wnt/β-catenin signaling via targeting FZD4 and LRP6 and thus suppresses the chemotactic migration of rMSCs toward HGF.

  3. Peptide linkers for the assembly of semiconductor quantum dot bioconjugates

    Science.gov (United States)

    Boeneman, Kelly; Mei, Bing C.; Deschamps, Jeffrey R.; Delehanty, James B.; Mattoussi, Hedi; Medintz, Igor

    2009-02-01

    The use of semiconductor luminescent quantum dots for the labeling of biomolecules is rapidly expanding, however it still requires facile methods to attach functional globular proteins to biologically optimized quantum dots. Here we discuss the development of controlled variable length peptidyl linkers to attach biomolecules to poly(ethylene) glycol (PEG) coated quantum dots for both in vitro and in vivo applications. The peptides chosen, β-sheets and alpha helices are appended to polyhistidine sequences and this allows for control of the ratio of peptide bioconjugated to QD and the distance from QD to the biomolecule. Recombinant DNA engineering, bacterial peptide expression and Ni-NTA purification of histidine labeled peptides are utilized to create the linkers. Peptide length is confirmed by in vitro fluorescent resonance energy transfer (FRET).

  4. Truncated glucagon-like peptide-1 (proglucagon 78-107 amide), an intestinal insulin-releasing peptide, has specific receptors on rat insulinoma cells (RIN 5AH)

    DEFF Research Database (Denmark)

    Orskov, C; Nielsen, Jens Høiriis

    1988-01-01

    We studied binding of 125I-labelled truncated-glucagon-like peptide-1 (proglucagon 78-107 amide) to a cloned rat insulin-producing cell line, RIN 5AH, in monolayer culture. Interaction of the peptide with pancreatic insulinoma cells was saturable and time dependent. Half-maximal binding was obtai......We studied binding of 125I-labelled truncated-glucagon-like peptide-1 (proglucagon 78-107 amide) to a cloned rat insulin-producing cell line, RIN 5AH, in monolayer culture. Interaction of the peptide with pancreatic insulinoma cells was saturable and time dependent. Half-maximal binding...

  5. Quantitative modeling of Escherichia coli chemotactic motion in environments varying in space and time.

    Directory of Open Access Journals (Sweden)

    Lili Jiang

    2010-04-01

    Full Text Available Escherichia coli chemotactic motion in spatiotemporally varying environments is studied by using a computational model based on a coarse-grained description of the intracellular signaling pathway dynamics. We find that the cell's chemotaxis drift velocity v(d is a constant in an exponential attractant concentration gradient [L] proportional, variantexp(Gx. v(d depends linearly on the exponential gradient G before it saturates when G is larger than a critical value G(C. We find that G(C is determined by the intracellular adaptation rate k(R with a simple scaling law: G(C infinity k(1/2(R. The linear dependence of v(d on G = d(ln[L]/dx directly demonstrates E. coli's ability in sensing the derivative of the logarithmic attractant concentration. The existence of the limiting gradient G(C and its scaling with k(R are explained by the underlying intracellular adaptation dynamics and the flagellar motor response characteristics. For individual cells, we find that the overall average run length in an exponential gradient is longer than that in a homogeneous environment, which is caused by the constant kinase activity shift (decrease. The forward runs (up the gradient are longer than the backward runs, as expected; and depending on the exact gradient, the (shorter backward runs can be comparable to runs in a spatially homogeneous environment, consistent with previous experiments. In (spatial ligand gradients that also vary in time, the chemotaxis motion is damped as the frequency omega of the time-varying spatial gradient becomes faster than a critical value omega(c, which is controlled by the cell's chemotaxis adaptation rate k(R. Finally, our model, with no adjustable parameters, agrees quantitatively with the classical capillary assay experiments where the attractant concentration changes both in space and time. Our model can thus be used to study E. coli chemotaxis behavior in arbitrary spatiotemporally varying environments. Further experiments are

  6. Probing Site-Specific Structural Information of Peptides at Model Membrane Interface In Situ.

    Science.gov (United States)

    Ding, Bei; Panahi, Afra; Ho, Jia-Jung; Laaser, Jennifer E; Brooks, Charles L; Zanni, Martin T; Chen, Zhan

    2015-08-19

    Isotope labeling is a powerful technique to probe detailed structures of biological molecules with a variety of analytical methods such as NMR and vibrational spectroscopies. It is important to obtain molecular structural information on biological molecules at interfaces such as cell membranes, but it is challenging to use the isotope labeling method to study interfacial biomolecules. Here, by individually (13)C═(16)O labeling ten residues of a peptide, Ovispirin-1, we have demonstrated for the first time that a site-specific environment of membrane associated peptide can be probed by the submonolayer surface sensitive sum frequency generation (SFG) vibrational spectroscopy in situ. With the peptide associated with a single lipid bilayer, the sinusoidal trend of the SFG line width and peak-center frequency suggests that the peptide is located at the interface beneath the lipid headgroup region. The constructive interferences between the isotope labeled peaks and the main peptide amide I peak contributed by the unlabeled components were used to determine the membrane orientation of the peptide. From the SFG spectral peak-center frequency, line width, and polarization dependence of the isotope labeled units, we deduced structural information on individual units of the peptide associated with a model cell membrane. We also performed molecular dynamics (MD) simulations to understand peptide-membrane interactions. The physical pictures described by simulation agree well with the SFG experimental result. This research demonstrates the feasibility and power of using isotope labeling SFG to probe molecular structures of interfacial biological molecules in situ in real time.

  7. Simulation of self-propelled chemotactic bacteria in a stokes flow*

    Directory of Open Access Journals (Sweden)

    Maury B.

    2010-12-01

    Full Text Available We prescrit a method to simulate the motion of self-propelled rigid particles in a twodimensional Stokesian fluid, taking into account chemotactic behaviour. Self-propulsion is modelled as a point force associated to each particle, placed at a certain distance from its gravity centre. The method for solving the fluid flow and the motion of the bacteria is based on a variational formulation on the whole domain, including fluid and particles: rigid motion is enforced by penalizing the strain rate tensor on the rigid domain, while incompressibility is treated by duality. This leads to a minimisation problem over unconstrained functional spaces which cari lie easily implemented from any finite element Stokes solver. In order to ensure robustness, a projection algorithm is used to deal with contacts between particles. The particles are meant to represent bacteria of the Escherichia coli type, which interact with their chemical environment through consumption of nutrients and orientation in some favorable direction. Our mode’ takes into account the interaction with oxygen. An advection-diffusion equation on the oxygen concentration is solved in the fluid domain, with a source term accounting for oxygen consumption by the bacteria. In addition, self-propulsion is deactivated for those particles which cannot consume enough oxygen. Finally, the mode’ includes random changes in the orientation of the individual bacteria, with a frequency that depends on the surrounding oxygen concentration, in order to favor the direction of the concentration gradient and thus to reproduce chemotactic behaviour. Numerical simulations implemented with FreeFem++ are presented. Nous présentons une méthode de simulation du mouvement de particules rigides autopropulsées dans un fluide de Stokes en dimension 2. en prenant en compte leur comportement chimiotactique. L’auto-propulsion est modélisée par une force (presque ponctuelle associée à chaque particule et plac

  8. Semiotic labelled deductive systems

    Energy Technology Data Exchange (ETDEWEB)

    Nossum, R.T. [Imperial College of Science, Technology and Medicine, London (United Kingdom)

    1996-12-31

    We review the class of Semiotic Models put forward by Pospelov, as well as the Labelled Deductive Systems developed by Gabbay, and construct an embedding of Semiotic Models into Labelled Deductive Systems.

  9. Mental Labels and Tattoos

    Science.gov (United States)

    Hyatt, I. Ralph

    1977-01-01

    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  10. Pesticide Product Label System

    Data.gov (United States)

    U.S. Environmental Protection Agency — The Pesticide Product Label System (PPLS) provides a collection of pesticide product labels (Adobe PDF format) that have been approved by EPA under Section 3 of the...

  11. In vitro inhibitory effects of Moringa oleifera leaf extract and its major components on chemiluminescence and chemotactic activity of phagocytes.

    Science.gov (United States)

    Vongsak, Boonyadist; Gritsanapan, Wandee; Wongkrajang, Yuvadee; Jantan, Ibrahim

    2013-11-01

    The ethanol extract of Moringa oleifera Lam. leaves and its major constituents, crypto-chlorogenic acid, quercetin 3-O-glucoside and kaempferol 3-O-glucoside, were investigated on the respiratory burst of human whole blood and isolated human polymorphonuclear leukocytes (PMNs) using a luminol-based chemiluminescence assay. The chemotactic migration of PMNs was also investigated using the Boyden chamber technique. The ethanol extract demonstrated inhibitory activities on the oxidative burst and the chemotactic migration of PMNs. Quercetin 3-O-glucoside, crypto-chlorogenic acid, and kaempferol 3-O-glucoside, isolated from the extract, expressed relatively strong inhibitory activity on the oxidative burst of PMNs with IC50 values of 4.1, 6.7 and 7.0 microM, respectively, comparable with that of aspirin. They also demonstrated strong inhibition of chemotatic migration of PMNs with IC50 values of 9.5, 15.9 and 18.2 microM, respectively. The results suggest that M. oleifera leaves could modulate the immune response of human phagocytes, linking to its ethnopharmacological use as an anti-inflammatory agent. The immunomodulating activity of the plant was mainly due to its major components.

  12. Multifunctional Prenylated Peptides for Live Cell Analysis

    Science.gov (United States)

    Wollack, James W.; Zeliadt, Nicholette A.; Mullen, Daniel G.; Amundson, Gregg; Geier, Suzanne; Falkum, Stacy; Wattenberg, Elizabeth V.; Barany, George; Distefano, Mark D.

    2009-01-01

    Protein prenylation is a common post-translational modification present in eukaryotic cells. Many key proteins involved in signal transduction pathways are prenylated and inhibition of prenylation can be useful as a therapeutic intervention. While significant progress has been made in understanding protein prenylation in vitro, we have been interested in studying this process in living cells, including the question of where prenylated molecules localize. Here, we describe the synthesis and live cell analysis of a series of fluorescently labeled multifunctional peptides, based on the C-terminus of the naturally prenylated protein CDC42. A synthetic route was developed that features a key Acm to Scm protecting group conversion. This strategy was compatible with acid-sensitive isoprenoid moieties, and allowed incorporation of an appropriate fluorophore as well as a cell-penetrating sequence (penetratin). These peptides are able to enter cells through different mechanisms, depending on the presence or absence of the penetratin vehicle and the nature of the prenyl group attached. Interestingly, prenylated peptides lacking penetratin are able to enter cells freely through an energy-independent process, and localize in a perinuclear fashion. This effect extends to a prenylated peptide that includes a full “CAAX box” sequence (specifically, CVLL). Hence, these peptides open the door for studies of protein prenylation in living cells, including enzymatic processing and intracellular peptide trafficking. Moreover, the synthetic strategy developed here should be useful for the assembly of other types of peptides that contain acid sensitive functionalities. PMID:19425596

  13. Labeling Schemes with Queries

    OpenAIRE

    2006-01-01

    We study the question of ``how robust are the known lower bounds of labeling schemes when one increases the number of consulted labels''. Let $f$ be a function on pairs of vertices. An $f$-labeling scheme for a family of graphs $\\cF$ labels the vertices of all graphs in $\\cF$ such that for every graph $G\\in\\cF$ and every two vertices $u,v\\in G$, the value $f(u,v)$ can be inferred by merely inspecting the labels of $u$ and $v$. This paper introduces a natural generalization: the notion of $f$-...

  14. Absence of in vitro innate immunomodulation by insect-derived short proline-rich antimicrobial peptides points to direct antibacterial action in vivo.

    Science.gov (United States)

    Fritsche, Stefanie; Knappe, Daniel; Berthold, Nicole; von Buttlar, Heiner; Hoffmann, Ralf; Alber, Gottfried

    2012-10-01

    Some antimicrobial peptides (AMPs) have been described to exert immunomodulatory effects, which may contribute to their in vivo antibacterial activity. Very recently, we could show that novel oncocin and apidaecin derivatives are potently antibacterially active in vivo. Therefore, we studied oncocin and apidaecin derivatives for their effects on murine dendritic cells (DC) and macrophages and compared them with well-known immunomodulatory activities of murine cathelicidin-related antimicrobial peptide (CRAMP). To characterize the immunomodulatory activity of the peptides on key cells of the innate immune system, we stimulated murine DC and macrophages with the oncocin and apidaecin derivatives alone, or in combination with lipopolysaccharide (LPS). We analyzed the secretion of pro-inflammatory cytokines, the expression of surface activation markers, and the chemotactic activity of the AMPs. In contrast to LPS, none of the oncocin and apidaecin derivatives alone has an influence on cytokine or surface marker expression by DC and macrophages. Furthermore, the tested oncocin and apidaecin derivatives do not modulate the immune response after LPS stimulation, whereas CRAMP shows a reduction of the LPS-mediated immune response as expected. All peptides tested are not chemotactic for DC. Together, lack of in vitro immunomodulatory effects by oncocin and apidaecin derivatives on key cells of the innate murine immune system suggests that their potent in vivo antibacterial activity relies on a direct antibacterial effect. This will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections.

  15. A LIVER-TUMOR ADHESION PEPTIDE FROM RANDOM PHAGE DISPLAY LIBRARY

    Institute of Scientific and Technical Information of China (English)

    WU Miao; DU Bing; WANG Lei; ZHOU Zhong-liang; QIAN Min

    2006-01-01

    Objective: To identify and localize the synthesized targeting peptide A54 to liver cancer cell line BEL-7402 in vivo and in vitro for confirming the potential clinical application of peptide A54 in hepatocarcinoma targeting therapy. Methods: Phage A54 was confirmed by ELISA. Biotin and FAM labeled A54 peptides were identified and localized by means of immunohistochemistry and immunocytochemistry. Results: A54 peptide could target the liver-tumor tissue in vivo and adhere to several liver-tumor cells in vitro. FAM-labeled A54 peptides were localized on the membrane surface of liver-tumor cells. Conclusion: Synthesized A54 peptide obtained from in vivo phage display technology still kept special ability to adhere liver-tumor cell in vivo and in vitro. The A54 peptide could be a candidate carrier for hepatocarcinoma targeting therapy.

  16. Human peptide transporters

    DEFF Research Database (Denmark)

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

    2002-01-01

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

  17. Epithelial Antimicrobial Peptides: Guardian of the Oral Cavity

    Directory of Open Access Journals (Sweden)

    Mayank Hans

    2014-01-01

    Full Text Available Gingival epithelium provides first line of defence from the microorganisms present in dental plaque. It not only provides a mechanical barrier but also has an active immune function too. Gingival epithelial cells participate in innate immunity by producing a range of antimicrobial peptides to protect the host against oral pathogens. These epithelial antimicrobial peptides (EAPs include the β-defensin family, cathelicidin (LL-37, calprotectin, and adrenomedullin. While some are constitutively expressed in gingival epithelial cells, others are induced upon exposure to microbial insults. It is likely that these EAPs have a role in determining the initiation and progression of oral diseases. EAPs are broad spectrum antimicrobials with a different but overlapping range of activity. Apart from antimicrobial activity, they participate in several other crucial roles in host tissues. Some of these, for instance, β-defensins, are chemotactic to immune cells. Others, such as calprotectin are important for wound healing and cell proliferation. Adrenomedullin, a multifunctional peptide, has its biological action in a wide range of tissues. Not only is it a potent vasodilator but also it has several endocrine effects. Knowing in detail the various bioactions of these EAPs may provide us with useful information regarding their utility as therapeutic agents.

  18. Succesful labelling schemes

    DEFF Research Database (Denmark)

    Juhl, Hans Jørn; Stacey, Julia

    2001-01-01

    It is usual practice to evaluate the success of a labelling scheme by looking at the awareness percentage, but in many cases this is not sufficient. The awareness percentage gives no indication of which of the consumer segments that are aware of and use labelling schemes and which do not. In the ......It is usual practice to evaluate the success of a labelling scheme by looking at the awareness percentage, but in many cases this is not sufficient. The awareness percentage gives no indication of which of the consumer segments that are aware of and use labelling schemes and which do not....... In the spring of 2001 MAPP carried out an extensive consumer study with special emphasis on the Nordic environmentally friendly label 'the swan'. The purpose was to find out how much consumers actually know and use various labelling schemes. 869 households were contacted and asked to fill in a questionnaire...... it into consideration when I go shopping. The respondent was asked to pick the most suitable answer, which described her use of each label. 29% - also called 'the labelling blind' - responded that they basically only knew the recycling label and the Government controlled organic label 'Ø-mærket'. Another segment of 6...

  19. Beta-adrenergic receptor agonists induce the release of granulocyte chemotactic protein-2, oncostatin M, and vascular endothelial growth factor from macrophages

    NARCIS (Netherlands)

    Verhoeckx, K.C.M.; Doornbos, R.P.; Witkamp, R.F.; Greef, de J.; Rodenburg, R.J.T.

    2006-01-01

    Vascular endothelial growth factor (VEGF), oncostatin M (OSM), and granulocyte chemotactic protein-2 (GCP-2/CXCL6) are up-regulated in U937 macrophages and peripheral blood macrophages exposed to LPS, beta-adrenergic receptor (ß2-AR) agonists (e.g. zilpaterol, and clenbuterol) and some other agents

  20. Beta-adrenergic receptor agonists induce the release of granulocyte chemotactic protein-2, oncostatin M, and vascular endothelial growth factor from macrophages

    NARCIS (Netherlands)

    Verhoeckx, K.C.; Doornbos, R.P.; Witkamp, R.F.; Greef, J. van der; Rodenburg, R.J.T.

    2006-01-01

    Vascular endothelial growth factor (VEGF), oncostatin M (OSM), and granulocyte chemotactic protein-2 (GCP-2/CXCL6) are up-regulated in U937 macrophages and peripheral blood macrophages exposed to LPS, beta-adrenergic receptor (beta2-AR) agonists (e.g. zilpaterol, and clenbuterol) and some other agen

  1. Beta-adrenergic receptor agonists induce the release of granulocyte chemotactic protein-2, oncostatin M, and vascular endothelial growth factor from macrophages

    NARCIS (Netherlands)

    Verhoeckx, K.C.M.; Doornbos, R.P.; Witkamp, R.F.; Greef, J. van der; Rodenburg, R.J.T.

    2006-01-01

    Vascular endothelial growth factor (VEGF), oncostatin M (OSM), and granulocyte chemotactic protein-2 (GCP-2/CXCL6) are up-regulated in U937 macrophages and peripheral blood macrophages exposed to LPS, beta-adrenergic receptor (β2-AR) agonists (e.g. zilpaterol, and clenbuterol) and some other agents

  2. Monocyte chemotactic protein-1 attenuates and high-fat diet exacerbates bone loss in mice with pulmonary metastasis of Lewis lung carcinoma

    Science.gov (United States)

    Bone can be adversely affected by obesity and cancer-associated complications including wasting. The objective of this study was to determine whether a high-fat diet and a deficiency in monocyte chemotactic protein-1 (MCP-1) altered bone structural defects found in male C57BL/6 mice with Lewis lung...

  3. Protein labelling with stable isotopes: strategies; Le marquage des proteines aux isotopes stables: strategies

    Energy Technology Data Exchange (ETDEWEB)

    Lirsac, P.N.; Gilles, N.; Jamin, N.; Toma, F.; Gabrielsen, O.; Boulain, J.C.; Menez, A. [CEA Centre d`Etudes de Saclay, 91 - Gif-sur-Yvette (France). Direction des Sciences du Vivant

    1994-12-31

    A protein labelling technique with stable isotopes has been developed at the CEA: a labelled complete medium has been developed, performing as well as the Luria medium, but differing from it because it contains not only free aminated acids and peptides, but also sugars (96% of D-glucopyrannose) and labelled nucleosides. These precursors are produced from a labelled photosynthetic micro-organisms biomass, obtained with micro-algae having incorporated carbon 13, nitrogen 15 and deuterium during their culture. Labelling costs are reduced. 1 fig., 1 tab., 3 refs.

  4. Migration of Chemotactic Bacteria Transverse to Flow in Response to a Benzoate Source Plume Created in a Saturated Sand-Packed Microcosm

    Science.gov (United States)

    Ford, R.; Boser, B.

    2012-12-01

    Bioremediation processes depend on contact between microbial populations and the groundwater contaminants that they biodegrade. Chemotaxis, the ability of bacteria to sense a chemical gradient and swim preferentially toward locations of higher concentration, can enhance the transport of bacteria toward contaminant sources that may not be readily accessible by advection and dispersion alone. A two-dimensional rectangular-shaped microcosm packed with quartz sand was used to quantify the effect of chemotaxis on the migration of bacteria within a saturated model aquifer system. Artificial groundwater was pumped through the microcosm at a rate of approximately 1 m/day. A plume of sodium benzoate was created by continuous injection into an upper port of the microcosm to generate a chemical gradient in the vertical direction transverse to flow. Chemotactic bacteria, Pseudomonas putida F1, or the nonchemotactic mutant, P. putida F1 CheA, were injected with a conservative tracer in a port several centimeters below the benzoate position. As the injectates traversed the one-meter length of the microcosm, samples were collected from a dozen effluent ports to determine vertical concentration distributions for the bacteria, benzoate and tracer. A moment analysis was implemented to estimate the center of mass, variance, and skewness of the concentration profiles. The transverse dispersion coefficient and the transverse dispersivity for chemotactic and nonchemotactic bacteria were also evaluated. Experiments performed with a continuous injection of bacteria showed that the center of mass for chemotactic bacteria was closer to the benzoate source on average than the nonchemotactic control (relative to the conservative tracer). These results demonstrated that chemotaxis can increase bacterial transport toward contaminants, potentially enhancing the effectiveness of in situ bioremediation. Experiments with 2 cm and 3 cm spacing between bacteria and benzoate injection locations were

  5. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco and Its Active Site for Chemotaxis

    Directory of Open Access Journals (Sweden)

    Farman Ullah Dawar

    2016-08-01

    Full Text Available Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA, a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS. The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics.

  6. Plasma Levels of Monocyte Chemotactic Protein 3 and Beta-Nerve Growth Factor Increase with Amnestic Mild Cognitive Impairment

    Institute of Scientific and Technical Information of China (English)

    Kang Soo Lee; Ji Hyung Chung; Kyung Hye Lee; Min-Jeong Shin; Byoung Hoon Oh; Soo Hyung Lee; Chang Hyung Hong

    2009-01-01

    A number of studies have investigated peripheral inflammatory indices, including plasma cytokines and related molecules according to subtypes of dementia, but not in mild cognitive impairment (MCI). In this study, we used multiplex cytokine assay to assess the plasma levels of 22 cytokines in patients with MCI subtyped as amnestic and non-amnestic, according to cognitive features. When comparing the levels of plasma growth factors, chemokines and cytokines, plasma levels of monocyte chemotactic protein 3 (MCP-3), and beta-nerve growth factor (β-NGF) in these two groups, they were found to be significantly higher in amnestic MCI patients than in non-amnestic MCI patients, after adjusting for age and gender. This suggests that plasma MCP-3 and β-NGF may be useful in differentiating subtypes of MCI. Cellular & Molecular Immunology.

  7. Multiscale dynamics of biological cells with chemotactic interactions: from a discrete stochastic model to a continuous description

    CERN Document Server

    Alber, M; Glimm, T; Lushnikov, P M; Alber, Mark; Chen, Nan; Glimm, Tilmann; Lushnikov, Pavel M.

    2006-01-01

    The Cellular Potts Model (CPM) has been used for simulating various biological phenomena such as differential adhesion, fruiting body formation of the slime mold Dictyostelium discoideum, angiogenesis, cancer invasion, chondrogenesis in embryonic vertebrate limbs, and many others. In this paper, we derive continuous limit of discrete one dimensional CPM with the chemotactic interactions between cells in the form of a Fokker-Planck equation for the evolution of the cell probability density function. This equation is then reduced to the classical macroscopic Keller-Segel model. In particular, all coefficients of the Keller-Segel model are obtained from parameters of the CPM. Theoretical results are verified numerically by comparing Monte Carlo simulations for the CPM with numerics for the Keller-Segel model.

  8. Crosstalk between medulloblastoma cells and endothelium triggers a strong chemotactic signal recruiting T lymphocytes to the tumor microenvironment.

    Directory of Open Access Journals (Sweden)

    Vita S Salsman

    Full Text Available Cancer cells can live and grow if they succeed in creating a favorable niche that often includes elements from the immune system. While T lymphocytes play an important role in the host response to tumor growth, the mechanism of their trafficking to the tumor remains poorly understood. We show here that T lymphocytes consistently infiltrate the primary brain cancer, medulloblastoma. We demonstrate, both in vitro and in vivo, that these T lymphocytes are attracted to tumor deposits only after the tumor cells have interacted with tumor vascular endothelium. Macrophage Migration Inhibitory Factor (MIF" is the key chemokine molecule secreted by tumor cells which induces the tumor vascular endothelial cells to secrete the potent T lymphocyte attractant "Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES." This in turn creates a chemotactic gradient for RANTES-receptor bearing T lymphocytes. Manipulation of this pathway could have important therapeutic implications.

  9. SITE-SPECIFIC LABELING OF A PROTEIN LYSINE RESIDUE BY NOVEL KINETIC LABELING COMBINATORIAL LIBRARIES

    Directory of Open Access Journals (Sweden)

    Allen Krantz

    2014-03-01

    Full Text Available The first example of a kinetic labeling library designed to enable the discovery of affinity labels is presented. Each library component (1 consists of a variable peptidyl component linked to a biotinyl moiety by a 4-mercaptobenzoyl linker in thioester format. We demonstrate that an affinity label can be uncovered by measuring reaction rates between library pools and the protein target, human serum albumin (HSA and identifying significant outliers. By choosing peptide functionality compatible with a potentially reactive thioester labeling entity, libraries can be screened in pools. It is noteworthy that a limited subset of amino acids (R, S, E, F, Y, l, M, W, and Q that compose the affinity moiety is sufficient to produce rate variances that guide the discovery process. After two rounds of deconvolution, J-FLYEE-NH2 (7-E emerges as a bona fide affinity label of HSA. Unlike known affinity labels, the affinity moiety is not retained in the protein product, but is extruded upon acylation of the protein. This feature affords a method of introducing various payloads, without extraneous elements, onto protein frameworks.

  10. 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.

  11. Imaging tumors with peptide-based radioligands

    Energy Technology Data Exchange (ETDEWEB)

    Behr, T. M.; Gotthardt, M.; Barth, A.; Behe, M. [Philipps-University of Marburg, Dept. of Nuclear Medicine, Marburg (Germany)

    2001-06-01

    Regulatory peptides are small, readily diffusable and potent natural substances with a wide spectrum of receptor-mediated actions in humans. High affinity receptors for these peptides are (over)-expressed in many neoplasms, and these receptors may represent, therefore, new molecular targets for cancer diagnosis and therapy. This review aims to give an overview of the peptide-based radiopharmaceuticals which are presently already commercially available or which are in advanced stages of their clinical testing so that their broader availability is anticipated soon. Physiologically, these peptides bind to and act through G protein-coupled receptors in the cell membrane. Historically, somatostatin analogs are the first class of receptor binding peptides having gained clinical application. In {sup 111}In-DTPA-(D-Phe{sup 1})-octreotide is the first and only radio peptide which has obtained regulatory approval in Europe and the United States to date. Extensive clinical studies involving several thousands of patients have shown that the major clinical application of somatostatin receptor scintigraphy is the detection and the staging of gastroenteropancreatic neuroendocrine tumors (carcinoids). In these tumors, octreotide scintigraphy is superior to any other staging method. However, its sensitivity and accuracy in other, more frequent neoplasms is limited. Radiolabeled vasoactive intestinal peptide (VIP) has been shown to visualize the majority of gastrointestinal adenocarcinomas, as well as some neuroendocrine tumors, including insulinomas (the latter being often missed by somatostatin receptor scintigraphy). Due to the outstanding diagnostic accuracy of the pentagastrin test in detecting the presence, persistence, or recurrence of medullary thyroid cancer (MTC), it was postulated the expression of the corresponding (i.e. cholecystokinin (CCK-)-B) receptor type in human MTC. This receptor is also widely expressed on human small-cell lung. Indeed, {sup 111}In-labeled DTPA

  12. Automated selected reaction monitoring software for accurate label-free protein quantification.

    Science.gov (United States)

    Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

    2012-07-06

    Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

  13. PeptideAtlas

    Data.gov (United States)

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

  14. A common precursor to two major crab neurosecretory peptides.

    Science.gov (United States)

    Stuenkel, E L

    1986-01-01

    The biosynthesis of proteins by the X-organ sinus gland (XOSG) neurosecretory system of the crab, Cardisoma carnifex was studied using the pulse-chase technique. Analysis of radioactive proteins following 2D-PAGE showed that during pulse incubations of less than or equal to 30 min a single predominant 14Kd prohormone was synthesized. With chase less than or equal to 3 hr the primary 14Kd protein was found to undergo differential and/or multiple post-translational modifications prior to its proteolytic cleavage. Increasing the chase to greater than 3 hr showed a shift in labeling from the 14Kd forms to 3 separate 6Kd proteins. Two of the 6Kd proteins were identified as crustacean hyperglycemic peptides (CHH). Similarity in protein labeling using [3H]leucine and [35S]cysteine suggest a second major peptide group, the H peptide, known to lack cysteine, is also contained within the 14Kd precursor. Peptide mapping of the 14Kd proteins and of unlabeled CHH and peptide H provide substantive evidence for this biosynthetic scheme. Thus, both the CHH and H peptide groups, which together constitute greater than 90% of the XOSG peptide content, in this species, arise from a common 14Kd precursor molecule.

  15. Advanced approaches for the characterization of a de novo designed antiparallel coiled coil peptide

    NARCIS (Netherlands)

    Pagel, K; Seeger, K; Seiwert, B; Villa, Alessandra; Mark, AE; Berger, S; Koksch, B

    2005-01-01

    We report here an advanced approach for the characterization of the folding pattern of a de novo designed antiparallel coiled coil peptide by high-resolution methods. Incorporation of two fluorescence labels at the C- and N-terminus of the peptide chain as well as modi. cation of two hydrophobic cor

  16. APD3: the antimicrobial peptide database as a tool for research and education.

    Science.gov (United States)

    Wang, Guangshun; Li, Xia; Wang, Zhe

    2016-01-04

    The antimicrobial peptide database (APD, http://aps.unmc.edu/AP/) is an original database initially online in 2003. The APD2 (2009 version) has been regularly updated and further expanded into the APD3. This database currently focuses on natural antimicrobial peptides (AMPs) with defined sequence and activity. It includes a total of 2619 AMPs with 261 bacteriocins from bacteria, 4 AMPs from archaea, 7 from protists, 13 from fungi, 321 from plants and 1972 animal host defense peptides. The APD3 contains 2169 antibacterial, 172 antiviral, 105 anti-HIV, 959 antifungal, 80 antiparasitic and 185 anticancer peptides. Newly annotated are AMPs with antibiofilm, antimalarial, anti-protist, insecticidal, spermicidal, chemotactic, wound healing, antioxidant and protease inhibiting properties. We also describe other searchable annotations, including target pathogens, molecule-binding partners, post-translational modifications and animal models. Amino acid profiles or signatures of natural AMPs are important for peptide classification, prediction and design. Finally, we summarize various database applications in research and education.

  17. Antimicrobial Peptides in 2014

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2015-03-01

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

  18. Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    2002-01-01

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

  19. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1998-01-01

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

  20. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2003-01-01

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

  1. Peptide Nucleic Acid Synthons

    DEFF Research Database (Denmark)

    2004-01-01

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

  2. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

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

  3. PH dependent adhesive peptides

    Science.gov (United States)

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

    2010-06-29

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

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

    Directory of Open Access Journals (Sweden)

    E. S. Umnyakova

    2016-01-01

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

  5. From Label to Practice

    DEFF Research Database (Denmark)

    Byrkjeflot, Haldor; Strandgaard, Jesper; Svejenova, Silviya

    2013-01-01

    This article examines the process of creation of new Nordic cuisine (NNC) as a culinary innovation, focusing on the main stages, actors, and mechanisms that shaped the new label and its practices and facilitated its diffusion in the region and internationally. Fast-paced diffusion was possible...... because NNC was conceived as an identity movement, triggered by active involvement of entrepreneurial leaders from the culinary profession, high-profile political supporters, legitimating scientists, disseminating media, and interpreting audiences. It was facilitated by three mechanisms: First, the use...... actors and institutions to develop practices associated with the NNC label. Third, organized dissemination allowed the excitement and engagement with the new label to spread quickly....

  6. FDA Online Label Repository

    Data.gov (United States)

    U.S. Department of Health & Human Services — The drug labels and other drug-specific information on this Web site represent the most recent drug listing information companies have submitted to the Food and Drug...

  7. Behind the Label "Alcoholic."

    Science.gov (United States)

    Wright, Deborah M.

    1989-01-01

    Relates individual's personal story of her childhood influenced by her parent's alcoholism, her own alcoholism as a young adult, and her experiences with counseling. Asks others not to reject her because of the label "alcoholic." (ABL)

  8. Vasoactive intestinal peptide signaling axis in human leukemia

    Institute of Scientific and Technical Information of China (English)

    Glenn; Paul; Dorsam; Keith; Benton; Jarrett; Failing; Sandeep; Batra

    2011-01-01

    The vasoactive intestinal peptide (VIP) signaling axis constitutes a master "communication coordinator" between cells of the nervous and immune systems.To date,VIP and its two main receptors expressed in T lymphocytes,vasoactive intestinal peptide receptor (VPAC)1 and VPAC2,mediate critical cellular functions regulating adaptive immunity,including arresting CD4 T cells in G 1 of the cell cycle,protection from apoptosis and a potent chemotactic recruiter of T cells to the mucosa associated lymphoid compartment of the gastrointestinal tissues.Since the discovery of VIP in 1970,followed by the cloning of VPAC1 and VPAC2 in the early 1990s,this signaling axis has been associated with common human cancers,including leukemia.This review highlights the present day knowledge of the VIP ligand and its receptor expression profile in T cell leukemia and cell lines.Also,there will be a discussion describing how the anti-leukemic DNA binding transcription factor,Ikaros,regulates VIP receptor expression in primary human CD4 T lymphocytes and T cell lymphoblastic cell lines (e.g.Hut-78).Lastly,future goals will be mentioned that are expected to uncover the role of how the VIP signaling axis contributes to human leukemogenesis,and to establish whether the VIP receptor signature expressed by leukemic blasts can provide therapeutic and/or diagnostic information.

  9. Analysis of illegal peptide biopharmaceuticals frequently encountered by controlling agencies.

    Science.gov (United States)

    Vanhee, Celine; Janvier, Steven; Desmedt, Bart; Moens, Goedele; Deconinck, Eric; De Beer, Jacques O; Courselle, Patricia

    2015-09-01

    derivatization or the use of expensive labelled peptides. This quantification method was successfully validated for a representative subset of 10 different peptides by using the "total error" approach in accordance with the validation requirements of ISO-17025.

  10. Epithelial transport of immunogenic and toxic gliadin peptides in vitro.

    Directory of Open Access Journals (Sweden)

    Christian Zimmermann

    Full Text Available Celiac disease is an autoimmune disorder caused by failure of oral tolerance against gluten in genetically predisposed individuals. The epithelial translocation of gluten-derived gliadin peptides is an important pathogenetic step; the underlying mechanisms, however, are poorly understood. Thus, we investigated the degradation and epithelial translocation of two different gliadin peptides, the toxic P31-43 and the immunogenic P56-68. As the size, and hence, the molecular weight of peptides might have an effect on the transport efficiency we chose two peptides of the same, rather short chain length.Fluorescence labeled P31-43 and P56-68 were synthesized and studied in a transwell system with human enterocytes. Fluorometric measurements were done to reveal antigen translocation and flow cytometry as well as confocal microscopy were used to investigate cellular uptake of peptides. Structural changes of these peptides were analysed by MALDI-TOF-MS. According to fluorescence intensities, significantly more P31-43 compared to P56-68 was transported through the enterocyte layer after 24 h incubation. In contrast to previous reports, however, mass spectrometric data do not only show a time-dependent cleavage of the immunogenic P56-68, but we observed for the first time the degradation of the toxic peptide P31-43 at the apical side of epithelial cells.Considering the degradation of gliadin peptides by enterocytes, measurement of fluorescence signals do not completely represent translocated intact gliadin peptides. From our experiments it is obvious that even short peptides can be digested prior to the translocation across the epithelial barrier. Thus, the chain length and the sensibility to degradations of gliadin peptides as well as the integrity of the epithelial barrier seem to be critical for the uptake of gliadin peptides and the subsequent inflammatory immune response.

  11. Chemotactic response with a constant delay-time mechanism in Ciona spermatozoa revealed by a high time resolution analysis of flagellar motility

    Directory of Open Access Journals (Sweden)

    Daisuke Miyashiro

    2015-01-01

    Full Text Available During their chemotactic swimming toward eggs, sperm cells detect their species-specific chemoattractant and sense concentration gradients by unknown mechanisms. After sensing the attractant, sperm cells commonly demonstrate a series of responses involving different swimming patterns by changing flagellar beats, gradually approaching a swimming path toward the eggs, which is the source of chemoattractants. Shiba et al. observed a rapid increase in intracellular Ca2+ concentrations in Ciona spermatozoa after sensing chemoattractants; however, the biochemical processes occurring inside the sperm cells are unclear. In the present study, we focused on the timing and sensing mechanism of chemical signal detection in Ciona. One of the most crucial problems to be solved is defining the initial epoch of chemotactic responses. We adopted a high rate of video recording (600 Hz for detailed analysis of sperm motion and a novel method for detecting subtle signs of beat forms and moving paths of sperm heads. From these analyses, we estimated a virtual sensing point of the attractant before initiation of motility responses and found that the time delay from sensing to motility responses was almost constant. To evaluate the efficiency of this constant delay model, we performed computer simulation of chemotactic behaviors of Ciona spermatozoa.

  12. Chemotactic response with a constant delay-time mechanism in Ciona spermatozoa revealed by a high time resolution analysis of flagellar motility

    Science.gov (United States)

    Miyashiro, Daisuke; Shiba, Kogiku; Miyashita, Tahahiro; Baba, Shoji A.; Yoshida, Manabu; Kamimura, Shinji

    2015-01-01

    ABSTRACT During their chemotactic swimming toward eggs, sperm cells detect their species-specific chemoattractant and sense concentration gradients by unknown mechanisms. After sensing the attractant, sperm cells commonly demonstrate a series of responses involving different swimming patterns by changing flagellar beats, gradually approaching a swimming path toward the eggs, which is the source of chemoattractants. Shiba et al. observed a rapid increase in intracellular Ca2+ concentrations in Ciona spermatozoa after sensing chemoattractants; however, the biochemical processes occurring inside the sperm cells are unclear. In the present study, we focused on the timing and sensing mechanism of chemical signal detection in Ciona. One of the most crucial problems to be solved is defining the initial epoch of chemotactic responses. We adopted a high rate of video recording (600 Hz) for detailed analysis of sperm motion and a novel method for detecting subtle signs of beat forms and moving paths of sperm heads. From these analyses, we estimated a virtual sensing point of the attractant before initiation of motility responses and found that the time delay from sensing to motility responses was almost constant. To evaluate the efficiency of this constant delay model, we performed computer simulation of chemotactic behaviors of Ciona spermatozoa. PMID:25572419

  13. Chemotactic response with a constant delay-time mechanism in Ciona spermatozoa revealed by a high time resolution analysis of flagellar motility.

    Science.gov (United States)

    Miyashiro, Daisuke; Shiba, Kogiku; Miyashita, Tahahiro; Baba, Shoji A; Yoshida, Manabu; Kamimura, Shinji

    2015-01-08

    During their chemotactic swimming toward eggs, sperm cells detect their species-specific chemoattractant and sense concentration gradients by unknown mechanisms. After sensing the attractant, sperm cells commonly demonstrate a series of responses involving different swimming patterns by changing flagellar beats, gradually approaching a swimming path toward the eggs, which is the source of chemoattractants. Shiba et al. observed a rapid increase in intracellular Ca(2+) concentrations in Ciona spermatozoa after sensing chemoattractants; however, the biochemical processes occurring inside the sperm cells are unclear. In the present study, we focused on the timing and sensing mechanism of chemical signal detection in Ciona. One of the most crucial problems to be solved is defining the initial epoch of chemotactic responses. We adopted a high rate of video recording (600 Hz) for detailed analysis of sperm motion and a novel method for detecting subtle signs of beat forms and moving paths of sperm heads. From these analyses, we estimated a virtual sensing point of the attractant before initiation of motility responses and found that the time delay from sensing to motility responses was almost constant. To evaluate the efficiency of this constant delay model, we performed computer simulation of chemotactic behaviors of Ciona spermatozoa.

  14. Stromal-derived factor-1α/CXCL12-CXCR4 chemotactic pathway promotes perineural invasion in pancreatic cancer.

    Science.gov (United States)

    Xu, Qinhong; Wang, Zheng; Chen, Xin; Duan, Wanxing; Lei, Jianjun; Zong, Liang; Li, Xuqi; Sheng, Liang; Ma, Jiguang; Han, Liang; Li, Wei; Zhang, Lun; Guo, Kun; Ma, Zhenhua; Wu, Zheng; Wu, Erxi; Ma, Qingyong

    2015-03-10

    Perineural invasion (PNI) is considered as an alternative route for the metastatic spread of pancreatic cancer cells; however, the molecular changes leading to PNI are still poorly understood. In this study, we show that the CXCL12/CXCR4 axis plays a pivotal role in the neurotropism of pancreatic cancer cells to local peripheral nerves. Immunohistochemical staining results revealed that CXCR4 elevation correlated with PNI in 78 pancreatic cancer samples. Both in vitro and in vivo PNI models were applied to investigate the function of the CXCL12/CXCR4 signaling in PNI progression and pathogenesis. The results showed that the activation of the CXCL12/CXCR4 axis significantly increased pancreatic cancer cells invasion and promoted the outgrowth of the dorsal root ganglia. CXCL12 derived from the peripheral nerves stimulated the invasion and chemotactic migration of CXCR4-positive cancer cells in a paracrine manner, eventually leading to PNI. In vivo analyses revealed that the abrogation of the activated signaling inhibited tumor growth and invasion of the sciatic nerve toward the spinal cord. These data indicate that the CXCL12/CXCR4 axis may be a novel therapeutic target to prevent the perineural dissemination of pancreatic cancer.

  15. Inhibition of chemiluminescence and chemotactic activity of phagocytes in vitro by the extracts of selected medicinal plants.

    Science.gov (United States)

    Jantan, Ibrahim; Harun, Nurul Hikmah; Septama, Abdi Wira; Murad, Shahnaz; Mesaik, M A

    2011-04-01

    The methanol extracts of 20 selected medicinal plants were investigated for their effects on the respiratory burst of human whole blood, isolated human polymorphonuclear leukocytes (PMNs) and isolated mice macrophages using a luminol/lucigenin-based chemiluminescence assay. We also tested the effect of the extracts on chemotactic migration of PMNs using the Boyden chamber technique. The extracts of Curcuma domestica L., Phyllanthus amarus Schum & Thonn and C. xanthorrhiza Roxb. were the samples producing the strongest oxidative burst of PMNs with luminol-based chemiluminescence, with IC(50) values ranging from 0.5 to 0.7 μg/ml. For macrophage cells, the extracts which showed strong suppressive activity for luminol-based chemiluminescence were C. xanthorrhiza and Garcinia mangostana L. Among the extracts studied, C. mangga Valton & Vazsjip, Piper nigrum L. and Labisia pumila var. alata showed strong inhibitory activity on lucigenin-amplified oxidative burst of PMNs, with IC(50) values ranging from 0.9 to 1.5 μg/ml. The extracts of Zingiber officinale Rosc., Alpinia galangal (L.) Willd and Averrhoa bilimbi Linn showed strong inhibition on the chemotaxic migration of cells, with IC(50) values comparable to that of ibuprofen (1.5 μg/ml). The results suggest that some of these plants were able to modulate the innate immune response of phagocytes at different steps, emphasizing their potential as a source of new immunomodulatory agents.

  16. Aurantio-obtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells.

    Science.gov (United States)

    Vishnuprasad, Chethala N; Tsuchiya, Tomoko; Kanegasaki, Shiro; Kim, Joon Ho; Han, Sung Soo

    2014-05-01

    Osteoporosis is one of the major metabolic bone diseases and is among the most challenging noncommunicable diseases to treat. Although there is an increasing interest in identifying bioactive molecules for the prevention and management of osteoporosis, such studies principally focus only on differentiation and mineralization of osteoblasts or inhibition of osteoclast activity. Stimulation of osteoblast migration must be a promising osteoanabolic strategy for improved metabolic bone disease therapy. In this study, we show that an anthraquinone derivative, aurantio-obtusin, stimulated chemotactic migration of MC3T3-E1 osteoblast cells in a concentration-dependent manner. The use of a real-time chemotaxis analyzing system, TAXIScan, facilitated the evaluation of both velocity and directionality of osteoblast migration in response to the compound. Besides migration, the compound stimulated osteoblast differentiation and mineralization. Taken together, the data presented in this paper demonstrate that aurantio-obtusin is a promising osteoanabolic compound of natural origin with potential therapeutic applications in the prevention of osteoporosis and other metabolic bone diseases.

  17. Cross-strand coupling of a beta-hairpin peptide stabilized with an Aib-Gly turn studied using isotope-edited IR spectroscopy.

    Science.gov (United States)

    Huang, Rong; Setnicka, Vladimir; Etienne, Marcus A; Kim, Joohyun; Kubelka, Jan; Hammer, Robert P; Keiderling, Timothy A

    2007-11-01

    Isotope-edited IR spectroscopy was used to study a series of singly and doubly 13C=O-labeled beta-hairpin peptides stabilized by an Aib-Gly turn sequence. The double-labeled peptides have amide I' IR spectra that show different degrees of vibrational coupling between the 13C-labeled amides due to variations in the local geometry of the peptide structure. The single-labeled peptides provide controls to determine frequencies characteristic of the diagonal force field (FF) contributions at each position for the uncoupled 13C=O modes. Separation of diagonal FF and coupling effects on the spectra are used to explain the cross-strand labeled spectral patterns. DFT calculations based on an idealized model beta-hairpin peptide correctly predict the vibrational coupling patterns. Extending these model results by consideration of frayed ends and the hairpin conformational flexibility yields an alternate interpretation of details of the spectra. Temperature-dependent isotopically labeled IR spectra reveal differences in the thermal stabilities of the individual isotopically labeled sites. This is the first example of using an IR-based isotopic labeling technique to differentiate structural transitions at specific sites along the peptide backbone in model beta-hairpin peptides.

  18. Design and Activity Determination of Cyclic RGD Peptide and Preparation of 99Tcm Labeled Cyclic RGD Dimer%RGD多肽类药物设计、活性测定及99Tcm-cRGD二聚体的制备

    Institute of Scientific and Technical Information of China (English)

    张丽; 张春丽; 王荣福; 闫平; 康磊; 郭凤琴; 魏海亮; 崔永刚; 卢霞

    2011-01-01

    通过V-life计算机模拟软件建立cRGD(cyclic Arg-Gly-Asp,cRGD)多肽类分子库,利用V-life软件中的DOCK功能对分子库内cRGD肽结构进行筛选评分,挑选出能与整合素αvβ3受体高特异性结合的cRGD 结构.将该结构进行改造后制备成二聚体,用99Tcm对该结构进行标记,制备成肿瘤分子探针.并对其标记条件、稳定性、水溶性和亲和力进行评价.结果表明,DOCK功能计算出评分最佳的cRGD分子结构为Cys-Arg-Gly-Asp-(D)Ser-Cys.将该结构进行改造制备成二聚体后,室温下、ρ(SnCl2·2H2O)=1 g/L、反应时间为30 min时,标记率可达(87.42±3.21)%,经Sephadex G10层析柱纯化后,其放化纯大于95%;在室温和37℃条件下,99Tcm-cRGD于生理盐水和正常人新鲜血清中均保持良好的标记稳定性;其脂水分配系数对数值lg P(正辛醇/生理盐水)为-1.96±0.01;与U87人神经胶质瘤细胞进行受体的放射性配基结合分析(radioligand binding assay of receptors,RBA)实验,其平衡解离常数(equilibrium dissociation constant,Kd)为(0.089±0.052)×10-9 L/mol.这表明,通过计算机模拟系统筛选出的cRGD肽可与整合素αvβ3特异性结合,是一种有前景的整合素αvβ3受体阳性肿瘤显像剂.%This paper was to design a cyclic RGD peptide tumor inhibitor with high affinity to integrin αvβ3 receptor by molecular docking technique. cRGD molecular library was built and an optimal structure of cRGD peptide with the lowest score that was Cys-Arg-Gly-Asp-(D)Ser-Cy s was screened out using the function of DOCK procedure of the V-life software. Based on the moiety a dimer linked by Tyr-(D)Ser-Lys-(D)Ser-Ser and with a side chain Gly-Gly-(D)Ala-Gly on Lys was synthesisized and 99Tcm-cRGD dimer was prepared. Its radiolabeled efficiency,stability, water-soluble and affinity in vitro were evaluated. Under the reaction condition of room temperature, 1 g/L SnCl2 · 2H2O and the 30 min of reaction time, labeling efficiency reachs (87

  19. European consumers and nutrition labelling

    DEFF Research Database (Denmark)

    Wills, Josephine M.; Grunert, Klaus G.; Celemín, Laura Fernández

    2009-01-01

    Nutrition labelling of food in Europe is not compulsory, unless a nutrition or health claim is made for the product. The European Commission is proposing mandatory nutrition labelling, even front of pack labelling with nutrition information. Yet, how widespread is nutrition labelling in the EU...

  20. Distance labeling schemes for trees

    DEFF Research Database (Denmark)

    Alstrup, Stephen; Gørtz, Inge Li; Bistrup Halvorsen, Esben;

    2016-01-01

    We consider distance labeling schemes for trees: given a tree with n nodes, label the nodes with binary strings such that, given the labels of any two nodes, one can determine, by looking only at the labels, the distance in the tree between the two nodes. A lower bound by Gavoille et al. [Gavoill...

  1. Study on biodistribution of 131 iodine labeled monoclonal antibody D-D3 against pro-gastrin-releasing peptide(31-98) in healthy Kunming mice%抗ProGRP(31-98)单克隆抗体D-D3的131I标记及其体内生物学分布研究

    Institute of Scientific and Technical Information of China (English)

    陈传新; 石怡珍; 杨仪; 唐军; 刘增礼; 徐巧玲

    2011-01-01

    Objective To study the 131I labeling methods, stability, and biological distribution pattern of D-D3 antibody against pro-gastrin-releasing peptide 31-98(ProGRP(31-98) ). Methods The radioiodination of D-D3 antibody was performed using the chloramine-T method. The radiochemical purity was determined through thin-layer chromotography. 131I-D-D3 was injected into the healthy Kunming mice via a tail vein, and the % ID/g for various organs was obtained, and then, the biodistribution and pharmacokinetics of 131 I-D-D3 antibody in healthy Kunming mice were studied. Results The 131 I-D-D3 labeling rate was (86.56 ± 3.8) %. The radiochemical purity of 131 I-D-D3 was (99.27 ± 0. 6)%. After 48 h incubating in 37 ℃ water bath, the radiochemical purity was (88.38 ± 0.4)%. While being mixed 24 h with healthy human serum, the radiochemical purity was still more than (64.43 ± 0.7)%. The metabolism of 131I-D-D3 in healthy Kunming mice was consistent with a two-compartment model with first-order absorption, T1/2α and T1/2β was 0.25,37.89 h, respectively. Conclusion The labeling efficiency and radiochemical purity of 131 I-D-D-D3 are high and stable. 131I-D-D3 is a promising radioimmunoimaging reagent for small cell lung cancer(SCLC).%目的 探讨抗胃泌素释放肽前体(ProGRP)(31-98)单克隆抗体D-D3的131I标记方法 及其在健康昆明小鼠体内的生物学分布规律与特点.方法 采用氯胺-T法用131I标记单克隆抗体D-D3,利用纸层析法测定其标记率、放化纯度和稳定性.取健康昆明小鼠50只,随机分为10组,每组5只,自昆明小鼠尾静脉注射131I-D-D3 1.48 kBq/100 μL(4 μCi/100 μL),各组小鼠分别于注射后5、15、30 min及1、2、4、8、12、24、48 h处死,取血液、心脏、肝脏、脾脏、肺脏、肾脏、胃、小肠、骨骼(右下肢)、肌肉(右下肢)和脑组织,称质量(g)后测放射性计数(cpm),计算各脏器组织的每克组织百分注射剂量率(%ID

  2. Optimization of combined temozolomide and peptide receptor radionuclide therapy (PRRT) in mice after multimodality molecular imaging studies

    NARCIS (Netherlands)

    S. Bison (Sander); J.C. Haeck (Joost); K. Bol (Karin); S. Koelewijn (Stuart); H.C. Groen (Harald); M.L. Melis (Marleen); J.F. Veenland (Jifke); M.R. Bernsen (Monique); M. de Jong (Marion)

    2015-01-01

    textabstractBackground: Successful treatments of patients with somatostatin receptor (SSTR)-overexpressing neuroendocrine tumours (NET) comprise somatostatin-analogue lutetium-177-labelled octreotate (177Lu-TATE) treatment, also referred to as peptide receptor radionuclide therapy (PRRT), and temozo

  3. Peptide Based Radiopharmaceuticals: Specific Construct Approach

    Energy Technology Data Exchange (ETDEWEB)

    Som, P; Rhodes, B A; Sharma, S S

    1997-10-21

    The objective of this project was to develop receptor based peptides for diagnostic imaging and therapy. A series of peptides related to cell adhesion molecules (CAM) and immune regulation were designed for radiolabeling with 99mTc and evaluated in animal models as potential diagnostic imaging agents for various disease conditions such as thrombus (clot), acute kidney failure, and inflection/inflammation imaging. The peptides for this project were designed by the industrial partner, Palatin Technologies, (formerly Rhomed, Inc.) using various peptide design approaches including a newly developed rational computer assisted drug design (CADD) approach termed MIDAS (Metal ion Induced Distinctive Array of Structures). In this approach, the biological function domain and the 99mTc complexing domain are fused together so that structurally these domains are indistinguishable. This approach allows construction of conformationally rigid metallo-peptide molecules (similar to cyclic peptides) that are metabolically stable in-vivo. All the newly designed peptides were screened in various in vitro receptor binding and functional assays to identify a lead compound. The lead compounds were formulated in a one-step 99mTc labeling kit form which were studied by BNL for detailed in-vivo imaging using various animals models of human disease. Two main peptides usingMIDAS approach evolved and were investigated: RGD peptide for acute renal failure and an immunomodulatory peptide derived from tuftsin (RMT-1) for infection/inflammation imaging. Various RGD based metallopeptides were designed, synthesized and assayed for their efficacy in inhibiting ADP-induced human platelet aggregation. Most of these peptides displayed biological activity in the 1-100 µM range. Based on previous work by others, RGD-I and RGD-II were evaluated in animal models of acute renal failure. These earlier studies showed that after acute ischemic injury the renal cortex displays

  4. Processing of laminin α chains generates peptides involved in wound healing and host defense.

    Science.gov (United States)

    Senyürek, Ilknur; Kempf, Wolfgang E; Klein, Gerd; Maurer, Andreas; Kalbacher, Hubert; Schäfer, Luisa; Wanke, Ines; Christ, Christina; Stevanovic, Stefan; Schaller, Martin; Rousselle, Patricia; Garbe, Claus; Biedermann, Tilo; Schittek, Birgit

    2014-01-01

    Laminins play a fundamental role in basement membrane architecture and function in human skin. The C-terminal laminin G domain-like (LG) modules of laminin α chains are modified by proteolysis to generate LG1-3 and secreted LG4-5 tandem modules. In this study, we provide evidence that skin-derived cells process and secrete biologically active peptides from the LG4-5 module of the laminin α3, α4 and α5 chain in vitro and in vivo. We show enhanced expression and processing of the LG4-5 module of laminin α3 in keratinocytes after infection and in chronic wounds in which the level of expression and further processing of the LG4-5 module correlated with the speed of wound healing. Furthermore, bacterial or host-derived proteases promote processing of laminin α3 LG4-5. On a functional level, we show that LG4-5-derived peptides play a role in wound healing. Moreover, we demonstrate that LG4-derived peptides from the α3, α4 and α5 chains have broad antimicrobial activity and possess strong chemotactic activity to mononuclear cells. Thus, the data strongly suggest a novel multifunctional role for laminin LG4-5-derived peptides in human skin and its involvement in physiological processes and pathological conditions such as inflammation, chronic wounds and skin infection.

  5. Topical peptides as cosmeceuticals

    Directory of Open Access Journals (Sweden)

    Varadraj Vasant Pai

    2017-01-01

    Full Text Available Peptides are known to have diverse biological roles, most prominently as signaling/regulatory molecules in a broad variety of physiological processes including defense, immunity, stress, growth, homeostasis and reproduction. These aspects have been used in the field of dermatology and cosmetology to produce short, stable and synthetic peptides for extracellular matrix synthesis, pigmentation, innate immunity and inflammation. The evolution of peptides over the century, which started with the discovery of penicillin, has now extended to their usage as cosmeceuticals in recent years. Cosmeceutical peptides may act as signal modulators of the extracellular matrix component, as structural peptides, carrier peptides and neurotransmitter function modulators. Transdermal delivery of peptides can be made more effective by penetration enhancers, chemical modification or encapsulation of peptides. The advantages of using peptides as cosmeceuticals include their involvement in many physiological functions of the skin, their selectivity, their lack of immunogenicity and absence of premarket regulatory requirements for their use. However, there are disadvantages: clinical evidence for efficacy is often weak, absorption may be poor due to low lipophilicity, high molecular weight and binding to other ingredients, and prices can be quite high.

  6. Semantic Role Labeling

    CERN Document Server

    Palmer, Martha; Xue, Nianwen

    2011-01-01

    This book is aimed at providing an overview of several aspects of semantic role labeling. Chapter 1 begins with linguistic background on the definition of semantic roles and the controversies surrounding them. Chapter 2 describes how the theories have led to structured lexicons such as FrameNet, VerbNet and the PropBank Frame Files that in turn provide the basis for large scale semantic annotation of corpora. This data has facilitated the development of automatic semantic role labeling systems based on supervised machine learning techniques. Chapter 3 presents the general principles of applyin

  7. Labeling of Patient Specimens

    Science.gov (United States)

    2011-01-26

    noted during the event that the actu.al number of near miss incidmts reported monthly was low due to laboratory personnel performing rounds each...specimens never leaves label and if moved it is labeled), All orders in system and all near misses and errors reported to patient safety Purchase/Install...Meeting 14 Aug 09, 1400 in lab break room thru out Develop TICK sheet to track near misses .JDI Ms. Clark Clinics will provide toPS 1st working day of

  8. The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74-103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice.

    Science.gov (United States)

    Vanheule, Vincent; Janssens, Rik; Boff, Daiane; Kitic, Nikola; Berghmans, Nele; Ronsse, Isabelle; Kungl, Andreas J; Amaral, Flavio Almeida; Teixeira, Mauro Martins; Van Damme, Jo; Proost, Paul; Mortier, Anneleen

    2015-08-28

    The ELR(-)CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74-103)) being the most potent. The COOH-terminal peptide CXCL9(74-103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74-103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74-103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74-103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions.

  9. Postoperative Changes in Aqueous Monocyte Chemotactic Protein-1 Levels and Bleb Morphology after Trabeculectomy vs. Ex-PRESS Shunt Surgery.

    Directory of Open Access Journals (Sweden)

    Kohei Shobayashi

    Full Text Available To evaluate the postoperative changes in blebs and levels of aqueous monocyte chemotactic protein-1 (MCP-1 after trabeculectomy vs. Ex-PRESS tube shunt surgery.Rabbits were subjected to trabeculectomy or Ex-PRESS tube shunt surgery and observed for up to 3 months. Intraocular pressure (IOP was measured using a rebound tonometer. The MCP-1 level was measured by enzyme-linked immunosorbent assay (ELISA. Bleb morphology was evaluated using photos and anterior-segment optical coherence tomography (OCT.There were no differences in bleb appearance or IOP at any time between the groups. Bleb wall density in the anterior-segment OCT image was significantly lower 1 week after surgery in the Ex-PRESS group than the trabeculectomy group. The MCP-1 level in control eyes was 304.1 ± 45.2 pg/mL. In the trabeculectomy group, the mean aqueous MCP-1 level was 1444.9, 1914.3, 1899.8, 516.4, 398.3, 427.3, 609.5, 1612.7, 386.2, and 167.9 pg/mL at 3, 6, and 12 h, and 1, 2, 5, 7, 14, 30, and 90 days after surgery, respectively. In the Ex-PRESS group, the corresponding values were 1744.0, 1372.0, 932.5, 711.7, 396.1, 487.3, 799.5, 1327.9, 293.6, and 184.0 pg/mL. There were no significant differences in the aqueous MCP-1 level between the groups at any time point.The postoperative changes were similar in the Ex-PRESS and trabeculectomy groups, except for bleb wall density in the anterior-segment OCT image. The postoperative aqueous MCP-1 level had bimodal peaks in both groups.

  10. Cathepsin K cleavage of SDF-1α inhibits its chemotactic activity towards glioblastoma stem-like cells.

    Science.gov (United States)

    Hira, Vashendriya V V; Verbovšek, Urška; Breznik, Barbara; Srdič, Matic; Novinec, Marko; Kakar, Hala; Wormer, Jill; der Swaan, Britt Van; Lenarčič, Brigita; Juliano, Luiz; Mehta, Shwetal; Van Noorden, Cornelis J F; Lah, Tamara T

    2017-03-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor with poor patient survival that is at least partly caused by malignant and therapy-resistant glioma stem-like cells (GSLCs) that are protected in GSLC niches. Previously, we have shown that the chemo-attractant stromal-derived factor-1α (SDF-1α), its C-X-C receptor type 4 (CXCR4) and the cysteine protease cathepsin K (CatK) are localized in GSLC niches in glioblastoma. Here, we investigated whether SDF-1α is a niche factor that through its interactions with CXCR4 and/or its second receptor CXCR7 on GSLCs facilitates their homing to niches. Furthermore, we aimed to prove that SDF-1α cleavage by CatK inactivates SDF-1α and inhibits the invasion of GSLCs. We performed mass spectrometric analysis of cleavage products of SDF-1α after proteolysis by CatK. We demonstrated that CatK cleaves SDF-1α at 3 sites in the N-terminus, which is the region of SDF-1α that binds to its receptors. Confocal imaging of human GBM tissue sections confirmed co-localization of SDF-1α and CatK in GSLC niches. In accordance, 2D and 3D invasion experiments using CXCR4/CXCR7-expressing GSLCs and GBM cells showed that SDF-1α had chemotactic activity whereas CatK cleavage products of SDF-1α did not. Besides, CXCR4 inhibitor plerixafor inhibited invasion of CXCR4/CXCR7-expressing GSLCs. In conclusion, CatK can cleave and inactivate SDF-1α. This implies that CatK activity facilitates migration of GSLCs out of niches. We propose that activation of CatK may be a promising strategy to prevent homing of GSLCs in niches and thus render these cells sensitive to chemotherapy and radiation.

  11. Structural pattern matching of nonribosomal peptides

    Directory of Open Access Journals (Sweden)

    Leclère Valérie

    2009-03-01

    Full Text Available Abstract Background Nonribosomal peptides (NRPs, bioactive secondary metabolites produced by many microorganisms, show a broad range of important biological activities (e.g. antibiotics, immunosuppressants, antitumor agents. NRPs are mainly composed of amino acids but their primary structure is not always linear and can contain cycles or branchings. Furthermore, there are several hundred different monomers that can be incorporated into NRPs. The NORINE database, the first resource entirely dedicated to NRPs, currently stores more than 700 NRPs annotated with their monomeric peptide structure encoded by undirected labeled graphs. This opens a way to a systematic analysis of structural patterns occurring in NRPs. Such studies can investigate the functional role of some monomeric chains, or analyse NRPs that have been computationally predicted from the synthetase protein sequence. A basic operation in such analyses is the search for a given structural pattern in the database. Results We developed an efficient method that allows for a quick search for a structural pattern in the NORINE database. The method identifies all peptides containing a pattern substructure of a given size. This amounts to solving a variant of the maximum common subgraph problem on pattern and peptide graphs, which is done by computing cliques in an appropriate compatibility graph. Conclusion The method has been incorporated into the NORINE database, available at http://bioinfo.lifl.fr/norine. Less than one second is needed to search for a pattern in the entire database.

  12. Affinity labeling of the active site and the reactive sulfhydryl associated with activation of rat liver phenylalanine hydroxylase

    Energy Technology Data Exchange (ETDEWEB)

    Gibbs, B.S.; Benkovic, S.J. (Pennsylvania State Univ., University Park (United States))

    1991-07-09

    A pterin analogue, 5-((3-azido-6-nitrobenzylidene)amino)-2,6-diamino-4-pyrimidinone (AN-BADP), was synthesized as a probe of the pterin binding site of phenylalanine hydroxylase. The photoaffinity label has been found to be a competitive inhibitor of the enzyme with respect to 6,7-dimethyltetrahydropterin, having a K{sub i} of 8.8{plus minus}1.1 {mu}M. The irreversible labeling of phenylalanine hydroxylase by the photoaffinity label upon irradiation is both concentration and time dependent. phenylalanine hydroxylase is covalently labeled with a stoichiometry of 0.87{plus minus}0.08 mol of label/enzyme subunit. 5-Deaza-6-methyltetrahydropterin protects against inactivation and both 5-deaza-6-methyltetrahydropterin and 6-methyltetrahydropterin protect against covalent labeling, indicating that labeling occurs at the pterin binding site. Three tryptic peptides were isolated from ({sup 3}H)ANBADP-photolabeled enzyme and sequenced. All peptides indicated the sequence Thr-Leu-Lys-Ala-Leu-Tyr-Lys (residues 192-198). The residues labeled with ({sup 3}H)ANBADP were Lys198 and Lys194, with the majority of the radioactivity being associated with Lys198. Tryptic and chymotryptic peptides were isolated from fluorescein-labeled enzyme and sequenced. The modified residue was identified as Cys236.

  13. Figuring Out Food Labels

    Science.gov (United States)

    ... should get 30%-40% of calories from fat. Saturated Fat and Trans Fat The amount of saturated fat appears beneath total fat. The FDA also requires ... to list trans fats separately on the label. Saturated fats and trans fats are often called "bad fats" ...

  14. Labelled Execution Systems

    Science.gov (United States)

    2012-05-07

    This is most starkly evident in the classical example used to demonstrate that, in the case of infinitely branching systems, a transfinite number of... transfinite number of iterations to converge, what can be shown to fail by appropriately embedding the labelled transition systems of [46, prop. 10.5

  15. Multi-label

    Directory of Open Access Journals (Sweden)

    Neda Abdelhamid

    2015-01-01

    Full Text Available Generating multi-label rules in associative classification (AC from single label data sets is considered a challenging task making the number of existing algorithms for this task rare. Current AC algorithms produce only the largest frequency class connected with a rule in the training data set and discard all other classes even though these classes have data representation with the rule’s body. In this paper, we deal with the above problem by proposing an AC algorithm called Enhanced Multi-label Classifiers based Associative Classification (eMCAC. This algorithm discovers rules associated with a set of classes from single label data that other current AC algorithms are unable to induce. Furthermore, eMCAC minimises the number of extracted rules using a classifier building method. The proposed algorithm has been tested on a real world application data set related to website phishing and the results reveal that eMCAC’s accuracy is highly competitive if contrasted with other known AC and classic classification algorithms in data mining. Lastly, the experimental results show that our algorithm is able to derive new rules from the phishing data sets that end-users can exploit in decision making.

  16. Waisda?: video labeling game

    NARCIS (Netherlands)

    Hildebrand, M.; Brinkerink, M.; Gligorov, R.; Steenbergen, M. van; Huijkman, J.; Oomen, J.

    2013-01-01

    The Waisda? video labeling game is a crowsourcing tool to collect user-generated metadata for video clips. It follows the paradigm of games-with-a-purpose, where two or more users play against each other by entering tags that describe the content of the video. Players score points by entering the sa

  17. Insulin C-peptide test

    Science.gov (United States)

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

  18. UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven; Fu, Kai; Ding, Shi-Jian

    2011-03-04

    We present UNiquant, a new software program for analyzing stable isotope labeling (SIL) based quantitative proteomics data. UNiquant surpassed the performance of two other platforms, MaxQuant and Mascot Distiller, using complex proteome mixtures having either known or unknown heavy/light ratios. UNiquant is compatible with a broad spectrum of search engines and SIL methods, providing outstanding peptide pair identification and accurate measurement of the relative peptide/protein abundance.

  19. Descriptors for antimicrobial peptides

    DEFF Research Database (Denmark)

    Jenssen, Håvard

    2011-01-01

    of antimicrobial drugs, and computational methods utilizing molecular descriptors can significantly accelerate the development of new peptide drug candidates. Areas covered: This paper gives a broad overview of peptide and amino-acid scale descriptors available for AMP modeling and highlights which...

  20. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

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

  1. Bacteriocin Inducer Peptides

    Science.gov (United States)

    Novel peptides produced by bacteriocin-producing bacteria stimulate the production of bacteriocins in vitro. The producer bacteria are cultured in the presence of a novel inducer bacteria and a peptide having a carboxy terminal sequence of VKGLT in order to achieve an increase in bacteriocin produc...

  2. Avian host defense peptides

    NARCIS (Netherlands)

    Cuperus, Tryntsje; Coorens, M.; van Dijk, A.; Haagsman, H.P.

    2013-01-01

    Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense

  3. APD: the Antimicrobial Peptide Database

    OpenAIRE

    Wang, Zhe; Wang, Guangshun

    2004-01-01

    An antimicrobial peptide database (APD) has been established based on an extensive literature search. It contains detailed information for 525 peptides (498 antibacterial, 155 antifungal, 28 antiviral and 18 antitumor). APD provides interactive interfaces for peptide query, prediction and design. It also provides statistical data for a select group of or all the peptides in the database. Peptide information can be searched using keywords such as peptide name, ID, length, net charge, hydrophob...

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    César de la Fuente-Núñez

    2014-10-01

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

  6. Novel alpha-MSH peptide analogs for melanoma targeting

    Science.gov (United States)

    Flook, Adam Michael

    Skin cancer is the one of the most diagnosed cancers in the United States with increasing incidence over the past two decades. There are three major forms of skin cancer but melanoma is the deadliest. It is estimated that 76,690 new diagnoses of melanoma and 9,480 deaths will occur in 2013. Melanoma accounts for approximately 1.6% of all cancer related deaths and is the 5 th leading diagnosed cancer in the United States. The mean survival rate of patients diagnosed with metastatic melanoma is six months, with five year survival rates of less than 5%. In this project, we describe the design and characterization of novel melanoma-targeting peptide analogs for use in diagnostic imaging of both primary and metastatic melanoma lesions. Novel alpha-MSH peptide conjugates were designed to target the melanocortin-1 receptor present and over-expressed on melanoma cells. These peptides were synthesized and their in-vitro melanocortin-1 receptor binding affinities were established in murine melanoma cells. Once binding affinities were determined, the peptides were radiolabeled with 99mTc utilizing a novel direct radiolabeling technique developed in our laboratory. The peptides were purified via reverse-phase high performance liquid chromatography and in-vivo melanoma targeting and pharmacokinetic properties were determined in B16/F1 melanoma-bearing female C57BL/6 mice. Biodistribution and SPECT/CT imaging studies were performed with the promising 99m Tc-labeled peptide conjugates. All alpha-MSH peptide conjugates tested showed low nanomolar binding affinity for the melanocortin-1 receptor. All peptides were readily radiolabeld with 99mTc with greater than 95% radiochemical purity. All 99mTc-labeled peptides displayed high specific in-vivo melanoma tumor uptake while maintaining low normal organ accumulation, and were excreted through the urinary system in a timely fashion. In addition, all tested 99mTc-labeld alpha-MSH peptides demonstrated clear visualization of in

  7. Tetraphenylporphyrin as a protein label for triple detection analytical systems

    Directory of Open Access Journals (Sweden)

    Kamila Konopińska

    2015-12-01

    Full Text Available Porphyrins and metalloporphyrins are promising new protein labels that can be detected using multiple techniques; improving the reliability of the analysis and broadening the range of the linear response. Here, we investigate the potential of 5,10,15,20-tetraphenyl-21H,23H-porphyrin (Tpp as a hybrid protein label. The electrochemical and optical properties of porphyrin conjugated with bovine serum albumin (BSA, chicken egg albumin (CEA and immunoglobulin G (IgG were determined and optimal conditions for Tpp-protein conjugation established. Model conjugates of carboxylated Tpp with BSA and short peptides were characterized using differential pulse voltammetry, UV–Vis spectrophotometry and spectrofluorimetry. These results reveal that Tpp is a promising molecule to be used in a triple detection protein labelling system.

  8. Short-term stimulation with interleukin (IL-4 enhances purified protein derivative-induced production of an eosinophil chemotactic lymphokine, but suppresses IL-5 production

    Directory of Open Access Journals (Sweden)

    Takehiko Nishiyama

    1998-01-01

    Full Text Available The effect of interleukin (IL-4 on eosinophil chemotactic lymphokine (ECL production from peripheral blood mononuclear cells (PBMC stimulated with purified protein derivative (PPD was examined. The PBMC stimulated with PPD in the absence of IL-4 failed to produce evident ECL. However, PPD-induced eosinophil chemotactic activity (ECA production was markedly enhanced in a dose-dependent manner by pretreatment of PBMC with IL-4. The most potent enhancement was induced by IL-4 at a concentration of 30 U in tuberculin-sensitive PBMC. Short-term pretreatment (30 min to 3 h was sufficient for the enhancement, whereas longer-term treatment was less effective. Eosinophil chemotactic lymphokine was found to be a CD4+ T cell-derived factor with an isoelectric point of approximately pH 7.0 and without heparin affinity, unlike chemokines such as RANTES and eotaxin. The effect of IL-4 on the production of other cytokines, such as interferon (IFN-γ, IL-5, RANTES (regulated on activation, normal T expressed and secreted, and granulocyte-macrophage colony stimulating factor (GM-CSF was also examined. Peripheral blood mononuclear cells produced all these cytokines when they were treated with PPD, even in the absence of IL-4. When PBMC were pretreated with IL-4, interestingly not only IFNy but also IL-5 production was suppressed by pretreatment with IL-4, although ECL production was enhanced by the pretreatment. In the case of RANTES and GM-CSF, significant amounts of these cytokines were produced, even without antigenic stimulation, and IL-4 pretreatment did not result in an enhancement of their production. It is thus suggested that IL-4, existing in allergic lesions, plays a crucial role in eosinophil accumulation mediated by the T cell-derived ECL.

  9. Decode the Sodium Label Lingo

    Science.gov (United States)

    ... For Preschooler For Gradeschooler For Teen Decode the Sodium Label Lingo Published January 24, 2013 Print Email Reading food labels can help you slash sodium. Here's how to decipher them. "Sodium free" or " ...

  10. CD147 and CD98 complex-mediated homotypic aggregation attenuates the CypA-induced chemotactic effect on Jurkat T cells.

    Science.gov (United States)

    Guo, Na; Zhang, Kui; Lv, Minghua; Miao, Jinlin; Chen, Zhinan; Zhu, Ping

    2015-02-01

    Homotypic cell aggregation plays important roles in physiological and pathological processes, including embryogenesis, immune responses, angiogenesis, tumor cell invasion and metastasis. CD147 has been implicated in most of these phenomena, and it was identified as a T cell activation-associated antigen due to its obvious up-regulation in activated T cells. However, the explicit function and mechanism of CD147 in T cells have not been fully elucidated. In this study, large and compact aggregates were observed in Jurkat T cells after treatment with the specific CD147 monoclonal antibody HAb18 or after the expression of CD147 was silenced by RNA interference, which indicated an inhibitory effect of CD147 in T cell homotypic aggregation. Knocking down CD147 expression resulted in a significant decrease in CD98, along with prominent cell aggregation, similar to that treated by CD98 and CD147 monoclonal antibodies. Furthermore, decreased cell chemotactic activity was observed following CD147- and CD98-mediated cell aggregation, and increased aggregation was correlated with a decrease in the chemotactic ability of the Jurkat T cells, suggesting that CD147- and CD98-mediated homotypic cell aggregation plays a negative role in T cell chemotaxis. Our data also showed that p-ERK, p-ZAP70, p-CD3ζ and p-LCK were significantly decreased in the CD147- and CD98-knocked down Jurkat T cells, which suggested that decreased CD147- and/or CD98-induced homotypic T cell aggregation and aggregation-inhibited chemotaxis might be associated with these signaling pathways. A role for CD147 in cell aggregation and chemotaxis was further indicated in primary CD4(+) T cells. Similarly, low expression of CD147 in primary T cells induced prominent cell aggregation and this aggregation attenuated primary T cell chemotactic ability in response to CypA. Our results have demonstrated the correlation between homotypic cell aggregation and the chemotactic response of T cells to CypA, and these data

  11. A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC(50) determination.

    Science.gov (United States)

    Yin, Liusong; Stern, Lawrence J

    2014-04-01

    HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.

  12. De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

    Directory of Open Access Journals (Sweden)

    Geisa P.C. Evaristo

    2015-09-01

    Full Text Available An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was achieved by complementary CID and ETD fragmentations of full-length peptides and of selected tryptic fragments. Heavy and light isotope dimethyl labeling assisted with annotation of sequence ion series. The identified primary structures are GCD[I/L]STCATHN[I/L]VNE[I/L]NKFDKSKPSSGGVGPESP-NH2 and SCNLSTCATHNLVNELNKFDKSKPSSGGVGPESF-NH2, i.e. two carboxyamidated 34 residue peptides with an aminoterminal intramolecular ring structure formed by a disulfide bridge between Cys2 and Cys7. Edman degradation analysis of the second peptide positively confirmed the exact sequence, resolving I/L discriminations. Both peptide sequences are novel and share homology with calcitonin, calcitonin gene related peptide (CGRP and adrenomedullin from other vertebrates. Detailed sequence analysis as well as the 34 residue length of both O. schmackeri peptides, suggest they do not fully qualify as either calcitonins (32 residues or CGRPs (37 amino acids and may justify their classification in a novel peptide family within the calcitonin gene related peptide superfamily. Smooth muscle contractility assays with synthetic replicas of the S–S linked peptides on rat tail artery, uterus, bladder and ileum did not reveal myotropic activity.

  13. Dynamic Proteomics: In Vivo Proteome-Wide Measurement of Protein Kinetics Using Metabolic Labeling.

    Science.gov (United States)

    Holmes, W E; Angel, T E; Li, K W; Hellerstein, M K

    2015-01-01

    Control of biosynthetic and catabolic rates of polymers, including proteins, stands at the center of phenotype, physiologic adaptation, and disease pathogenesis. Advances in stable isotope-labeling concepts and mass spectrometric instrumentation now allow accurate in vivo measurement of protein synthesis and turnover rates, both for targeted proteins and for unbiased screening across the proteome. We describe here the underlying principles and operational protocols for measuring protein dynamics, focusing on metabolic labeling with (2)H2O (heavy water) combined with tandem mass spectrometric analysis of mass isotopomer abundances in trypsin-generated peptides. The core principles of combinatorial analysis (mass isotopomer distribution analysis or MIDA) are reviewed in detail, including practical advantages, limitations, and technical procedures to ensure optimal kinetic results. Technical factors include heavy water labeling protocols, optimal duration of labeling, clean up and simplification of sample matrices, accurate quantitation of mass isotopomer abundances in peptides, criteria for adequacy of mass spectrometric abundance measurements, and calculation algorithms. Some applications are described, including the noninvasive "virtual biopsy" strategy for measuring molecular flux rates in tissues through measurements in body fluids. In addition, application of heavy water labeling to measure flux lipidomics is noted. In summary, the combination of stable isotope labeling, particularly from (2)H2O, with tandem mass spectrometric analysis of mass isotopomer abundances in peptides, provides a powerful approach for characterizing the dynamics of proteins across the global proteome. Many applications in research and clinical medicine have been achieved and many others can be envisioned.

  14. Labelling GM-free Products

    DEFF Research Database (Denmark)

    Punt, Maarten; Venus, Thomas; Wesseler, Justus

    2016-01-01

    Food suppliers in the EU must comply with labelling regulations for genetically modified organisms (GMOs). However, excluded from mandatory labelling are food products derived from animals fed with GM feed (mainly GM soybean in the EU). Because of this labelling exemption, consumers are unable to...

  15. Scaffolding Visitors' Learning through Labels

    Science.gov (United States)

    Wang, Joyce; Yoon, Susan

    2013-01-01

    In museum literature, labels have been found to increase visitor learning and contribute to greater cognitive gains. In this study, we seek to understand how various labels support the visitors' learning experience, and specifically in regards to conceptual and cognitive learning. We investigated the increasing use of three types of labels (visual…

  16. Food Labels Tell the Story!

    Science.gov (United States)

    ... My World From the Label to the Table! Food Labels Tell the Story! What is in food? Food provides your body with all of the ... your food choices. Nutrition Facts—the Labels on Food Products Beginning in 1994, the US government began ...

  17. Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.

    Science.gov (United States)

    Pashkova, Anna; Chen, Hsuan-Shen; Rejtar, Tomas; Zang, Xin; Giese, Roger; Andreev, Victor; Moskovets, Eugene; Karger, Barry L

    2005-04-01

    The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.

  18. Histidine-containing peptide catalysts developed by a facile library screening method.

    Science.gov (United States)

    Akagawa, Kengo; Sakai, Nobutaka; Kudo, Kazuaki

    2015-02-02

    Although peptide catalysts have a high potential for the use as organocatalysts, the optimization of peptide sequences is laborious and time-consuming. To address this issue, a facile screening method for finding efficient aminocatalysts from a peptide library has been developed. In the screening for the Michael addition of a malonate to an enal, a dye-labeled product is immobilized on resin-bound peptides through reductive amination to visualize active catalysts. This procedure allows for the monitoring of the reactivity of entire peptides without modifying the resin beads beforehand. Peptides containing histidine at an appropriate position were identified by this method. A novel function of the histidyl residue, which enhances the binding of a substrate to the catalyst by capturing an iminium intermediate, was indicated.

  19. Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

    Science.gov (United States)

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2013-01-15

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  20. Simultaneous enrichment of cysteine-containing peptides and phosphopeptides using a cysteine-specific phosphonate adaptable tag (CysPAT) in combination with titanium dioxide (TiO2) chromatography

    DEFF Research Database (Denmark)

    Huang, Honggang; Pedersen, Martin Haar; Ibañez-Vea, Maria

    2016-01-01

    to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO2) and subsequent mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to protein lysates from...

  1. Identifying structural features of fibrillar islet amyloid polypeptide using site-directed spin labeling.

    Science.gov (United States)

    Jayasinghe, Sajith A; Langen, Ralf

    2004-11-12

    Pancreatic amyloid deposits, composed primarily of the 37-residue islet amyloid polypeptide (IAPP), are a characteristic feature found in more than 90% of patients with type II diabetes. Although IAPP amyloid deposits are associated with areas of pancreatic islet beta-cell dysfunction and depletion and are thought to play a role in disease, their structure is unknown. We used electron paramagnetic resonance spectroscopy to analyze eight spin-labeled derivatives of IAPP in an effort to determine structural features of the peptide. In solution, all eight derivatives gave rise to electron paramagnetic resonance spectra with sharp lines indicative of rapid motion on the sub-nanosecond time scale. These spectra are consistent with a rapidly tumbling and highly dynamic peptide. In contrast, spectra for the fibrillar form exhibit reduced mobility and the presence of strong intermolecular spin-spin interactions. The latter implies that the peptide subunits are ordered and that the same residues from neighboring peptides are in close proximity to one another. Our data are consistent with a parallel arrangement of IAPP peptides within the amyloid fibril. Analysis of spin label mobility indicates a high degree of order throughout the peptide, although the N-terminal region is slightly less ordered. Possible similarities with respect to the domain organization and parallelism of Alzheimer's amyloid beta peptide fibrils are discussed.

  2. Map labeling and its generalizations

    Energy Technology Data Exchange (ETDEWEB)

    Doddi, S. [New Mexico Univ., Albuquerque, NM (United States). Dept. of Computer Science]|[Los Alamos National Lab., NM (United States); Marathe, M.V. [Los Alamos National Lab., NM (United States); Mirzaian, A. [York Univ., Toronto, ON (Canada). Dept. of Computer Science; Moret, B.M.E. [New Mexico Univ., Albuquerque, NM (United States). Dept. of Computer Science; Zhu, B. [City Univ. of Hong Kong (Hong Kong). Dept. of Computer Science]|[Los Alamos National Lab., NM (United States)

    1997-01-01

    Map labeling is of fundamental importance in cartography and geographical information systems and is one of the areas targeted for research by the ACM Computational Geometry Impact Task Force. Previous work on map labeling has focused on the problem of placing maximal uniform, axis-aligned, disjoint rectangles on the plane so that each point feature to be labeled lies at the corner of one rectangle. Here, we consider a number of variants of the map labeling problem. We obtain three general types of results. First, we devise constant-factor polynomial-time-approximation algorithms for labeling point features by rectangular labels, where the feature may lie anywhere on the boundary of its label region and where labeling rectangles may be placed in any orientation. These results generalize to the case of elliptical labels. Secondly, we consider the problem of labeling a map consisting of disjoint rectilinear fine segments. We obtain constant-factor polynomial-time approximation algorithms for the general problem and an optimal algorithm for the special case where all segments are horizontal. Finally, we formulate a bicriteria version of the map-labeling problem and provide bicriteria polynomial- time approximation schemes for a number of such problems.

  3. Chemokine-Derived Peptides: Novel Antimicrobial and Antineoplasic Agents

    Science.gov (United States)

    Valdivia-Silva, Julio; Medina-Tamayo, Jaciel; Garcia-Zepeda, Eduardo A.

    2015-01-01

    Chemokines are a burgeoning family of chemotactic cytokines displaying a broad array of functions such as regulation of homeostatic leukocyte traffic and development, as well as activating the innate immune system. Their role in controlling early and late inflammatory stages is now well recognized. An improper balance either in chemokine synthesis or chemokine receptor expression contributes to various pathological disorders making chemokines and their receptors a useful therapeutic target. Research in this area is progressing rapidly, and development of novel agents based on chemokine/chemokine receptors antagonist functions are emerging as attractive alternative drugs. Some of these novel agents include generation of chemokine-derived peptides (CDP) with potential agonist and antagonist effects on inflammation, cancer and against bacterial infections. CDP have been generated mainly from N- and C-terminus chemokine sequences with subsequent modifications such as truncations or elongations. In this review, we present a glimpse of the different pharmacological actions reported for CDP and our current understanding regarding the potential use of CDP alone or as part of the novel therapies proposed in the treatment of microbial infections and cancer. PMID:26062132

  4. Chemokine-Derived Peptides: Novel Antimicrobial and Antineoplasic Agents

    Directory of Open Access Journals (Sweden)

    Julio Valdivia-Silva

    2015-06-01

    Full Text Available Chemokines are a burgeoning family of chemotactic cytokines displaying a broad array of functions such as regulation of homeostatic leukocyte traffic and development, as well as activating the innate immune system. Their role in controlling early and late inflammatory stages is now well recognized. An improper balance either in chemokine synthesis or chemokine receptor expression contributes to various pathological disorders making chemokines and their receptors a useful therapeutic target. Research in this area is progressing rapidly, and development of novel agents based on chemokine/ chemokine receptors antagonist functions are emerging as attractive alternative drugs. Some of these novel agents include generation of chemokine-derived peptides (CDP with potential agonist and antagonist effects on inflammation, cancer and against bacterial infections. CDP have been generated mainly from N- and C-terminus chemokine sequences with subsequent modifications such as truncations or elongations. In this review, we present a glimpse of the different pharmacological actions reported for CDP and our current understanding regarding the potential use of CDP alone or as part of the novel therapies proposed in the treatment of microbial infections and cancer.

  5. Linerless label device and method

    KAUST Repository

    Binladen, Abdulkari

    2016-01-14

    This apparatus and method for applying a linerless label to an end user product includes a device with a printer for printing on a face surface of a linerless label, and a release coat applicator for applying a release coat to the face surface of the label; another device including an unwinder unit (103) to unwind a roll of printed linerless label; a belt (108); a glue applicator (102) for applying glue to the belt; a nip roller (106) for contacting and applying pressure to the face surface of the linerless label such that the glue on the belt transfers to the back surface of the linerless label; at least one slitting knife 105) positioned downstream the belt and a rewinder unit (104) positioned downstream the slitting knife; and a third device which die cuts and applies the linerless label to an end user object.

  6. Anti-antimicrobial Peptides

    Science.gov (United States)

    Ryan, Lloyd; Lamarre, Baptiste; Diu, Ting; Ravi, Jascindra; Judge, Peter J.; Temple, Adam; Carr, Matthew; Cerasoli, Eleonora; Su, Bo; Jenkinson, Howard F.; Martyna, Glenn; Crain, Jason; Watts, Anthony; Ryadnov, Maxim G.

    2013-01-01

    Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance. PMID:23737519

  7. Tumor penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tambet eTeesalu

    2013-08-01

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

  8. Real-time, high-throughput measurements of peptide-MHC-I dissociation using a scintillation proximity assay

    DEFF Research Database (Denmark)

    Harndahl, Mikkel; Rasmussen, Michael; Røder, Gustav Andreas;

    2011-01-01

    timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal...... requirements. To avoid labeling of the highly variable peptide, we labeled the invariant ß2m and monitored its dissociation by a scintillation proximity assay, which has no separation steps and allows for real-time quantitative measurement of dissociation. Validating this work-around to create a virtually...

  9. {sup 18}F-labeled radiopharmaceuticals for PET in oncology, excluding FDG

    Energy Technology Data Exchange (ETDEWEB)

    Varagnolo, L.; Stokkel, M.P.M.; Mazzi, U.; Pauwels, E.K.J

    2000-02-01

    This article reviews possible use of {sup 18}F-labelled radiopharmaceuticals in oncology with positron emission tomography. The characteristics of various {sup 18}F-labelled compounds are proteins and peptides, those that bind to {center_dot} receptors, agents to assess hypoxia, and agents to evaluate gene therapy are highlighted. Furthermore, different {sup 18}F-labelled tissue specific agents are indicated for the detection and monitoring of various malignancies: melanoma, brain tumours, breast cancer, prostate cancer and colorectal cancer. {sup 18}F-fluorodeoxyglucose has been excluded from this summary.

  10. Immunotherapy with Allergen Peptides

    OpenAIRE

    Larché Mark

    2007-01-01

    Specific allergen immunotherapy (SIT) is disease-modifying and efficacious. However, the use of whole allergen preparations is associated with frequent allergic adverse events during treatment. Many novel approaches are being designed to reduce the allergenicity of immunotherapy preparations whilst maintaining immunogenicity. One approach is the use of short synthetic peptides which representing dominant T cell epitopes of the allergen. Short peptides exhibit markedly reduced capacity to cro...

  11. Antimicrobial Peptides in Echinoderms

    OpenAIRE

    Li, C; Haug, T; K Stensvåg

    2010-01-01

    Antimicrobial peptides (AMPs) are important immune effector molecules for invertebrates, including echinoderms, which lack a vertebrate-type adaptive immune system. Here we summarize the knowledge of such peptides in echinoderms. Strongylocins are a novel family of cysteine-rich AMPs, recently identified in the sea urchins, Strongylocentrotus droebachiensis and S. purpuratus. Although these molecules present diverse amino acid sequences, they share an identical cysteine arrangement pattern, d...

  12. Preparation of Radiopharmaceuticals Labeled with Metal Radionuclides

    Energy Technology Data Exchange (ETDEWEB)

    Welch, M.J.

    2012-02-16

    The overall goal of this project was to develop methods for the production of metal-based radionuclides, to develop metal-based radiopharmaceuticals and in a limited number of cases, to translate these agents to the clinical situation. Initial work concentrated on the application of the radionuclides of Cu, Cu-60, Cu-61 and Cu-64, as well as application of Ga-68 radiopharmaceuticals. Initially Cu-64 was produced at the Missouri University Research Reactor and experiments carried out at Washington University. A limited number of studies were carried out utilizing Cu-62, a generator produced radionuclide produced by Mallinckrodt Inc. (now Covidien). In these studies, copper-62-labeled pyruvaldehyde Bis(N{sup 4}-methylthiosemicarbazonato)-copper(II) was studied as an agent for cerebral myocardial perfusion. A remote system for the production of this radiopharmaceutical was developed and a limited number of patient studies carried out with this agent. Various other copper radiopharmaceuticals were investigated, these included copper labeled blood imaging agents as well as Cu-64 labeled antibodies. Cu-64 labeled antibodies targeting colon cancer were translated to the human situation. Cu-64 was also used to label peptides (Cu-64 octriatide) and this is one of the first applications of a peptide radiolabeled with a positron emitting metal radionuclide. Investigations were then pursued on the preparation of the copper radionuclides on a small biomedical cyclotron. A system for the production of high specific activity Cu-64 was developed and initially the Cu-64 was utilized to study the hypoxic imaging agent Cu-64 ATSM. Utilizing the same target system, other positron emitting metal radionuclides were produced, these were Y-86 and Ga-66. Radiopharmaceuticals were labeled utilizing both of these radionuclides. Many studies were carried out in animal models on the uptake of Cu-ATSM in hypoxic tissue. The hypothesis is that Cu-ATSM retention in vivo is dependent upon the

  13. Antimicrobial peptide LL-37 promotes antigen-specific immune responses in mice by enhancing Th17-skewed mucosal and systemic immunities.

    Science.gov (United States)

    Kim, Sae-Hae; Yang, In-Young; Kim, Ju; Lee, Kyung-Yeol; Jang, Yong-Suk

    2015-05-01

    The human antimicrobial peptide LL-37 is known to have chemotactic and modulatory activities on various cells including monocytes, T cells, and epithelial cells. Given that LL-37 enhances chemotactic attraction and modulates the activity of DCs, it is conceivable that it might play a role as an immune adjuvant by skewing the immune environment toward immunostimulatory conditions. In this study, we characterized the mucosal adjuvant activity of LL-37 using model and pathogenic Ags. When LL-37-conjugated Ag was administered orally to mice, a tolerogenic Peyer's patch environment was altered to cell populations containing IL-6-secreting CD11c(+), CD11c(+) CD70(+), and Th17 cells capable of evoking a subsequent LL-37-conjugated Ag-specific immune response in both systemic and mucosal immune compartments. In addition, we showed presentation of formyl peptide receptor, an LL-37 receptor, on M cells, which may aid the initiation of an LL-37-mediated enhanced immune response through targeting and transcytosis of the conjugated Ag. Based on our findings, we conclude that LL-37 has potential as an oral mucosal adjuvant, not only by enhancing the delivery of LL-37-conjugated Ag to M cells, but also by triggering T-cell-mediated Ag-specific immune responses through modulation of the mucosal immune environment.

  14. Radio labeling with pre-assigned frequencies

    NARCIS (Netherlands)

    Bodlaender, H.L.; Broersma, H.J.; Fomin, F.V.; Pyatkin, A.V.; Woeginer, G.J.

    2007-01-01

    A radio labeling of a graph G is an assignment of pairwise distinct, positive integer labels to the vertices of G such that labels of adjacent vertices differ by at least 2. The radio labeling problem (RL) consists in determining a radio labeling that minimizes the maximum label that is used (the so

  15. Towards Multi Label Text Classification through Label Propagation

    Directory of Open Access Journals (Sweden)

    Shweta C. Dharmadhikari

    2012-06-01

    Full Text Available Classifying text data has been an active area of research for a long time. Text document is multifaceted object and often inherently ambiguous by nature. Multi-label learning deals with such ambiguous object. Classification of such ambiguous text objects often makes task of classifier difficult while assigning relevant classes to input document. Traditional single label and multi class text classification paradigms cannot efficiently classify such multifaceted text corpus. Through our paper we are proposing a novel label propagation approach based on semi supervised learning for Multi Label Text Classification. Our proposed approach models the relationship between class labels and also effectively represents input text documents. We are using semi supervised learning technique for effective utilization of labeled and unlabeled data for classification. Our proposed approach promises better classification accuracy and handling of complexity and elaborated on the basis of standard datasets such as Enron, Slashdot and Bibtex.

  16. Selective labeling of polypeptides using protein farnesyltransferase via rapid oxime ligation.

    Science.gov (United States)

    Rashidian, Mohammad; Dozier, Jonathan K; Lenevich, Stepan; Distefano, Mark D

    2010-12-21

    An aldehyde-containing alternative substrate for protein farnesyltransferase was prepared and shown to be enzymatically incorporated into a peptide and a protein. The protein was subsequently immobilized onto aminooxy-functionalized agarose beads or labeled with a fluorophore. This method for protein modification provides an alternative to the commonly employed Cu(I)-catalyzed click reaction.

  17. Rapid peptide metabolism: A major component of soil nitrogen cycling?

    Science.gov (United States)

    Farrell, Mark; Hill, Paul W.; Wanniarachchi, Sudas D.; Farrar, John; Bardgett, Richard D.; Jones, Davey L.

    2011-09-01

    Proteinaceous and peptidic nitrogen is a potential direct nutrient source for both plants and microbes in the soil, without prior degradation to amino acids and mineralization. We used a series of five sites along an elevation gradient from 15 m a.s.l. to 710 m a.s.l. along which primary productivity decreases to investigate peptide utilization rates by soil microbes. Using 14C-labeled L-alanine, L-dialanine, and L-trialanine in a series of incubation experiments, we show that peptides are directly and rapidly assimilated by soil microbes, and that they are utilized for both biomass production and respiration. Alanine, dialanine, and trialanine were mineralized rapidly by soil microbes from the five sites along the gradient. Across all five sites, dialanine and trialanine were mineralized faster than alanine. In competition experiments, a 100-fold excess of alanine had no effect on the rate of trialanine mineralization in four of the five sites, and the same excess of trialanine had no effect on alanine mineralization. This is indicative of uptake of the intact peptide by the soil microbial community. Our findings have implications for understanding terrestrial nitrogen cycling because they point to a short-circuit whereby large peptides and proteins need only be extracellularly cleaved to short chain length peptides before direct assimilation by microbes.

  18. Natriuretic Peptides, Diagnostic and Prognostic Biomarkers

    NARCIS (Netherlands)

    J.H.W. Rutten (Joost)

    2010-01-01

    textabstractIn humans, the natriuretic peptide family consists of three different types of peptides: atrial natriuretic peptide (synonym: atrial natriuretic factor), B-type natriuretic peptide (synonym: brain natriuretic peptide) and C-natriuretic peptide.1 Atrial natriuretic peptide (ANP) was the f

  19. Investigation of peptide based surface functionalization for copper ions detection using an ultrasensitive mechanical microresonator

    DEFF Research Database (Denmark)

    Cagliani, Alberto; Fischer, Lee MacKenzie; Rasmussen, Jakob Lyager

    2011-01-01

    In the framework of developing a portable label-free sensor for multi arrayed detection of heavy metals in drinking water, we present a mechanical resonator-based copper ions sensor, which uses a recently synthesized peptide Cysteine–Glycine–Glycine–Histidine (CGGH) and the l-Cysteine (Cys) peptide...... devices to detect a concentration of 10μM of copper in water, we regenerate the surface by removing the copper ions from the functionalization layer using EDTA....

  20. 78 FR 66826 - Prior Label Approval System: Generic Label Approval

    Science.gov (United States)

    2013-11-07

    ... submitted for evaluation and approval before use under the final rule are: (1) Labels for chicken produced..., such as ``no antibiotics administered'' or ``vegetarian fed''; (4) instructional or...

  1. The Suzuki–Miyaura Cross-Coupling as a Versatile Tool for Peptide Diversification and Cyclization

    Directory of Open Access Journals (Sweden)

    Tom Willemse

    2017-02-01

    Full Text Available The (site-selective derivatization of amino acids and peptides represents an attractive field with potential applications in the establishment of structure–activity relationships and labeling of bioactive compounds. In this respect, bioorthogonal cross-coupling reactions provide valuable means for ready access to peptide analogues with diversified structure and function. Due to the complex and chiral nature of peptides, mild reaction conditions are preferred; hence, a suitable cross-coupling reaction is required for the chemical modification of these challenging substrates. The Suzuki reaction, involving organoboron species, is appropriate given the stability and environmentally benign nature of these reactants and their amenability to be applied in (partial aqueous reaction conditions, an expected requirement upon the derivatization of peptides. Concerning the halogenated reaction partner, residues bearing halogen moieties can either be introduced directly as halogenated amino acids during solid-phase peptide synthesis (SPPS or genetically encoded into larger proteins. A reversed approach building in boron in the peptidic backbone is also possible. Furthermore, based on this complementarity, cyclic peptides can be prepared by halogenation, and borylation of two amino acid side chains present within the same peptidic substrate. Here, the Suzuki–Miyaura reaction is a tool to induce the desired cyclization. In this review, we discuss diverse amino acid and peptide-based applications explored by means of this extremely versatile cross-coupling reaction. With the advent of peptide-based drugs, versatile bioorthogonal conversions on these substrates have become highly valuable.

  2. Peptidomic Analysis of Amniotic Fluid for Identification of Putative Bioactive Peptides in Ventricular Septal Defect

    Directory of Open Access Journals (Sweden)

    Xing Li

    2016-05-01

    Full Text Available Background: Ventricular septal defect (VSD is one of the most common congenital heart diseases and to date the role of peptides in human amniotic fluid in the pathogenesis of VSD have been rarely investigated. Methods: To gain insight into the mechanisms of protein and peptides in cardiovascular development, we constructed a comparative peptidomic profiling of human amniotic fluid between normal and VSD fetuses using a stable isobaric labeling strategy involving tandem mass tag reagents, followed by nano liquid chromatography tandem mass spectrometry. Results: We identified and quantified 692 non-redundant peptides, 183 of which were differentially expressed in the amniotic fluid of healthy and VSD fetuses; 69 peptides were up regulated and 114 peptides were down regulated. These peptides were imported into the Ingenuity Pathway Analysis (IPA and identified putative roles in cardiovascular system morphogenesis and cardiogenesis. Conclusion: We concluded that 35 peptides located within the functional domains of their precursor proteins could be candidate bioactive peptides for VSD. The identified peptide changes in amniotic fluid of VSD fetuses may advance our current understanding of congenital heart disease and these peptides may be involved in the etiology of VSD.

  3. Natriuretic Peptides, Diagnostic and Prognostic Biomarkers

    OpenAIRE

    Rutten, Joost

    2010-01-01

    textabstractIn humans, the natriuretic peptide family consists of three different types of peptides: atrial natriuretic peptide (synonym: atrial natriuretic factor), B-type natriuretic peptide (synonym: brain natriuretic peptide) and C-natriuretic peptide.1 Atrial natriuretic peptide (ANP) was the fi rst natriuretic peptide to be discovered and in humans ANP is predominantly formed in the cardiomyocytes of the atria.2 B-type natriuretic peptide (BNP) was fi rst discovered in porcine brain hen...

  4. Selective antimicrobial activity and mode of action of adepantins, glycine-rich peptide antibiotics based on anuran antimicrobial peptide sequences.

    Science.gov (United States)

    Ilić, Nada; Novković, Mario; Guida, Filomena; Xhindoli, Daniela; Benincasa, Monica; Tossi, Alessandro; Juretić, Davor

    2013-03-01

    A challenge when designing membrane-active peptide antibiotics with therapeutic potential is how to ensure a useful antibacterial activity whilst avoiding unacceptable cytotoxicity for host cells. Understanding their mode of interaction with membranes and the reasons underlying their ability to distinguish between bacterial and eukaryotic cytoplasmic cells is crucial for any rational attempt to improve this selectivity. We have approached this problem by analysing natural helical antimicrobial peptides of anuran origin, using a structure-activity database to determine an antimicrobial selectivity index (SI) relating the minimal inhibitory concentration against Escherichia coli to the haemolytic activity (SI=HC(50)/MIC). A parameter that correlated strongly with SI, derived from the lengthwise asymmetry of the peptides' hydrophobicity (sequence moment), was then used in the "Designer" algorithm to propose novel, highly selective peptides. Amongst these are the 'adepantins', peptides rich in glycines and lysines that are highly selective for Gram-negative bacteria, have an exceptionally low haemolytic activity, and are less than 50% homologous to any other natural or synthetic antimicrobial peptide. In particular, they showed a very high SI for E. coli (up to 400) whilst maintaining an antimicrobial activity in the 0.5-4μM range. Experiments with monomeric, dimeric and fluorescently labelled versions of the adepantins, using different bacterial strains, host cells and model membrane systems provided insight into their mechanism of action.

  5. Diversity-Oriented Peptide Stapling

    DEFF Research Database (Denmark)

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

    2017-01-01

    The introduction of macrocyclic constraints in peptides (peptide stapling) is an important tool within peptide medicinal chemistry for stabilising and pre-organising peptides in a desired conformation. In recent years, the copper-catalysed azide-alkyne cycloaddition (CuAAC) has emerged...... as a powerful method for peptide stapling. However, to date CuAAC stapling has not provided a simple method for obtaining peptides that are easily diversified further. In the present study, we report a new diversity-oriented peptide stapling (DOPS) methodology based on CuAAC chemistry. Stapling of peptides...... incorporating two azide-modified amino acids with 1,3,5-triethynylbenzene efficiently provides (i, i+7)- and (i, i+9)-stapled peptides with a single free alkyne positioned on the staple, that can be further conjugated or dimerised. A unique feature of the present method is that it provides easy access...

  6. Novel antimicrobial peptides with high anticancer activity and selectivity.

    Directory of Open Access Journals (Sweden)

    Hung-Lun Chu

    Full Text Available We describe a strategy to boost anticancer activity and reduce normal cell toxicity of short antimicrobial peptides by adding positive charge amino acids and non-nature bulky amino acid β-naphthylalanine residues to their termini. Among the designed peptides, K4R2-Nal2-S1 displayed better salt resistance and less toxicity to hRBCs and human fibroblast than Nal2-S1 and K6-Nal2-S1. Fluorescence microscopic studies indicated that the FITC-labeled K4R2-Nal2-S1 preferentially binds cancer cells and causes apoptotic cell death. Moreover, a significant inhibition in human lung tumor growth was observed in the xenograft mice treated with K4R2-Nal2-S1. Our strategy provides new opportunities in the development of highly effective and selective antimicrobial and anticancer peptide-based therapeutics.

  7. GEO label: The General Framework for Labeling and Certification

    Science.gov (United States)

    Bye, B. L.; McCallum, I.; Maso, J.

    2012-04-01

    The Group on Earth Observations (GEO) is coordinating efforts to build a Global Earth Observation System of Systems, or GEOSS. As part of a strategy to increase the involvement of the science and technology community in GEOSS, both as users and developers of GEOSS itself, GEO decided to develop a GEO label concept related to the scientific relevance, quality, acceptance and societal needs for services and data sets of GEOSS. The development of a GEO label is included in the GEO work plan and several projects address the challenges of developing a GEO label concept. Within the different projects developing the GEO label, various perspectives and approaches are being applied. In order to arrive at a generally accepted GEO label concept, a common understanding and basic knowledge of labeling is necessary. Assessment of quality of internationally standardized Earth observation data products implies possible certification. A general understanding of the framework for international standards and certification will also contribute to a more coherent discussion and more efficient development of a GEO label. We will describe the general labeling and certification framework emphasizing the relation to the three elements of the GEO label: quality, user acceptance and relevance. Based on a survey of international labels done by the EGIDA project, we have analyzed the legal framework and organization of labels and certification. We will discuss the frameworks for certification, user ratings, registration and analysis of user requirements. Quality assessment is a particular focus of the analysis and is based on the work done by the GeoViQua project. A GEO label will function both as a data distribution strategy and as a general management system for data. Through a label users can compare different data sets and get access to more information about the relevant data, including quality. A label will provide traceability of data both in the interest of users as well as data

  8. Electron transfer in peptides.

    Science.gov (United States)

    Shah, Afzal; Adhikari, Bimalendu; Martic, Sanela; Munir, Azeema; Shahzad, Suniya; Ahmad, Khurshid; Kraatz, Heinz-Bernhard

    2015-02-21

    In this review, we discuss the factors that influence electron transfer in peptides. We summarize experimental results from solution and surface studies and highlight the ongoing debate on the mechanistic aspects of this fundamental reaction. Here, we provide a balanced approach that remains unbiased and does not favor one mechanistic view over another. Support for a putative hopping mechanism in which an electron transfers in a stepwise manner is contrasted with experimental results that support electron tunneling or even some form of ballistic transfer or a pathway transfer for an electron between donor and acceptor sites. In some cases, experimental evidence suggests that a change in the electron transfer mechanism occurs as a result of donor-acceptor separation. However, this common understanding of the switch between tunneling and hopping as a function of chain length is not sufficient for explaining electron transfer in peptides. Apart from chain length, several other factors such as the extent of the secondary structure, backbone conformation, dipole orientation, the presence of special amino acids, hydrogen bonding, and the dynamic properties of a peptide also influence the rate and mode of electron transfer in peptides. Electron transfer plays a key role in physical, chemical and biological systems, so its control is a fundamental task in bioelectrochemical systems, the design of peptide based sensors and molecular junctions. Therefore, this topic is at the heart of a number of biological and technological processes and thus remains of vital interest.

  9. Sustainability labels on food products

    DEFF Research Database (Denmark)

    Grunert, Klaus G; Hieke, Sophie; Wills, Josephine

    2014-01-01

    This study investigates the relationship between consumer motivation, understanding and use of sustainability labels on food products (both environmental and ethical labels), which are increasingly appearing on food products. Data was collected by means of an online survey implemented in the UK......, human values as measured by the Schwartz value domains, and country differences. The results imply that sustainability labels currently do not play a major role in consumers’ food choices, and future use of these labels will depend on the extent to which consumers’ general concern about sustainability...

  10. Studies on distribution and imaging of new 99Tcm-labelled RGD peptide in synovial pannus of mice%采用99Tcm标记的新精氨酸-甘氨酸-天冬氨酸多肽序列显示胶原诱导性关节炎大鼠滑膜血管翳形成的研究

    Institute of Scientific and Technical Information of China (English)

    达古拉; 李鸿斌; 王勇

    2013-01-01

    arginine-glycineaspartic(RGD)peptide(99Tcm-3P4-RGD2)in collagen-induced arthritis(CIA),and to investigate the possibility of new markers for monitoring the angiogenesis of rheumatoid arthritis(RA)before and after treatment by single photon emission computed tomography(SPECT)labelled with 99Tcm-3P4-RGD2.Methods Type Ⅱ chicken collagen was injected into the tails of Wistar rats to establish arthritis models.The CIA-Wistar rats were randomly divided into three group:the Avastin group,the recombinant human tumor necrosis factor-α receptor Ⅱ group,and the blank control group.The changes of the arthritis index,joint pad thickness and radiological of T/NT before and after treatment were observed.The level of vascular endothelial growth factor(VEGF)in the serum was measured by enzyme-linked immuno sorbent assay(ELISA).The back metapedes pathological section was analyzed by Hematoxylin and Eosin stain(HE)and Safranin O stain as well as arthritic pathology index was also scored.The synovial expression level of VEGF was measured by immuno-histochemistry.The stain level of VEGF was assayed by semiquantitative approach.T test was applied for the comparison of two intergroup samples.Results Abnormal radioactive concentration appeared in the arthritis of the CIA-Wistar rats(1.40±0.17,1.32±0.20,1.30±0.08,P<0.05).Through the treatment of Avastin and recombinant human tumor necrosis factor-α receptor Ⅱ,the radiological of T/NT decreased significantly(0.43±0.14,0.40±0.12,t=17.710,16.812,P<0.05).After the treatment with Avastin and recombinant human tumor necrosis factor-α receptor Ⅱ,the severity of synovial pannus,joint pad thickness,arthritis index and the expression of VEGF were all decreased.The radiological of T/NT was positively correlated with the indices mentioned above(r=0.753,0.800,0.892,all P<0.01 respectively with the the rhTNFR:Fc group)(r=0.701,P=0.001;r=0.502,P=0.024;r=0.481,P=0.032 individually with the rhTNFR:Fc group).Conclusion When 99Tcm-3P4-RGD2 is

  11. Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli

    Science.gov (United States)

    Di Paolo, Diana; Afanzar, Oshri; Armitage, Judith P.; Berry, Richard M.

    2016-01-01

    For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time. This article is part of the themed issue ‘The new bacteriology’. PMID:27672145

  12. 76 FR 75809 - Prior Label Approval System: Generic Label Approval

    Science.gov (United States)

    2011-12-05

    ... Food Standards and Labeling Policy Book; does not bear any special claims, such as quality claims... addition, deletion, or amendment of recipe suggestions for the product; j. Any change in punctuation; k... Policy Book, and questions and answers on various topics, such as irradiation and the labeling...

  13. Autoradiographic localization of a gluten peptide during organ culture of human duodenal mucosa

    Energy Technology Data Exchange (ETDEWEB)

    Fluge, G.; Aksnes, L.

    1983-01-01

    An 125I-labeled subfraction of Frazer's fraction III (molecular weight, 8,000) was added to the culture medium during organ culture of duodenal biopsies from two patients with celiac disease in exacerbation. The isotope-labeled gluten peptide was localized by autoradiography after 6, 12, and 24 h of culture. At 6 h, labeling was located mainly in the basal layers of the biopsies. The tissue was well preserved. After 12 h in culture, the labeling had spread to the lamina propria and the crypts. A few grains were located over enterocytes and desquamated cells. Moderate histological signs of toxicity were observed. After 24 h, there was marked toxic deterioration, comparable to that seen after culture with alpha-gliadin. Labeling had spread throughout the entire section. There seemed to be no specificity of the binding, for the entire section was affected. Culture with the identical gluten fraction, in the radionegative state, produced histological deterioration comparable to that seen after exposure to the isotope-labeled peptide. Gluten peptides are presented to the target cells in a unique way during organ culture, different from in vivo conditions. This may influence the results when the organ culture method is used to investigate the pathogenesis of celiac disease.

  14. Quality Evaluation Model for Map Labeling

    Institute of Scientific and Technical Information of China (English)

    FAN Hong; ZHANG Zuxun; DU Daosheng

    2005-01-01

    This paper discusses and sums up the basic criterions of guaranteeing the labeling quality and abstracts the four basic factors including the conflict for a label with a label, overlay for label with the features, position's priority and the association for a label with its feature. By establishing the scoring system, a formalized four-factors quality evaluation model is constructed. Last, this paper introduces the experimental result of the quality evaluation model applied to the automatic map labeling system-MapLabel.

  15. Immunotherapy with Allergen Peptides

    Directory of Open Access Journals (Sweden)

    Larché Mark

    2007-06-01

    Full Text Available Specific allergen immunotherapy (SIT is disease-modifying and efficacious. However, the use of whole allergen preparations is associated with frequent allergic adverse events during treatment. Many novel approaches are being designed to reduce the allergenicity of immunotherapy preparations whilst maintaining immunogenicity. One approach is the use of short synthetic peptides which representing dominant T cell epitopes of the allergen. Short peptides exhibit markedly reduced capacity to cross link IgE and activate mast cells and basophils, due to lack of tertiary structure. Murine pre-clinical studies have established the feasibility of this approach and clinical studies are currently in progress in both allergic and autoimmune diseases.

  16. Therapeutic HIV Peptide Vaccine

    DEFF Research Database (Denmark)

    Fomsgaard, Anders

    2015-01-01

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

  17. β-PEPTIDES CYCLOBUTANIQUES

    OpenAIRE

    Fernandez, Carlos

    2008-01-01

    The synthesis of β-amino acids, structural analogues of?-Amino acids, is an issue essential in the development of oligopeptides. A lot of work has been conducted on the behavior of β-peptide (sequence of β-amino acids) as well as peptides mixed (mixed β-and β- amino acids). As a result, the conformational preference of β-amino acids will induce the appearance of a three-dimensional structure of the oligopeptide ordered. Thus, several types of helices, sheets and elbows were observed in β-olig...

  18. Invertebrate FMRFamide related peptides.

    Science.gov (United States)

    Krajniak, Kevin G

    2013-06-01

    In 1977 the neuropeptide FMRFamide was isolated from the clam, Macrocallista nimbosa. Since then several hundred FMRFamide-related peptides (FaRPs) have been isolated from invertebrate animals. Precursors to the FaRPs likely arose in the cnidarians. With the transition to a bilateral body plan FaRPs became a fixture in the invertebrate phyla. They have come to play a critical role as neurotransmitters, neuromodulators, and neurohormones. FaRPs regulate a variety of body functions including, feeding, digestion, circulation, reproduction, movement. The evolution of the molecular form and function of these omnipresent peptides will be considered.

  19. Dicyclopropylmethyl peptide backbone protectant.

    Science.gov (United States)

    Carpino, Louis A; Nasr, Khaled; Abdel-Maksoud, Adel Ali; El-Faham, Ayman; Ionescu, Dumitru; Henklein, Peter; Wenschuh, Holger; Beyermann, Michael; Krause, Eberhard; Bienert, Michael

    2009-08-20

    The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an amide bond protectant for peptide synthesis. Examples which demonstrate the amelioration of aggregation effects include syntheses of the alanine decapeptide and the prion peptide (106-126). Avoidance of cyclization to the aminosuccinimide followed substitution of Fmoc-(Dcpm)Gly-OH for Fmoc-Gly-OH in the assembly of sequences containing the sensitive Asp-Gly unit.

  20. Distance labeling schemes for trees

    DEFF Research Database (Denmark)

    Alstrup, Stephen; Gørtz, Inge Li; Bistrup Halvorsen, Esben;

    2016-01-01

    variants such as, for example, small distances in trees [Alstrup et al., SODA, 2003]. We improve the known upper and lower bounds of exact distance labeling by showing that 1/4 log2(n) bits are needed and that 1/2 log2(n) bits are sufficient. We also give (1 + ε)-stretch labeling schemes using Theta...

  1. Nutrition Marketing on Food Labels

    Science.gov (United States)

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

  2. Opposite modulation of cell migration by distinct subregions of urokinase connecting peptide.

    Science.gov (United States)

    Franco, Paola; Carotenuto, Alfonso; Marcozzi, Cristina; Votta, Giuseppina; Sarno, Ciro; Iaccarino, Ingram; Brancaccio, Diego; De Vincenzo, Anna; Novellino, Ettore; Grieco, Paolo; Stoppelli, Maria Patrizia

    2013-05-10

    Functional analysis of isolated protein domains may uncover cryptic activities otherwise missed. The serine protease urokinase (uPA) has a clear-cut motogen activity that is catalytically independent and resides in its amino-terminal growth factor domain (GFD, residues 1-49) and connecting peptide region (CP, residues 132-158). To functionally dissect the CP region, we analysed the biological activity of two synthetic peptides corresponding to the N-terminal [uPA-(135-143), residues 135-143] and C-terminal [uPA-(144-158), residues 144-158] CP subregions. Most of the chemotactic activity of connecting peptide-derived peptide (CPp, [uPA-(135-158)]) for embryonic kidney HEK293/uPAR-25 cells is retained by uPA-(144-158) at nanomolar concentrations. In contrast, uPA-(135-143) inhibits basal, CPp -, vitronectin- and fibronectin-induced cell migration. Radioreceptor binding assays on intact HEK293 cells revealed that uPA-(135-143) and uPA-(144-158) are both able to compete with [(125)I]-CPp, albeit with different binding affinities. The consequences of phospho-mimicking, S138E substitution, were studied using [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. Unlike CPp, [138E]uPA-(135-158) and [138E]uPA-(135-143) exhibit remarkable inhibitory properties. Finally, analysis of the conformational preferences of the peptides allowed to identify secondary structure elements exclusively characterising the stimulatory CPp and uPA-(144-158) versus the inhibitory uPA-(135-143), [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. In conclusion, these data shed light on the cryptic activities of uPA connecting peptide, revealing the occurrence of two adjacent regions, both competing for binding to cell surface but conveying opposite signalling on cell migration.

  3. Characteristic Time Scales of Transport Processes for Chemotactic Bacteria in Groundwater: Analysis of Pore-scale to Field-scale Experimental Data

    Science.gov (United States)

    Ford, R. M.

    2010-12-01

    Many processes contribute to the transport of microorganisms in groundwater environments. One process of interest is chemotaxis, whereby motile bacteria are able to detect and swim toward increasing concentrations of industrial hydrocarbons that they perceive as food sources. By enabling bacteria to migrate to the sources of pollutants that they degrade, chemotaxis has the potential to enhance bioremediation efforts, especially in less permeable zones where contamination may persist. To determine the field conditions under which chemotaxis might be exploited in a bioremediation scheme requires an understanding of the characteristic time scales in the system. We defined a dimensionless chemotaxis number that compares the time over which a bacterial population is exposed to a chemical gradient to the time required for a bacterial population to migrate a significant distance in response to a chemical gradient. The exposure time and the response time are dependent upon the experimental conditions and properties of the bacteria and chemical attractant. Experimental data was analyzed for a range of groundwater flow rates over a wide scope of experimental systems including a single-pore with NAPL source, a microfluidic channel with and without a porous matrix, a laboratory column, a bench-scale microcosm and a field-scale study. Chemical gradients were created transverse to the flow direction. Distributions of chemotactic and nonchemotactic bacteria were compared to determine the extent of migration due to chemotaxis. Under some conditions at higher flow rates, the effect of chemotaxis was diminished to the point of not being detected. The goal of the study was to determine a critical value for the dimensionless chemotaxis number (which is independent of scale) that can be used as a design criterion to ascertain a priori the conditions under which a chemotactic response will impact bacterial transport relative to other processes such as advection and dispersion.

  4. The strength of the chemotactic response to a CCR5 binding chemokine is determined by the level of cell surface CCR5 density.

    Science.gov (United States)

    Desmetz, Caroline; Lin, Yea-Lih; Mettling, Clément; Portalès, Pierre; Rabesandratana, Herisoa; Clot, Jacques; Corbeau, Pierre

    2006-12-01

    We have shown that the intensity of expression of the C-C chemokine receptor CCR5 at the single CD4(+) cell level strongly determines the efficiency of its function as a coreceptor for human immunodeficiency virus type 1. By analogy, we examined if the number of CCR5 molecules at the cell surface might determine its chemotactic response to CCR5 ligands. To test this hypothesis, we measured by flow cytometry the migration of primary human T cells towards the CCR5-binding chemokine CCL5 in vitro. First, we observed a dose-dependent blockage of this migration exerted by an anti-CCR5 monoclonal antibody. Second, we sorted peripheral blood mononuclear cells into five subpopulations expressing various cell surface CCR5 densities, and observed a correlation between the intensity of migration towards CCL5 and the level of CCR5 expression on these subpopulations. Third, we transduced CCR5(+) peripheral blood mononuclear cells with the CCR5 gene, and observed that the CCR5 over-expression induced an over-migration towards CCL5. Finally, we observed in healthy donors a correlation between the chemotactic response of peripheral blood CD8(+) T cell to CCL5 and their level of surface CCR5 expression. T-cell surface CCR5 density, which is constant over time for a given individual, but varies drastically among individuals, might therefore be an important personal determinant of T-cell migration in many biological situations where CCR5-binding chemokines play a role, such as graft rejection, T helper 1-mediated auto-immune diseases, and infectious diseases involving CCR5. Moreover, our data highlight the therapeutic potential of CCR5 antagonists in these situations.

  5. Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution

    Science.gov (United States)

    Silva, R. A. G. D.; Kubelka, Jan; Bour, Petr; Decatur, Sean M.; Keiderling, Timothy A.

    2000-01-01

    Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form α-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the α-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis. PMID:10880566

  6. Pulsed EPR in the method of spin labels and probes

    Energy Technology Data Exchange (ETDEWEB)

    Dzuba, Sergei A [Institute of Chemical Kinetics and Combustion, Siberian Branch of the Russian Academy of Sciences, Novosibirsk (Russian Federation)

    2007-08-31

    Various pulsed EPR in the method of spin labels and probes based on electron spin echo spectroscopy (spin echo envelope modulation through electron-nuclear interactions, electron-electron double resonance, echo detected EPR, etc.) are considered. These methods provide information on the conformations of complex biomolecules, nanostructure of matter, depth of water penetration into biological membranes, supramolecular structure of multicomponent systems (membrane-peptide, etc.), co-operative orientational dynamics of molecules and dynamic low-temperature transitions in disordered molecular media and biosystems.

  7. Semi-Synthesis of Labeled Proteins for Spectroscopic Applications

    Directory of Open Access Journals (Sweden)

    Luca Domenico D'Andrea

    2013-01-01

    Full Text Available Since the introduction of SPPS by Merrifield in the 60s, peptide chemists have considered the possibility of preparing large proteins. The introduction of native chemical ligation in the 90s and then of expressed protein ligation have opened the way to the preparation of synthetic proteins without size limitations. This review focuses on semi-synthetic strategies useful to prepare proteins decorated with spectroscopic probes, like fluorescent labels and stable isotopes, and their biophysical applications. We show that expressed protein ligation, combining the advantages of organic chemistry with the easy and size limitless recombinant protein expression, is an excellent strategy for the chemical synthesis of labeled proteins, enabling a single protein to be functionalized at one or even more distinct positions with different probes.

  8. A better carbon footprint label

    DEFF Research Database (Denmark)

    Thøgersen, John; Nielsen, Kristian S.

    2016-01-01

    Based on insights from behavioral economics, it is suggested to extend carbon footprint labeling with information about relative performance, using the well-known “traffic light” color scheme to communicate relative performance. To test this proposition, the impact of a carbon footprint label...... on Danish consumers’ choice of ground coffee was tested in a 3 price levels x 3 levels of carbon emission x 3 certifying organizations x 2 organic labeling conditions discrete choice experiment. Participants were randomly assigned to two slightly different variants of the experiment: In one condition......, participants saw the original Carbon Trust label and in the other condition they saw the same label, but with traffic light colors added to communicate the product’s relative performance in terms of carbon footprint. All included attributes were found to have a significant impact on consumer choices...

  9. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

    . An inefficient post-translational prohormone maturation will also affect the biology of the cardiac natriuretic peptide system. This review aims at summarizing the myocardial synthesis of natriuretic peptides focusing on B-type natriuretic peptide, where new data has disclosed cardiac myocytes as highly...

  10. 21 CFR 201.71 - Magnesium labeling.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  11. 16 CFR 460.12 - Labels.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  12. 21 CFR 610.60 - Container label.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Container label. 610.60 Section 610.60 Food and... GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.60 Container label. (a) Full label. The following items shall appear on the label affixed to each container of a product capable of bearing a...

  13. Optimization of iTRAQ labelling coupled to OFFGEL fractionation as a proteomic workflow to the analysis of microsomal proteins of Medicago truncatula roots

    Directory of Open Access Journals (Sweden)

    Abdallah Cosette

    2012-06-01

    Full Text Available Abstract Background Shotgun proteomics represents an attractive technical framework for the study of membrane proteins that are generally difficult to resolve using two-dimensional gel electrophoresis. The use of iTRAQ, a set of amine-specific isobaric tags, is currently the labelling method of choice allowing multiplexing of up to eight samples and the relative quantification of multiple peptides for each protein. Recently the hyphenation of different separation techniques with mass spectrometry was used in the analysis of iTRAQ labelled samples. OFFGEL electrophoresis has proved its effectiveness in isoelectric point-based peptide and protein separation in solution. Here we describe the first application of iTRAQ-OFFGEL-LC-MS/MS on microsomal proteins from plant material. The investigation of the iTRAQ labelling effect on peptide electrofocusing in OFFGEL fractionator was carried out on Medicago truncatula membrane protein digests. Results In-filter protein digestion, with easy recovery of a peptide fraction compatible with iTRAQ labelling, was successfully used in this study. The focusing quality in OFFGEL electrophoresis was maintained for iTRAQ labelled peptides with a higher than expected number of identified peptides in basic OFFGEL-fractions. We furthermore observed, by comparing the isoelectric point (pI fractionation of unlabelled versus labelled samples, a non-negligible pI shifts mainly to higher values. Conclusions The present work describes a feasible and novel protocol for in-solution protein digestion in which the filter unit permits protein retention and buffer removal. The data demonstrates an impact of iTRAQ labelling on peptide electrofocusing behaviour in OFFGEL fractionation compared to their native counterpart by the induction of a substantial, generally basic pI shift. Explanations for the occasionally observed acidic shifts are likewise presented.

  14. Comparison of Two Kinds of 64Cu Labelled Octreotide Analogues

    Directory of Open Access Journals (Sweden)

    HAN Zhen-yi1;LIANG Ji-xin1;HU Ji2;LUO Hong-yi1;QING Jing2;CHEN Yu-qing2;LI Guang2;LI Hong-yu1,2

    2016-10-01

    Full Text Available Octreotide analogues DOTA-TOC and DOTA-TATE were labeled with 64Cu. The influences of the ratio of peptide mass to 64Cu activity, pH value, temperature and reaction time on labeling yield were investigated. The optimum labeling was determined. In vitro stability tests in saline and 10% bovine serum had been carried out. Biodistribution of the two radiolabelled compounds in normal mice and Micro PET imaging in nude mice bearing U87MG tumor had been evaluated. The results showed that the labeling yields of 64Cu-DOTA-TOC and 64Cu-DOTA-TATE were higher than 95%. Two kinds of octreotide analogues labeled with 64Cu were quite stable in saline and decomposed slowly in 10% bovine serum at 37 ℃. Biodistribution results in normal mice showed that two 64Cu labelled tracers had similar profiles. Both of the compounds washed out from the blood quickly. High uptake of radioactivity in liver and kidneys indicated the tracers were excreted via both hepatobiliary system and renal system. At the same time, compared to 64Cu-DOTA-TOC, higher radioactivity accumulation of 64Cu-DOTA-TATE in liver and kidneys was observed. Micro PET images of U87MG tumor-bearing nude mice with 64Cu-DOTA-TOC and 64Cu-DOTA-TATE showed the tumors very clearly. The radioactivity uptake of 64Cu-DOTA-TATE in tumor was higher than that of 64Cu-DOTA-TOC. This work has paved the way for further preclinical and clinical application of 64Cu-DOTA-TOC and 64Cu-DOTA-TATE as PET tumor imaging agents.

  15. Peptide vectors for gene delivery: from single peptides to multifunctional peptide nanocarriers.

    Science.gov (United States)

    Raad, Markus de; Teunissen, Erik A; Mastrobattista, Enrico

    2014-07-01

    The therapeutic use of nucleic acids relies on the availability of sophisticated delivery systems for targeted and intracellular delivery of these molecules. Such a gene delivery should possess essential characteristics to overcome several extracellular and intracellular barriers. Peptides offer an attractive platform for nonviral gene delivery, as several functional peptide classes exist capable of overcoming these barriers. However, none of these functional peptide classes contain all the essential characteristics required to overcome all of the barriers associated with successful gene delivery. Combining functional peptides into multifunctional peptide vectors will be pivotal for improving peptide-based gene delivery systems. By using combinatorial strategies and high-throughput screening, the identification of multifunctional peptide vectors will accelerate the optimization of peptide-based gene delivery systems.

  16. Synthesis Of Labeled Metabolites

    Science.gov (United States)

    Martinez, Rodolfo A.; Silks, III, Louis A.; Unkefer, Clifford J.; Atcher, Robert

    2004-03-23

    The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

  17. Labeled Cocaine Analogs

    Science.gov (United States)

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-01-26

    Novel compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)naphthyl Y in .beta. configuration is Y.sub.1 or Y.sub.2, where Y.sub.1 is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, and Y.sub.2 is 2-methanesulfonyloxy ethoxy, 3-methanesulfonyloxy propoxy, 4-methanesulfonyloxy butoxy, 2-methanesulfonyloxy cyclopropoxy, 2 or 3-methanesulfonyloxy cyclobutoxy, 1'methanesulfonyloxy isopropoxy, 1'-fluoro, 3'-methanesulfonyloxy isopropoxy, 1'-methanesulfonyloxy, 3'-fluoro isopropoxy, 1'-methanesulfonyloxy isobutoxy, or 4'-methanesulfonyloxy isobutoxy bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  18. Biochemical functionalization of peptide nanotubes with phage displayed peptides

    Science.gov (United States)

    Swaminathan, Swathi; Cui, Yue

    2016-09-01

    The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

  19. A Probabilistic Framework for Peptide and Protein Quantification from Data-Dependent and Data-Independent LC-MS Proteomics Experiments

    DEFF Research Database (Denmark)

    Richardson, Katherine; Denny, R.; Hughes, C.

    2012-01-01

    A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data...

  20. Immune regulatory activities of fowlicidin-1, a cathelicidin host defense peptide.

    Science.gov (United States)

    Bommineni, Yugendar R; Pham, Giang H; Sunkara, Lakshmi T; Achanta, Mallika; Zhang, Guolong

    2014-05-01

    Appropriate modulation of immunity is beneficial in antimicrobial therapy and vaccine development. Host defense peptides (HDPs) constitute critically important components of innate immunity with both antimicrobial and immune regulatory activities. We previously showed that a chicken HDP, namely fowlicidin-1(6-26), has potent antibacterial activities in vitro and in vivo. Here we further revealed that fowl-1(6-26) possesses strong immunomodulatory properties. The peptide is chemotactic specifically to neutrophils, but not monocytes or lymphocytes, after injected into the mouse peritoneum. Fowl-1(6-26) also has the capacity to activate macrophages by inducing the expression of inflammatory mediators including IL-1β, CCL2, and CCL3. However, unlike bacterial lipopolysaccharide that triggers massive production of inflammatory cytokines and chemokines, fowl-1(6-26) only marginally increased their expression in mouse RAW264.7 macrophages. Additionally, fowl-1(6-26) enhanced the surface expression of MHC II and CD86 on RAW264.7 cells, suggesting that it may facilitate development of adaptive immune response. Indeed, co-immunization of mice with chicken ovalbumin (OVA) and fowl-1(6-26) augmented both OVA-specific IgG1 and IgG2a titers, relative to OVA alone. We further showed that fowl-1(6-26) is capable of preventing a methicillin-resistant Staphylococcus aureus (MRSA) infection due to its enhancement of host defense. All mice survived from an otherwise lethal infection when the peptide was administered 1-2 days prior to MRSA infection, and 50% mice were protected if receiving the peptide 4 days before infection. Taken together, with a strong capacity to stimulate innate and adaptive immunity, fowl-1(6-26) may have potential to be developed as a novel antimicrobial and a vaccine adjuvant.

  1. Structure elucidation of uniformly 13C labeled small molecule natural products.

    Science.gov (United States)

    Reibarkh, Mikhail; Wyche, Thomas P; Saurí, Josep; Bugni, Tim S; Martin, Gary E; Williamson, R Thomas

    2015-12-01

    Utilization of isotopically labeled proteins and peptides is a routinely employed approach in biomolecular NMR investigations. The widespread availability of inexpensive, uniformly (13) C-enriched glucose now makes it possible to produce uniformly (13) C-labeled natural products by microbial fermentation. In this feature article, the authors describe an experimental approach for the rapid structural characterization of uniformly (13) C-labeled natural products based on the Constant-Time HSQC (CT-HSQC) experiment. Rigorous theoretical evaluation of the CT-HSQC experiment allowed the applicability of the experiment to be expanded from the traditional, narrow scope of labeled amino acids to encompass virtually any small molecule or U-(13) C labeled natural product. A suite of experiments including CT-HSQC, (13) C-(13) C COSY, and COSYLR experiments is sufficient for the structure elucidation of uniformly (13) C-labeled small molecules and natural products. Differences in NMR approaches for structure elucidation of natural abundance and uniformly (13) C-labeled molecules are also discussed. The present work provides a researcher working in this area of natural products chemistry with NMR structure elucidation tools for investigating (13) C-labeled small molecules and natural products.

  2. Invention of stimulus-responsive peptide-bond-cleaving residue (Spr) and its application to chemical biology tools.

    Science.gov (United States)

    Shigenaga, Akira; Yamamoto, Jun; Kohiki, Taiki; Inokuma, Tsubasa; Otaka, Akira

    2017-01-19

    Elucidation of biological functions of peptides and proteins is essential for understanding peptide/protein-related biological events and developing drugs. Caged peptides and proteins that release a parent active peptide/protein by photo-irradiation have successfully been employed to elucidate the functions. Whereas the usual caged peptide/protein enables conversion of an inactive form to an active form (OFF-to-ON conversion) by photo-induced deprotection, photo-triggered main chain cleavage is reported to be applicable to ON-to-OFF conversion. These peptides and proteins are photo-responsive; however, if peptides and proteins could respond to other stimuli such as disease-related environment or enzymes, their range of application should be widened. To convert the photo-responsive peptide/protein into other stimulus-responsive peptide/protein, quite laborious de novo design and synthesis of the stimulus-responsive unit are required. In this context, we designed a stimulus-responsive peptide-bond-cleaving residue (Spr) in which the stimuli available for the main chain cleavage vary according to the choice of protecting groups on the residue. In this review, design and synthesis of Spr are introduced, and challenges to apply Spr to other fields to enable, for example, functional control, localization control, delivery of cargos, labeling of a protein of interest in living cells, and identification of target proteins of bioactive ligands are discussed. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  3. Algorithms for Labeling Focus Regions.

    Science.gov (United States)

    Fink, M; Haunert, Jan-Henrik; Schulz, A; Spoerhase, J; Wolff, A

    2012-12-01

    In this paper, we investigate the problem of labeling point sites in focus regions of maps or diagrams. This problem occurs, for example, when the user of a mapping service wants to see the names of restaurants or other POIs in a crowded downtown area but keep the overview over a larger area. Our approach is to place the labels at the boundary of the focus region and connect each site with its label by a linear connection, which is called a leader. In this way, we move labels from the focus region to the less valuable context region surrounding it. In order to make the leader layout well readable, we present algorithms that rule out crossings between leaders and optimize other characteristics such as total leader length and distance between labels. This yields a new variant of the boundary labeling problem, which has been studied in the literature. Other than in traditional boundary labeling, where leaders are usually schematized polylines, we focus on leaders that are either straight-line segments or Bezier curves. Further, we present algorithms that, given the sites, find a position of the focus region that optimizes the above characteristics. We also consider a variant of the problem where we have more sites than space for labels. In this situation, we assume that the sites are prioritized by the user. Alternatively, we take a new facility-location perspective which yields a clustering of the sites. We label one representative of each cluster. If the user wishes, we apply our approach to the sites within a cluster, giving details on demand.

  4. How to Read a Nutrition Facts Label

    Medline Plus

    Full Text Available ... las etiquetas de datos nutricionales (video) Most packaged foods come with a Nutrition Facts label. These labels ... Watch this video for tips on figuring out food labels so you can make healthy choices. For ...

  5. Radiolabeled CCK/gastrin peptides for imaging and therapy of CCK2 receptor-expressing tumors

    NARCIS (Netherlands)

    Roosenburg, S.; Laverman, P.; Delft, F.L. van; Boerman, O.C.

    2011-01-01

    Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, like medullary thyroid carcinomas, small cell lung cancers and stromal ovarian cancers. The specific receptor-binding property of the endogenous ligands for these receptors can be exploited by labeling peptides with a radio

  6. Applying fluorescence correlation spectroscopy to investigate peptide-induced membrane disruption

    DEFF Research Database (Denmark)

    Kristensen, Kasper; Henriksen, Jonas Rosager; Andresen, Thomas Lars

    2017-01-01

    to quantify leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles, thereby providing a tool for estimating the size of peptide-induced membrane disruptions. If fluorescently labeled lipids are incorporated into the membranes of the vesicles, FCS can also be used to obtain...

  7. Peptide-evoked release of amylase from isolated acini of the rat parotid gland

    DEFF Research Database (Denmark)

    Goll, R; Poulsen, J H; Schmidt, P

    1994-01-01

    in extracts of the gland. The immunoreactivity of these peptides could be located to varicose nerve fibers in the gland. Binding of labeled SP to the isolated acini exhibited the characteristics of a genuine agonist/receptor interaction, and the rank order of displacement potencies indicated the presence...

  8. Multi-frequency, multi-technique pulsed EPR investigation of the copper binding site of murine amyloid β peptide.

    Science.gov (United States)

    Kim, Donghun; Bang, Jeong Kyu; Kim, Sun Hee

    2015-01-26

    Copper-amyloid peptides are proposed to be the cause of Alzheimer's disease, presumably by oxidative stress. However, mice do not produce amyloid plaques and thus do not suffer from Alzheimer's disease. Although much effort has been focused on the structural characterization of the copper- human amyloid peptides, little is known regarding the copper-binding mode in murine amyloid peptides. Thus, we investigated the structure of copper-murine amyloid peptides through multi-frequency, multi-technique pulsed EPR spectroscopy in conjunction with specific isotope labeling. Based on our pulsed EPR results, we found that Ala2, Glu3, His6, and His14 are directly coordinated with the copper ion in murine amyloid β peptides at pH 8.5. This is the first detailed structural characterization of the copper-binding mode in murine amyloid β peptides. This work may advance the knowledge required for developing inhibitors of Alzheimer's disease.

  9. Cyanine-based probe\\tag-peptide pair for fluorescence protein imaging and fluorescence protein imaging methods

    Science.gov (United States)

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2010-08-17

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  10. Labelling of benzocaine with tritium

    Energy Technology Data Exchange (ETDEWEB)

    Malik, Sohail (Washington Univ., Seattle, WA (United States))

    1994-10-01

    A convenient method is described to label a local anesthetic, benzocaine, with tritium. The bromoester of para-aminobenzoic acid (PABA) was prepared from para-nitrotoluene and was reduced with tritium. The generation of isotopic hydrogen and labelling of benzocaine was achieved in one-step. A mixture of sodium borohydride (NaB[sup 3]H[sub 4]) with cobalt (II) chloride was used to generate tritium gas. 5% Pd/C was used as a catalyst. This constitutes the first report of tritium labelled benzocaine. (author).

  11. New labels for radiation therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kubota, Susumu; Mukai, Minoru; Kato, Hirotoshi (National Inst. of Radiological Sciences, Chiba (Japan))

    1992-12-01

    In simulating radiotherapy, the bone and trachea identified by plain X-P and the other organs, such as the esophagus and bladder, outlined by contrast medium have so far been used as labels. However, irradiation with a high therapeutic ratio is required for an intracorporeal insertion of artificial labels that are identified by X-ray fluoroscopy. For this purpose, metal clips and seed dummies are available, although they cause artifacts in CT scans. Therefore, the authors are using an acupuncture needle and lipiodol for tracing as new artificial labels, since both are identified by X-ray fluoroscopy and CT scan and create few artifacts. (J.P.N.).

  12. APD: the Antimicrobial Peptide Database.

    Science.gov (United States)

    Wang, Zhe; Wang, Guangshun

    2004-01-01

    An antimicrobial peptide database (APD) has been established based on an extensive literature search. It contains detailed information for 525 peptides (498 antibacterial, 155 antifungal, 28 antiviral and 18 antitumor). APD provides interactive interfaces for peptide query, prediction and design. It also provides statistical data for a select group of or all the peptides in the database. Peptide information can be searched using keywords such as peptide name, ID, length, net charge, hydrophobic percentage, key residue, unique sequence motif, structure and activity. APD is a useful tool for studying the structure-function relationship of antimicrobial peptides. The database can be accessed via a web-based browser at the URL: http://aps.unmc.edu/AP/main.html.

  13. Iodinated Australian brown snake venom peptides as potential thrombus imaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Jena, M.J.; Eu, P.; Kelly, M.J. [Alfred Hospital, Prahran, VIC (Australia). Dept. of Nuclear Medicine

    1997-09-01

    Full text: A 54 kDa protein component isolated from the venom of Australian brown snake, Pseudonaja textilis textilis, which was found to activate prothrombin, was investigated for its potential as a thrombus imaging agent. Haematological affinity of the iodinated peptide was tested in vitro. The peptide was labelled with {sup 125}l using the lodogen method. Fresh blood samples were allowed to clot over a 24-hour period. The clots were then washed three times with 0.01 M phosphate buffered saline (PBS) and incubated for 30 min with purified labelled peptide or free radioiodine. For each sample, the percentage uptake in the clot was then calculated to determine specific and non-specific uptake. The degree of binding to cellular components of blood (CCB) was established by incubating fresh heparinised blood for 30 min with labelled peptide. Preliminary studies indicate that the radiolabelled peptide has a statistically significant specific affinity for formed thrombus, with only minor uptake in cellular blood components. These results suggest that further investigation of this agent for thrombus imaging is warranted. 1 tab.

  14. General Compact Labeling Schemes for Dynamic Trees

    OpenAIRE

    2006-01-01

    Let $F$ be a function on pairs of vertices. An {\\em $F$- labeling scheme} is composed of a {\\em marker} algorithm for labeling the vertices of a graph with short labels, coupled with a {\\em decoder} algorithm allowing one to compute $F(u,v)$ of any two vertices $u$ and $v$ directly from their labels. As applications for labeling schemes concern mainly large and dynamically changing networks, it is of interest to study {\\em distributed dynamic} labeling schemes. This paper investigates labelin...

  15. Antimicrobial Peptides (AMPs

    Directory of Open Access Journals (Sweden)

    Mehrzad Sadredinamin

    2016-04-01

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

  16. Antimicrobial peptides in Echinoderms

    Directory of Open Access Journals (Sweden)

    C Li

    2010-05-01

    Full Text Available Antimicrobial peptides (AMPs are important immune effector molecules for invertebrates, including echinoderms, which lack a vertebrate-type adaptive immune system. Here we summarize the knowledge of such peptides in echinoderms. Strongylocins are a novel family of cysteine-rich AMPs, recently identified in the sea urchins, Strongylocentrotus droebachiensis and S. purpuratus. Although these molecules present diverse amino acid sequences, they share an identical cysteine arrangement pattern, dissimilar to other known AMPs. A family of heterodimeric AMPs, named centrocins, are also present in S. droebachiensis. Lysozymes and fragments of larger proteins, such as beta-thymocins, actin, histone 2A and filamin A have also been shown to display antimicrobial activities in echinoderms. Future studies on AMPs should be aimed in revealing how echinoderms use these AMPs in the immune response against microbial pathogens.

  17. Avian host defense peptides.

    Science.gov (United States)

    Cuperus, Tryntsje; Coorens, Maarten; van Dijk, Albert; Haagsman, Henk P

    2013-11-01

    Host defense peptides (HDPs) are important effector molecules of the innate immune system of vertebrates. These antimicrobial peptides are also present in invertebrates, plants and fungi. HDPs display broad-spectrum antimicrobial activities and fulfill an important role in the first line of defense of many organisms. It is becoming increasingly clear that in the animal kingdom the functions of HDPs are not confined to direct antimicrobial actions. Research in mammals has indicated that HDPs have many immunomodulatory functions and are also involved in other physiological processes ranging from development to wound healing. During the past five years our knowledge about avian HDPs has increased considerably. This review addresses our current knowledge on the evolution, regulation and biological functions of HDPs of birds.

  18. New Ideas on Labeling Schemes

    DEFF Research Database (Denmark)

    Rotbart, Noy Galil

    evaluation of fully dynamic labeling schemes. Due to a connection between adjacency labeling schemes and the graph theoretical study of induced universal graphs, we study these in depth and show novel results for bounded degree graphs and power-law graphs. We also survey and make progress on the related......With ever increasing size of graphs, many distributed graph systems emerged to store, preprocess and analyze them. While such systems ease up congestion on servers, they incur certain penalties compared to centralized data structure. First, the total storage required to store a graph...... in a distributed fashion increases. Second, attempting to answer queries on vertices of a graph stored in a distributed fashion can be significantly more complicated. In order to lay theoretical foundations to the first penalty mentioned a large body of work concentrated on labeling schemes. A labeling scheme...

  19. Off-Label Drug Use

    Science.gov (United States)

    ... it’s not legal for drug companies to market (advertise or promote) their drugs for off-label uses. ... Statistics Center Volunteer Learning Center Follow Us Twitter Facebook Instagram Cancer Information, Answers, and Hope. Available Every ...

  20. Peptides and Food Intake

    OpenAIRE

    Carmen Sobrino Crespo; Aranzazu Perianes Cachero; Lilian Puebla Jiménez; Vicente eBarrios; Eduardo eArilla

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the r...

  1. Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

    Science.gov (United States)

    Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

    2010-09-15

    The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase.

  2. [C-peptide physiological effects].

    Science.gov (United States)

    Shpakov, A O; Granstrem, O K

    2013-02-01

    In the recent years there were numerous evidences that C-peptide, which was previously considered as a product of insulin biosynthesis, is one of the key regulators of physiological processes. C-peptide via heterotrimeric G(i/o) protein-coupled receptors activates a wide range of intracellular effector proteins and transcription factors and, thus, controls the inflammatory and neurotrophic processes, pain sensitivity, cognitive function, macro- and microcirculation, glomerular filtration. These effects of C-peptide are mainly expressed in its absolute or relative deficiency occurred in type 1 diabetes mellitus and they are less pronounced when the level of C-peptide is close to normal. Replacement therapy with C-peptide prevents many complications of type 1 diabetes, such as atherosclerosis, diabetic peripheral neuropathy, and nephropathy. C-peptide interacts with the insulin hexamer complexes and induces their dissociation and, as a result, regulates the functional activity of the insulin signaling system. At the same time, C-peptide at the concentrations above physiological may demonstrate pro-inflammatory effects on the endothelial cells and cause atherosclerotic changes in the vessels, which should be considered in the study of pathogenic mechanisms of complications of type 2 diabetes mellitus, where the level of C peptide is increased, as well as in the development of approaches for C-peptide application in clinic. This review is devoted contemporary achievements and unsolved problems in the study of C-peptide, as an important regulator of physiological and biochemical processes.

  3. Photoaffinity labeling of cytochrome P4501A1 with azidocumene: identification of cumene hydroperoxide binding region.

    Science.gov (United States)

    Cvrk, T; Strobel, H W

    1998-01-01

    Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which cumene hydroperoxide binds, we prepared an analog of cumene hydroperoxide for use as a photoaffinity label. p-Azido-isopro-pylbenzene (azidocumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [3H]-azidocumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492-503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494-512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501-504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501-L502-K503, is the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A

  4. Odd-Even Sequence Effect of Surface-Mediated Peptide Assemblies Observed by Scanning Tunneling Microscopy

    Institute of Scientific and Technical Information of China (English)

    郭元元; 王晨轩; 侯静菲; 杨爱华; 张雪梅; 王宜冰; 张敏; 杨延莲; 王琛

    2012-01-01

    The peptide assembly structures of polyglutamine (PolyQ) have been studied by using scanning tunneling microscopy (STM) with high spatial resolution in ambient conditions. 4,4'-Bipyridyl (4Bpy) was introduced into the PolyQ7 and PolyQ8 peptide assemblies for labeling the C-termini of the peptides. The fine structures of the 4Bpy-PolyQ7 and 4Bpy-PolyQ8 co-assemblies are observed, and the statistics of the apparent peptide strand length reveal different length distributions for PolyQ7 and PolyQs. One predominant apparent peptide strand length is ob- served for PolyQ7 reflecting one predominant peptide conformation in assembly structures, while three major ap- parent strand lengths can be identified with PolyQ8 reflecting three co-existing peptide conformations in peptide as- semblies. Such drastic difference in assembling characteristics can be considered as a reflection of asymmetric ad- sorption effect ofpeptides relating to odd-even residue numbers of PolyQ7 and PolyQ8,

  5. CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Rosemeire A. Silva

    2016-08-01

    Full Text Available The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr−/− mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05, when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr−/− mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC was synthesized and labeled with 111InCl3 in >95% yield as determined by high performance liquid chromatography (HPLC, to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases.

  6. Rapid and Reliable Detection of Antimicrobial Peptide Penetration into Gram-Negative Bacteria Based on Fluorescence Quenching▿

    OpenAIRE

    Benincasa, Monica; Pacor, Sabrina; Gennaro, Renato; Scocchi, Marco

    2009-01-01

    In this paper, we describe a rapid flow cytometry method to identify antimicrobial peptides that are internalized into bacterial cells and differentiate them from those that are membrane active. The method was applied to fluorescently labeled Bac71-35 and polymyxin B, whose mechanisms of action are, respectively, based on cell penetration and on membrane binding and permeabilization. Identification of peptides with the former mechanism is of considerable interest for the intracellular deliver...

  7. Rapid and Reliable Detection of Antimicrobial Peptide Penetration into Gram-Negative Bacteria Based on Fluorescence Quenching▿

    Science.gov (United States)

    Benincasa, Monica; Pacor, Sabrina; Gennaro, Renato; Scocchi, Marco

    2009-01-01

    In this paper, we describe a rapid flow cytometry method to identify antimicrobial peptides that are internalized into bacterial cells and differentiate them from those that are membrane active. The method was applied to fluorescently labeled Bac71-35 and polymyxin B, whose mechanisms of action are, respectively, based on cell penetration and on membrane binding and permeabilization. Identification of peptides with the former mechanism is of considerable interest for the intracellular delivery of membrane-impermeant drugs. PMID:19470515

  8. {sup 123}I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor, p21{sup WAF-1/Cip-1}

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Meiduo [University Health Network, Division of Nuclear Medicine, Toronto, ON (Canada); University of Toronto, Department of Pharmaceutical Sciences, Toronto, ON (Canada); Chen, Paul; Wang, Judy; Scollard, Deborah A. [University Health Network, Division of Nuclear Medicine, Toronto, ON (Canada); Vallis, Katherine A. [University Health Network, Department of Radiation Oncology, Toronto, ON (Canada); University of Toronto, Department of Medical Biophysics, Toronto, ON (Canada); Reilly, Raymond M. [University Health Network, Division of Nuclear Medicine, Toronto, ON (Canada); University of Toronto, Department of Pharmaceutical Sciences, Toronto, ON (Canada); University of Toronto, Department of Medical Imaging, Toronto, ON (Canada); University of Toronto, Leslie Dan Faculty of Pharmacy, Toronto, ON (Canada)

    2007-03-15

    To evaluate the internalization and nuclear translocation of {sup 123}I-tat-peptide radioimmunoconjugates in MDA-MB-468 breast cancer cells and their ability to interact with the cyclin-dependent kinase inhibitor, p21{sup WAF-1/Cip-1}. Peptides [GRKKRRQRRRPPQGYGC] harboring the nuclear-localizing sequence from HIV tat domain were conjugated to anti-p21{sup WAF-1/Cip-1} antibodies. Immunoreactivity was assessed by Western blot using lysate from MDA-MB-468 cells exposed to EGF to induce p21{sup WAF-1/Cip-1}. Internalization and nuclear translocation were measured. The ability of tat-anti-p21{sup WAF-1/Cip-1} to block G{sub 1}-S phase arrest in MDA-MB-468 cells caused by EGF-induced p21{sup WAF-1/Cip-1} was evaluated. Tumor and normal tissue uptake were determined at 48 h p.i. in athymic mice implanted s.c. with MDA-MB-468 xenografts injected intratumorally with EGF. There was 13.4{+-}0.2% of radioactivity internalized by MDA-MB-468 cells incubated with {sup 123}I-tat-anti-p21{sup WAF-1/Cip-1} and 34.6{+-}3.1% imported into the nucleus. Tat-anti-p21{sup WAF-1/Cip-1}(8 {mu}M) decreased the proportion of EGF-treated cells in G{sub 1} phase from 81.9{+-}0.7% to 46.1{+-}0.7% (p<0.001), almost restoring the G{sub 1} phase fraction to that of unexposed cells (25.8{+-}0.2%). Non-specific tat-mouse IgG did not block EGF-induced G{sub 1}-S phase arrest. Tumor uptake of radioactivity was higher in mice injected with EGF to induce p21{sup WAF-1/Cip-1} than in mice not receiving EGF (3.1{+-}0.4% versus 1.8{+-}0.2% ID/g; p=0.04). Western blot analysis of tumors revealed a threefold increase in the p21{sup WAF-1/Cip-1}/{beta}-actin ratio. We conclude that intracellular and nuclear epitopes in cancer cells can be functionally targeted with tat-radioimmunoconjugates to exploit many more epitopes for imaging and radiotherapeutic applications than have previously been accessible. (orig.)

  9. A Map of General and Specialized Chromatin Readers in Mouse Tissues Generated by Label-free Interaction Proteomics

    DEFF Research Database (Denmark)

    Eberl, H.C.; Mann, M.; Spruijt, C.G.;

    2013-01-01

    . Here we describe a sensitive, label-free histone peptide pull-down technology with extracts of different mouse tissues. Applying this workflow to the classical activating and repressive epigenetic marks on histone H3, H3K4me3, and H3K9me3, we identified known and putative readers in extracts from brain...

  10. Proteome Scale-Protein Turnover Analysis Using High Resolution Mass Spectrometric Data from Stable-Isotope Labeled Plants.

    Science.gov (United States)

    Fan, Kai-Ting; Rendahl, Aaron K; Chen, Wen-Ping; Freund, Dana M; Gray, William M; Cohen, Jerry D; Hegeman, Adrian D

    2016-03-04

    Protein turnover is an important aspect of the regulation of cellular processes for organisms when responding to developmental or environmental cues. The measurement of protein turnover in plants, in contrast to that of rapidly growing unicellular organismal cultures, is made more complicated by the high degree of amino acid recycling, resulting in significant transient isotope incorporation distributions that must be dealt with computationally for high throughput analysis to be practical. An algorithm in R, ProteinTurnover, was developed to calculate protein turnover with transient stable isotope incorporation distributions in a high throughput automated manner using high resolution MS and MS/MS proteomic analysis of stable isotopically labeled plant material. ProteinTurnover extracts isotopic distribution information from raw MS data for peptides identified by MS/MS from data sets of either isotopic label dilution or incorporation experiments. Variable isotopic incorporation distributions were modeled using binomial and beta-binomial distributions to deconvolute the natural abundance, newly synthesized/partial-labeled, and fully labeled peptide distributions. Maximum likelihood estimation was performed to calculate the distribution abundance proportion of old and newly synthesized peptides. The half-life or turnover rate of each peptide was calculated from changes in the distribution abundance proportions using nonlinear regression. We applied ProteinTurnover to obtain half-lives of proteins from enriched soluble and membrane fractions from Arabidopsis roots.

  11. Broad characterization of endogenous peptides in the tree shrew visual system.

    Science.gov (United States)

    Ranc, Vaclav; Petruzziello, Filomena; Kretz, Robert; Argandoña, Enrike G; Zhang, Xiaozhe; Rainer, Gregor

    2012-05-17

    Endogenous neuropeptides, acting as neurotransmitters or hormones in the brain, carry out important functions including neural plasticity, metabolism and angiogenesis. Previous neuropeptide studies have focused on peptide-rich brain regions such as the striatum or hypothalamus. Here we present an investigation of peptides in the visual system, composed of brain regions that are generally less rich in peptides, with the aim of providing the first broad overview of peptides involved in mammalian visual functions. We target three important parts of the visual system: the primary visual cortex (V1), lateral geniculate nucleus (LGN) and superior colliculus (SC). Our study is performed in the tree shrew, a close relative of primates. Using a combination of data dependent acquisition and targeted LC-MS/MS based neuropeptidomics; we identified a total of 52 peptides from the tree shrew visual system. A total of 26 peptides, for example GAV and neuropeptide K were identified in the visual system for the first time. Out of the total 52 peptides, 27 peptides with high signal-to-noise-ratio (>10) in extracted ion chromatograms (EIC) were subjected to label-free quantitation. We observed generally lower abundance of peptides in the LGN compared to V1 and SC. Consistently, a number of individual peptides showed high abundance in V1 (such as neuropeptide Y or somatostatin 28) and in SC (such as somatostatin 28 AA1-12). This study provides the first in-depth characterization of peptides in the mammalian visual system. These findings now permit the investigation of neuropeptide-regulated mechanisms of visual perception.

  12. How to Read a Nutrition Facts Label

    Medline Plus

    Full Text Available ... to 2-Year-Old How to Read a Nutrition Facts Label (Video) KidsHealth > For Parents > How to Read a Nutrition Facts Label (Video) Print A A A en ... nutricionales (video) Most packaged foods come with a Nutrition Facts label. These labels have a lot of ...

  13. 21 CFR 606.121 - Container label.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Container label. 606.121 Section 606.121 Food and... Container label. (a) The container label requirements are designed to facilitate the use of a uniform container label for blood and blood components (except Source Plasma) by all blood establishments. (b)...

  14. Time-Resolved Fluorescence Anisotropy Study of the Interaction Between DNA and a Peptide Truncated from the p53 Protein Core Domain.

    Science.gov (United States)

    Liu, Chengxuan; Liang, Gaiting; Liu, Zhen; Zu, Lily

    2014-03-01

    Time-resolved fluorescence anisotropy spectroscopy was applied to study the interaction between a peptide truncated from the binding site of tumor suppressor p53 protein and the DNAs covalently labeled with 6-carboxyfluorescein (FAM) dye. Fluorescence intensity quenching and changes of anisotropy decay lifetime were monitored when FAM labeled DNA formed complex with the peptide. The results demonstrated that the sequence of DNA could not define the binding specificity between the peptide and DNA. But the anisotropy decay of FAM can be used to examine the binding affinity of the peptide to DNA. The fluorescent dynamics of FAM can also be used to represent the rigidity of the complex formed between the peptide and DNA.

  15. Isotope labeled internal standards (ILIS) as a basis for quality control in clinical studies using plasma samples.

    Science.gov (United States)

    Rezeli, Melinda; Végvári, Akos; Marko-Varga, György; Laurell, Thomas

    2010-04-18

    For clinical proteomic studies, the quality of the biofluid samples such as human blood plasma is extremely important. In this study we have investigated the stability of human plasma samples by spiking stable isotope-labeled peptides into the plasma and monitoring their degradation under different storage conditions. FPA-1, C4A and C3f were synthesized with isotopically labeled amino acids, and used as reference peptides. The mixture of internal calibrants was spiked into plasma at the starting point of investigation, mimicking the time of collection for future biobanking efforts, and their qualitative and quantitative changes were analyzed over time by using both MALDI-MS (LTQ Orbitrap XL) and nanoLC-ESI-MS (LTQ XL ETD). We have found that all three synthetic peptides were stable in plasma at -20 and -80 degrees C during the examined 2-month period. However, different proteolytic degradation profiles of the peptides were observed at room temperature. We anticipate that the use of these isotope-labeled peptides as internal standards (ILIS) provides a quality control for long-term storage and proteomic plasma analysis.

  16. Improving Peptide Applications Using Nanotechnology.

    Science.gov (United States)

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

    2016-01-01

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

  17. Antitumor Peptides from Marine Organisms

    Directory of Open Access Journals (Sweden)

    Mi Sun

    2011-10-01

    Full Text Available The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new antitumor agents in the field of the development of marine bioactive substances. In this review, the progress on studies of antitumor peptides from marine sources is provided. The biological properties and mechanisms of action of different marine peptides are described; information about their molecular diversity is also presented. Novel peptides that induce apoptosis signal pathway, affect the tubulin-microtubule equilibrium and inhibit angiogenesis are presented in association with their pharmacological properties. It is intended to provide useful information for further research in the fields of marine antitumor peptides.

  18. The Pig PeptideAtlas

    DEFF Research Database (Denmark)

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

    2016-01-01

    underrepresented in existing repositories. We here present a significantly improved build of the Pig PeptideAtlas, which includes pig proteome data from 25 tissues and three body fluid types mapped to 7139 canonical proteins. The content of the Pig PeptideAtlas reflects actively ongoing research within...... the veterinary proteomics domain, and this article demonstrates how the expression of isoform-unique peptides can be observed across distinct tissues and body fluids. The Pig PeptideAtlas is a unique resource for use in animal proteome research, particularly biomarker discovery and for preliminary design of SRM...

  19. Solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Jensen, Knud Jørgen

    2013-01-01

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

  20. An Optimal Labeling Scheme for Ancestry Queries

    OpenAIRE

    2009-01-01

    An ancestry labeling scheme assigns labels (bit strings) to the nodes of rooted trees such that ancestry queries between any two nodes in a tree can be answered merely by looking at their corresponding labels. The quality of an ancestry labeling scheme is measured by its label size, that is the maximal number of bits in a label of a tree node. In addition to its theoretical appeal, the design of efficient ancestry labeling schemes is motivated by applications in web search engines. For this p...

  1. A brief history of cell labelling

    Energy Technology Data Exchange (ETDEWEB)

    Peters, A.M. [Royal Sussex Country Hospital, Brighton (United Kingdom)

    2005-12-15

    The term cell labelling is usually used in the context of labelled leukocytes for imaging inflammation and labelled platelets for imaging thrombosis. Erythrocyte labelling for in vitro measurements of red cell life span, in vivo measurements of splenic red cell pooling, radionuclide ventriculography and imaging sites of bleeding has developed rather separately and has a different history. Labelled platelets and leukocytes were originally developed for cell kinetic studies. Since the current-day applications of labelled platelets and leukocytes depend on a clear understanding of cell kinetics, these classical studies are important and relevant to the history of cell labelling.

  2. Fluorescent amino acid undergoing excited state intramolecular proton transfer for site-specific probing and imaging of peptide interactions.

    Science.gov (United States)

    Sholokh, Marianna; Zamotaiev, Oleksandr M; Das, Ranjan; Postupalenko, Viktoriia Y; Richert, Ludovic; Dujardin, Denis; Zaporozhets, Olga A; Pivovarenko, Vasyl G; Klymchenko, Andrey S; Mély, Yves

    2015-02-12

    Fluorescent amino acids bearing environment-sensitive fluorophores are highly valuable tools for site-selective probing of peptide/ligand interactions. Herein, we synthesized a fluorescent l-amino acid bearing the 4'-methoxy-3-hydroxyflavone fluorophore (M3HFaa) that shows dual emission, as a result of an excited state intramolecular proton transfer (ESIPT). The dual emission of M3HFaa was found to be substantially more sensitive to hydration as compared to previous analogues. By replacing the Ala30 and Trp37 residues of a HIV-1 nucleocapsid peptide, M3HFaa was observed to preserve the peptide structure and functions. Interaction of the labeled peptides with nucleic acids and lipid vesicles produced a strong switch in their dual emission, favoring the emission of the ESIPT product. This switch was associated with the appearance of long-lived fluorescence lifetimes for the ESIPT product, as a consequence of the rigid environment in the complexes that restricted the relative motions of the M3HFaa aromatic moieties. The strongest restriction and thus the longest fluorescence lifetimes were observed at position 37 in complexes with nucleic acids, where the probe likely stacks with the nucleobases. Based on the dependence of the lifetime values on the nature of the ligand and the labeled position, two-photon fluorescence lifetime imaging was used to identify the binding partners of the labeled peptides microinjected into living cells. Thus, M3HFaa appears as a sensitive tool for monitoring site selectively peptide interactions in solution and living cells.

  3. Photoaffinity Labeling of Plasma Proteins

    Directory of Open Access Journals (Sweden)

    Masaki Otagiri

    2013-11-01

    Full Text Available Photoaffinity labeling is a powerful technique for identifying a target protein. A high degree of labeling specificity can be achieved with this method in comparison to chemical labeling. Human serum albumin (HSA and α1-acid glycoprotein (AGP are two plasma proteins that bind a variety of endogenous and exogenous substances. The ligand binding mechanism of these two proteins is complex. Fatty acids, which are known to be transported in plasma by HSA, cause conformational changes and participate in allosteric ligand binding to HSA. HSA undergoes an N-B transition, a conformational change at alkaline pH, that has been reported to result in increased ligand binding. Attempts have been made to investigate the impact of fatty acids and the N-B transition on ligand binding in HSA using ketoprofen and flunitrazepam as photolabeling agents. Meanwhile, plasma AGP is a mixture of genetic variants of the protein. The photolabeling of AGP with flunitrazepam has been utilized to shed light on the topology of the protein ligand binding site. Furthermore, a review of photoaffinity labeling performed on other major plasma proteins will also be discussed. Using a photoreactive natural ligand as a photolabeling agent to identify target protein in the plasma would reduce non-specific labeling.

  4. Anticancer peptides from bacteria

    Directory of Open Access Journals (Sweden)

    Tomasz M. Karpiński

    2013-08-01

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

  5. Peptides and Food Intake

    Science.gov (United States)

    Sobrino Crespo, Carmen; Perianes Cachero, Aránzazu; Puebla Jiménez, Lilian; Barrios, Vicente; Arilla Ferreiro, Eduardo

    2014-01-01

    The mechanisms for controlling food intake involve mainly an interplay between gut, brain, and adipose tissue (AT), among the major organs. Parasympathetic, sympathetic, and other systems are required for communication between the brain satiety center, gut, and AT. These neuronal circuits include a variety of peptides and hormones, being ghrelin the only orexigenic molecule known, whereas the plethora of other factors are inhibitors of appetite, suggesting its physiological relevance in the regulation of food intake and energy homeostasis. Nutrients generated by food digestion have been proposed to activate G-protein-coupled receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the release of gut hormones into the circulation such as glucagon-like peptide-1 (GLP-1), oxyntomodulin, pancreatic polypeptides, peptide tyrosine tyrosine, and cholecystokinin, which inhibit appetite. Ghrelin is a peptide secreted from the stomach and, in contrast to other gut hormones, plasma levels decrease after a meal and potently stimulate food intake. Other circulating factors such as insulin and leptin relay information regarding long-term energy stores. Both hormones circulate at proportional levels to body fat content, enter the CNS proportionally to their plasma levels, and reduce food intake. Circulating hormones can influence the activity of the arcuate nucleus (ARC) neurons of the hypothalamus, after passing across the median eminence. Circulating factors such as gut hormones may also influence the nucleus of the tractus solitarius (NTS) through the adjacent circumventricular organ. On the other hand, gastrointestinal vagal afferents converge in the NTS of the brainstem. Neural projections from the NTS, in turn, carry signals to the hypothalamus. The ARC acts as an integrative center, with two major subpopulations of neurons influencing appetite, one of them coexpressing neuropeptide Y and agouti-related protein (AgRP) that increases food

  6. 49 CFR 172.430 - POISON label.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  7. In vivo fluorescence-based endoscopic detection of colon dysplasia in the mouse using a novel peptide probe.

    Directory of Open Access Journals (Sweden)

    Sharon J Miller

    Full Text Available Colorectal cancer (CRC is a major cause of cancer-related deaths in much of the world. Most CRCs arise from pre-malignant (dysplastic lesions, such as adenomatous polyps, and current endoscopic screening approaches with white light do not detect all dysplastic lesions. Thus, new strategies to identify such lesions, including non-polypoid lesions, are needed. We aim to identify and validate novel peptides that specifically target dysplastic colonic epithelium in vivo. We used phage display to identify a novel peptide that binds to dysplastic colonic mucosa in vivo in a genetically engineered mouse model of colo-rectal tumorigenesis, based on somatic Apc (adenomatous polyposis coli gene inactivation. Binding was confirmed using confocal microscopy on biopsied adenomas and excised adenomas incubated with peptide ex vivo. Studies of mice where a mutant Kras allele was somatically activated in the colon to generate hyperplastic epithelium were also performed for comparison. Several rounds of in vivo T7 library biopanning isolated a peptide, QPIHPNNM. The fluorescent-labeled peptide bound to dysplastic lesions on endoscopic analysis. Quantitative assessment revealed the fluorescent-labeled peptide (target/background: 2.17±0.61 binds ∼2-fold greater to the colonic adenomas when compared to the control peptide (target/background: 1.14±0.15, p<0.01. The peptide did not bind to the non-dysplastic (hyperplastic epithelium of the Kras mice. This work is first to image fluorescence-labeled peptide binding in vivo that is specific towards colonic dysplasia on wide-area surveillance. This finding highlights an innovative strategy for targeted detection to localize pre-malignant lesions that can be generalized to the epithelium of hollow organs.

  8. Preparation of Fluorescent Microcystin Derivatives by Direct Arginine Labelling and Their Biological Evaluation.

    Science.gov (United States)

    Grundler, Verena; Faltermann, Susanne; Fent, Karl; Gademann, Karl

    2015-07-27

    Microcystin is the most prevalent toxin produced by cyanobacteria and poses a severe threat to livestock, humans and entire ecosystems. We report the preparation of a series of fluorescent microcystin derivatives by direct arginine labelling of the unprotected peptides at the guanidinium side chain. This new method allows a simple late-stage diversification strategy for native peptides devoid of protecting groups under mild conditions. A series of fluorophores were conjugated to microcystin-LR in good to very good yield. The fluorescent probes displayed biological activity comparable to that of unlabelled microcystin, in both phosphatase inhibition assays and toxicity tests on the crustacean Thamnocephalus platyurus. In addition, we demonstrate that the fluorescent probes penetrated Huh7 cells. Whole-animal imaging was performed on T. platyurus: labelled compound was mainly observed in the digestive tract.

  9. Tritium labeling of gonadotropin releasing hormone in its proline and histidine residues

    Energy Technology Data Exchange (ETDEWEB)

    Klauschenz, E.; Bienert, M.; Egler, H.; Pleiss, U.; Niedrich, H.; Nikolics, K.

    3,4-dehydroproline9-GnRH prepared by solid phase peptide synthesis was tritiated catalytically under various conditions yielding 3H-GnRH with specific radioactivities in the range from 35-60 Ci/mmol and full LH releasing activity in vitro. Using palladium/alumina catalyst, the tritiation of the double bond occurs within ten minutes. Investigation of the tritium distribution between the amino acid residues showed a remarkably high incorporation of tritium into the histidine residue (11 to 37%). On the basis of this observation, the tritium labeling of GnRH and angiotensin I by direct catalytic hydrogen-tritium exchange was found to be useful for the labeling of these peptides at remarkably high specific radioactivity.

  10. Exploring Protein-Peptide Binding Specificity through Computational Peptide Screening.

    Directory of Open Access Journals (Sweden)

    Arnab Bhattacherjee

    2013-10-01

    Full Text Available The binding of short disordered peptide stretches to globular protein domains is important for a wide range of cellular processes, including signal transduction, protein transport, and immune response. The often promiscuous nature of these interactions and the conformational flexibility of the peptide chain, sometimes even when bound, make the binding specificity of this type of protein interaction a challenge to understand. Here we develop and test a Monte Carlo-based procedure for calculating protein-peptide binding thermodynamics for many sequences in a single run. The method explores both peptide sequence and conformational space simultaneously by simulating a joint probability distribution which, in particular, makes searching through peptide sequence space computationally efficient. To test our method, we apply it to 3 different peptide-binding protein domains and test its ability to capture the experimentally determined specificity profiles. Insight into the molecular underpinnings of the observed specificities is obtained by analyzing the peptide conformational ensembles of a large number of binding-competent sequences. We also explore the possibility of using our method to discover new peptide-binding pockets on protein structures.

  11. A theoretical justification for single molecule peptide sequencing.

    Directory of Open Access Journals (Sweden)

    Jagannath Swaminathan

    2015-02-01

    Full Text Available The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification; while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology.

  12. Vasoactive intestinal peptide stimulates tracheal submucosal gland secretion in ferret

    Energy Technology Data Exchange (ETDEWEB)

    Peatfield, A.C.; Barnes, P.J.; Bratcher, C.; Nadel, J.A.; Davis, B.

    1983-07-01

    We studied the effect of vasoactive intestinal peptide (VIP) on the output of 35S-labeled macromolecules from ferret tracheal explants either placed in beakers or suspended in modified Ussing chambers. In Ussing chamber experiments, the radiolabel precursor, sodium (35S)sulfate, and all drugs were placed on the submucosal side of the tissue. Washings were collected at 30-min intervals from the luminal side and were dialyzed to remove unbound 35S, leaving radiolabeled macromolecules. Vasoactive intestinal peptide at 3 X 10(-7) M stimulated bound 35S output by a mean of + 252.6% (n . 14). The VIP response was dose-dependent with a near maximal response and a half maximal response at approximately 10(-6) M and 10(-8), M, respectively. The VIP effect was not inhibited by a mixture of tetrodotoxin, atropine, I-propranolol, and phentolamine. Vasoactive intestinal peptide had no effect on the electrical properties of the of the tissues. We conclude that VIP stimulates output of sulfated-macromolecules from ferret tracheal submucosal glands without stimulating ion transport. Our studies also suggest that VIP acts on submucosal glands via specific VIP receptors. Vasoactive intestinal peptide has been shown to increase intracellular levels of cyclic AMP, and we suggest that this may be the mechanism for its effect on the output of macromolecules. This mechanism may be important in the neural regulation of submucosal gland secretion.

  13. Label-free selection of RNA aptamers for metabolic engineering.

    Science.gov (United States)

    Hwang, Chuhern; Carothers, James M

    2016-08-15

    RNA aptamers can be assembled into genetic regulatory devices that sense and respond to levels of specific cellular metabolites and thus serve an integral part of designing dynamic control into engineered metabolic pathways. Here, we describe a practical method for generating specific and high affinity aptamers to enable the wider use of in vitro selection and a broader application of aptamers for metabolic engineering. Conventional selection methods involving either radioactive labeling of RNA or the use of label-free methods such as SPR to track aptamer enrichment require resources that are not widely accessible to research groups. We present a label-free selection method that uses small volume spectrophotometers to track RNA enrichment paired with previously characterized affinity chromatography methods. Borrowing techniques used in solid phase peptide synthesis, we present an approach for immobilizing a wide range of metabolites to an amino PEGA matrix. As an illustration, we detail laboratory techniques employed to generate aptamers that bind p-aminophenylalanine, a metabolic precursor for bio-based production of plastics and the pristinamycin family of antibiotics. We focused on the development of methods for ligand immobilization, selection via affinity chromatography, and nucleic acid quantification that can be performed with common laboratory equipment.

  14. Fluorescent labeling of antibody fragments using split GFP.

    Directory of Open Access Journals (Sweden)

    Fortunato Ferrara

    Full Text Available Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs using the split green fluorescent protein (GFP system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11, is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.

  15. One-step 18F labeling of biomolecules using organotrifluoroborates

    Science.gov (United States)

    Liu, Zhibo; Lin, Kuo-Shyan; Bénard, François; Pourghiasian, Maral; Kiesewetter, Dale O; Perrin, David M; Chen, Xiaoyuan

    2017-01-01

    Herein we present a general protocol for the functionalization of biomolecules with an organotrifluoroborate moiety so that they can be radiolabeled with aqueous 18F fluoride (18F−) and used for positron emission tomography (PET) imaging. Among the β+-emitting radionuclides, fluorine-18 (18F) is the isotope of choice for PET, and it is produced, on-demand, in many hospitals worldwide. Organotrifluoroborates can be 18F-labeled in one step in aqueous conditions via 18F–19F isotope exchange. This protocol features a recently designed ammoniomethyltrifluoroborate, and it describes the following: (i) a synthetic strategy that affords modular synthesis of radiolabeling precursors via a copper-catalyzed ‘click’ reaction; and (ii) a one-step 18F-labeling method that obviates the need for HPLC purification. Within 30 min, 18F-labeled PET imaging probes, such as peptides, can be synthesized in good chemical and radiochemical purity (>98%), satisfactory radiochemical yield of 20–35% (n > 20, non-decay corrected) and high specific activity of 40–111 GBq/µmol (1.1–3.0 Ci/µmol). The entire procedure, including the precursor preparation and 18F radiolabeling, takes 7–10 d. PMID:26313478

  16. Circulating histamine and neutrophil chemotactic activity during allergen-induced asthma: the effect of inhaled antihistamines and anti-allergic compounds.

    Science.gov (United States)

    Morgan, D J; Moodley, I; Cundell, D R; Sheinman, B D; Smart, W; Davies, R J

    1985-07-01

    Plasma histamine and serum neutrophil chemotactic activity (S-NCA) were measured in ten atopic asthmatic patients on four separate occasions after allergen bronchial provocation testing (BPT). Single doses of inhaled sodium cromoglycate (SCG; 20 mg), clemastine (0.5 mg), ketotifen (0.5 mg) and isotonic saline (0.9% NaCl) placebo were administered 30 min before bronchial provocation testing in random order and double-blind. The airflow obstruction after BPT was monitored by measurement of forced expiratory volume in 1 s (FEV1). Plasma histamine was measured by the double-isotope radioenzymatic assay and S-NCA by a modified Boyden chamber technique. A highly significant decrease in FEV1 after BPT occurred on the placebo pre-treatment visit (P less than 0.001). Prior administration of inhaled SCG, clemastine and ketotifen significantly reduced the decrease in airflow obstruction seen after BPT when compared with placebo treatment (P less than 0.01, P less than 0.02, P less than 0.05 respectively). No significant alteration in plasma histamine was detected during allergen-induced airflow obstruction. Levels of S-NCA were significantly higher 5, 10 and 15 min after BPT when compared with the pre-challenge level (P less than 0.01, P less than 0.01, P less than 0.001 respectively). These levels were not significantly decreased when airflow obstruction was inhibited by the prior inhalation of SCG, clemastine or ketotifen.

  17. Fibrinogen labelling with I-131

    Energy Technology Data Exchange (ETDEWEB)

    Seminario, C.; Capillo, T.; Montanez, J. (Instituto Peruano de Energia Nuclear, Lima)

    1983-05-01

    Of the different techniques of labelling liophylized human fibrinogen, the technique of mono-chloride with modified iodine was selected. The labelling of the molecule was performed in alkali media of buffalo glycine in which the solution of stable iodine will react as well as on a later stage will the radioactive isotope. The labelling processes which were undertaken with different activities had an efficiency of over 40%; when purification with resins amberlite was carried through, in none of the cases were the impurities over 5%. Daily controls till the seventh day showed that the average values of radiochemical purity decrease were lower than 1%. The specific activity as well as the concentration of I/sup 131/, the fibrinogen and other characteristics come up to the norms of the pharmacopoeia which are applied.

  18. Learning With Auxiliary Less-Noisy Labels.

    Science.gov (United States)

    Duan, Yunyan; Wu, Ou

    2016-04-06

    Obtaining a sufficient number of accurate labels to form a training set for learning a classifier can be difficult due to the limited access to reliable label resources. Instead, in real-world applications, less-accurate labels, such as labels from nonexpert labelers, are often used. However, learning with less-accurate labels can lead to serious performance deterioration because of the high noise rate. Although several learning methods (e.g., noise-tolerant classifiers) have been advanced to increase classification performance in the presence of label noise, only a few of them take the noise rate into account and utilize both noisy but easily accessible labels and less-noisy labels, a small amount of which can be obtained with an acceptable added time cost and expense. In this brief, we propose a learning method, in which not only noisy labels but also auxiliary less-noisy labels, which are available in a small portion of the training data, are taken into account. Based on a flipping probability noise model and a logistic regression classifier, this method estimates the noise rate parameters, infers ground-truth labels, and learns the classifier simultaneously in a maximum likelihood manner. The proposed method yields three learning algorithms, which correspond to three prior knowledge states regarding the less-noisy labels. The experiments show that the proposed method is tolerant to label noise, and outperforms classifiers that do not explicitly consider the auxiliary less-noisy labels.

  19. Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples

    DEFF Research Database (Denmark)

    Truhlar, Stephanie M E; Cervantes, Carla F; Torpey, Justin W

    2008-01-01

    Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label...... during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined...... by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used...

  20. Labeling and imaging of human mesenchymal stem cells with quantum dot bioconjugates during proliferation and osteogenic differentiation in long term.

    Science.gov (United States)

    Shah, B; Clark, P; Stroscio, M; Mao, J

    2006-01-01

    Quantum dots (QDs) are semiconductor nanocrystals that serve as promising alternatives to organic dyes for cell labeling. Because of their unique spectral, physical and chemical properties, QDs are useful for concurrently monitoring several intercellular and intracellular interactions in live normal cells and cancer cells over periods ranging from less than a second to over several days (several divisions of cells). Here, peptide CGGGRGD is immobilized on CdSe-ZnS QDs coated with carboxyl groups by cross linking with amine groups. These conjugates are directed by the peptide to bind with selected integrins on the membrane of human Mesenchymal stem cells. Upon overnight incubation with optimal concentration, QDs effectively labeled all the cells. Here, we report long-term labeling of human bone-marrow-derived mesenchymal stem cells (hMSCs) with RGD-conjugated QDs during self replication and differentiation into osteogenic cell lineages.

  1. Denture labeling: A new approach

    Directory of Open Access Journals (Sweden)

    Pardeep K Bansal

    2011-01-01

    Full Text Available The need for denture labeling is important for forensic and social reasons in case patients need to be identified individually. The importance of denture marking has long been acknowledged by the dental profession. Over the years, various denture marking systems have been reported in the literature, but none till date fulfills all the prescribed ADA specifications. A simple, easy, inexpensive procedure for marking accurate identification marks on dentures with a lead foil is described here. The label caring the patient information is incorporated in the acrylic resin during the denture processing.

  2. Electrical detection of the biological interaction of a charged peptide via gallium arsenide junction-field-effect transistors

    OpenAIRE

    Lee, Kangho; Nair, Pradeep R.; Alam, Muhammad A.; Janes, David B.; Wampler, Heeyeon P; Zemlyanov, Dmitry; Ivanisevic, Albena

    2008-01-01

    GaAs junction-field-effect transistors (JFETs) are utilized to achieve label-free detection of biological interaction between a probe transactivating transcriptional activator (TAT) peptide and the target trans-activation-responsive (TAR) RNA. The TAT peptide is a short sequence derived from the human immunodeficiency virus-type 1 TAT protein. The GaAs JFETs are modified with a mixed adlayer of 1-octadecanethiol (ODT) and TAT peptide, with the ODT passivating the GaAs surface from polar ions ...

  3. The effect of peptide adsorption on signal linearity and a simple approach to improve reliability of quantification☆

    Science.gov (United States)

    Warwood, Stacey; Byron, Adam; Humphries, Martin J.; Knight, David

    2013-01-01

    Peptide quantification using MS often relies on the comparison of peptide signal intensities between different samples, which is based on the assumption that observed signal intensity has a linear relationship to peptide abundance. A typical proteomics experiment is subject to multiple sources of variance, so we focussed here on properties affecting peptide linearity under simple, well-defined conditions. Peptides from a standard protein digest were analysed by multiple reaction monitoring (MRM) MS to determine peptide linearity over a range of concentrations. We show that many peptides do not display a linear relationship between signal intensity and amount under standard conditions. Increasing the organic content of the sample solvent increased peptide linearity by increasing the accuracy and precision of quantification, which suggests that peptide non-linearity is due to concentration-dependent surface adsorption. Using multiple peptides at various dilutions, we show that peptide non-linearity is related to observed retention time and predicted hydrophobicity. Whereas the effect of adsorption on peptide storage has been investigated previously, here we demonstrate the deleterious effect of peptide adsorption on the quantification of fresh samples, highlight aspects of sample preparation that can minimise the effect, and suggest bioinformatic approaches to enhance the selection of peptides for quantification. Biological significance Accurate quantification is central to many aspects of science, especially those examining dynamic processes or comparing molecular stoichiometries. In biological research, the quantification of proteins is an important yet challenging objective. Large-scale quantification of proteins using MS often depends on the comparison of peptide intensities with only a single-level calibrant (as in stable isotope labelling and absolute quantification approaches) or no calibrants at all (as in label-free approaches). For these approaches to be

  4. Application of stable isotopes to the NMR conformational study of peptides and membrane proteins; Application des isotopes stables a l`etude conformationnelle par RMN de peptides et proteines membranaires

    Energy Technology Data Exchange (ETDEWEB)

    Neumann, J.M.; Macquaire, F.; Cordier, F.; Musat, G.; Baleux, F. [CEA Centre d`Etudes de Saclay, 91 - Gif-sur-Yvette (France). Dept. de Biologie Cellulaire et Moleculaire; Huynh-Dinh, T.; Roux, M.; Sanson, A. [Centre National de la Recherche Scientifique (CNRS), 75 - Paris (France)

    1994-12-31

    The nuclear magnetic resonance spectral analysis of the lipid-peptide complexes generally necessitates isotopic enrichment, specifically or not, of the lipidic or peptidic partner. The isotope labelling depends on the membrane model and the associated NMR techniques: high resolution {sup 1}H NMR of peptides or proteins in the presence of per-deuterated phospholipidic micells, high resolution (micells) or ``solid`` type {sup 2}H NMR of the lipid partner, ``solid`` type NMR ({sup 15}N, {sup 13}C) of the peptide partner in a bi-layer. Application examples are given: utilization of stable isotopes for NMR study of lipopeptide structure and dynamic, of folding-up and functional linking at the annexines membrane interface, and of phospholipid conformation and dynamics in the lipids-ions-peptides interactions. 3 figs.

  5. Rat MHC-linked peptide transporter alleles strongly influence peptide binding by HLA-B27 but not B27-associated inflammatory disease.

    Science.gov (United States)

    Simmons, W A; Leong, L Y; Satumtira, N; Butcher, G W; Howard, J C; Richardson, J A; Slaughter, C A; Hammer, R E; Taurog, J D

    1996-02-15

    Rats transgenic for the human MHC molecule HLA-B27 were used to study the effect of two alleles, cima and cimb, which are associated with peptide transport by the MHC-encoded Tap2 transporter, on the function of HLA-B27 as a restriction element for CTL recognition of the male H-Y minor H Ag and on the multisystem inflammatory disease characteristic of B27 transgenic rats. Anti-H-Y CTL generated in cima B27 transgenic rats lysed male B27 cimb/b targets significantly less well than cima/a or cima/b targets. Addition of exogenous H-Y peptides to male B27 cimb/b targets increased susceptibility to lysis to the level of cima/a targets. Male B27 cimb/b cells were less efficient than cima/a cells in competitively inhibiting CTL lysis of female B27 cima/a targets sensitized with exogenous H-Y peptides. 3H-Labeled peptides eluted from B27 molecules of lymphoblasts from rats of two cimb and three cima RT1 haplotypes showed that the cimb peptide pool favors comparatively longer and/or more hydrophobic peptides. These results indicate that RT1-linked Tap2 polymorphism in the rat strongly influences peptide loading of HLA-B27. Nonetheless, the prevalence and severity of multisystem inflammatory lesions were comparable in backcross rats bearing either cima/b or cimb/b. It thus appears either that binding of specific peptides to B27 is unimportant in the pathogenesis of B27-associated disease or that the critical peptides, unlike H-Y and many others, are not influenced by Tap transporter polymorphism.

  6. Endocrine cells producing regulatory peptides.

    Science.gov (United States)

    Solcia, E; Usellini, L; Buffa, R; Rindi, G; Villani, L; Zampatti, C; Silini, E

    1987-07-15

    Recent data on the immunolocalization of regulatory peptides and related propeptide sequences in endocrine cells and tumors of the gastrointestinal tract, pancreas, lung, thyroid, pituitary (ACTH and opioids), adrenals and paraganglia have been revised and discussed. Gastrin, xenopsin, cholecystokinin (CCK), somatostatin, motilin, secretin, GIP (gastric inhibitory polypeptide), neurotensin, glicentin/glucagon-37 and PYY (peptide tyrosine tyrosine) are the main products of gastrointestinal endocrine cells; glucagon, CRF (corticotropin releasing factor), somatostatin, PP (pancreatic polypeptide) and GRF (growth hormone releasing factor), in addition to insulin, are produced in pancreatic islet cells; bombesin-related peptides are the main markers of pulmonary endocrine cells; calcitonin and CGRP (calcitonin gene-related peptide) occur in thyroid and extrathyroid C cells; ACTH and endorphins in anterior and intermediate lobe pituitary cells, alpha-MSH and CLIP (corticotropin-like intermediate lobe peptide) in intermediate lobe cells; met- and leu-enkephalins and related peptides in adrenal medullary and paraganglionic cells as well as in some gut (enterochromaffin) cells; NPY (neuropeptide Y) in adrenaline-type adrenal medullary cells, etc.. Both tissue-appropriate and tissue-inappropriate regulatory peptides are produced by endocrine tumours, with inappropriate peptides mostly produced by malignant tumours.

  7. Endogenous opioid peptides and epilepsy

    NARCIS (Netherlands)

    J. Haffmans (Judith)

    1985-01-01

    textabstractIn recent years a large number of pept:ides, many of which were originall.y characterized in non-neural tissues, have been reported to be present in the central nervous system ( CNS) . The detection of these peptides within the CNS has raised many questions regarding their source and mec

  8. Urinary Peptides in Rett Syndrome.

    Science.gov (United States)

    Solaas, K. M.; Skjeldal, O.; Gardner, M. L. G.; Kase, B. F.; Reichelt, K. L.

    2002-01-01

    A study found a significantly higher level of peptides in the urine of 53 girls with Rett syndrome compared with controls. The elevation was similar to that in 35 girls with infantile autism. Levels of peptides were lower in girls with classic Rett syndrome than those with congenital Rett syndrome. (Contains references.) (Author/CR)

  9. Peptide Antibiotics for ESKAPE Pathogens

    DEFF Research Database (Denmark)

    Thomsen, Thomas Thyge

    and toxicity by utilizing of the fruit fly Drosophila melanogaster as a whole animal model. This was carried out by testing of antimicrobial peptides targeting Gram-positive bacteria exemplified by the important human pathogen methicillin resistant S. aureus (MRSA). The peptide BP214 was developed from...

  10. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

    Cardiac-derived peptide hormones were identified more than 25 years ago. An astonishing amount of clinical studies have established cardiac natriuretic peptides and their molecular precursors as useful markers of heart disease. In contrast to the clinical applications, the biogenesis of cardiac...

  11. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

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

  12. Connected Component Labeling Using Components Neighbors-Scan Labeling Approach

    Directory of Open Access Journals (Sweden)

    Akmal Rakhmadi

    2010-01-01

    Full Text Available Problem statement: Many approaches have been proposed in previous such as the classic sequential connected components labeling algorithm which is relies on two subsequent raster-scans of a binary image. This method produced good performance in terms of accuracy, but because of the implementation of the image processing systems now requires faster process of the computer, the speed of this technique’s process has become an important issue. Approach: A computational approach, called components neighbors-scan labeling algorithm for connected component labeling was presented in this study. This algorithm required scanning through an image only once to label connected components. The algorithm started by scanning from the head of the component’s group, before tracing all the components neighbors by using the main component’s information. This algorithm had desirable characteristics, it is simple while promoted accuracy and low time consuming. By using a table of components, this approach also gave other advantages as the information for the next higher process. Results: The approach had been tested with a collection of binary images. In practically all cases, the technique had successfully given the desired result. Averagely, from the results the algorithm increased the speed around 67.4% from the two times scanning method. Conclusion: Conclusion from the comparison with the previous method, the approach of components neighbors-scan for connected component labeling promoted speed, accuracy and simplicity. The results showed that the approach has a good performance in terms of accuracy, the time consumed and the simplicity of the algorithm.

  13. Earle K. Plyler Prize for Molecular Spectroscopy and Dynamics Lecture: 2D IR Spectroscopy of Peptide Conformation

    Science.gov (United States)

    Tokmakoff, Andrei

    2012-02-01

    Descriptions of protein and peptide conformation are colored by the methods we use to study them. Protein x-ray and NMR structures often lead to impressions of rigid or well-defined conformations, even though these are dynamic molecules. The conformational fluctuations and disorder of proteins and peptides is more difficult to quantify. This presentation will describe an approach toward characterizing and quantifying structural heterogeneity and disorder in peptides using 2D IR spectroscopy. Using amide I vibrational spectroscopy, isotope labeling strategies, and computational modeling based on molecular dynamics simulations and Markov state models allows us to characterize distinct peptide conformers and conformational variation. The examples illustrated include the beta-hairpin tripzip2 and elastin-like peptides.

  14. Food quality labels from the producers’ perspective

    Directory of Open Access Journals (Sweden)

    Šárka Velčovská

    2016-09-01

    Full Text Available The paper deals with analysing the food producer attitudes towards quality labels. The Klasa label, as the most known and the most frequently used food quality label in the Czech Republic, have become the subject of investigation. The aim of the research was to identify the benefits and problems arising from the certification process and the label use. Primary data were collected in online survey based on standardized questionnaire. In census, 86 respondents from the total 218 producers with the Klasa label in the Czech Republic completed the questionnaire. The most of producers (72% have a longer experience with the label, they are using the label for more than four years. The producers’ expectations from the label were fulfilled only partially. A poor state marketing support and missing marketing strategy were identified as general problems of the label. Specific perceived problems are formalities connected with the certification process and certification of poor-quality products. Correlation analysis, t-test and Pearson chi-square test were calculated to discover relations between variables. The results of the study can be beneficial to both, food producers as well as administrator of the label. Identified problems could help them to improve marketing strategy of the label in order to manage the label in effective way and use all benefits arising from the certification. Administrator of the label should make the certification process more effective and transparent, promotion should be focused on the explanation to consumers what the Klasa label guarantees.

  15. Matrix-assisted laser desorption/ionization time-of-flight and nano-electrospray ionization ion trap mass spectrometric characterization of 1-cyano-2-substituted-benz[f]isoindole derivatives of peptides for fluorescence detection

    DEFF Research Database (Denmark)

    Linnemayr, K; Brückner, A; Körner, R;

    1999-01-01

    A series of hexa- to decapeptides (molecular mass range 800-1200) were labeled with naphthalene-2,3-dicarboxaldehyde, which preferentially reacts with the primary amino groups of a peptide. A highly stable peptide conjugate is formed, which allows selective analysis by fluorescence at excitation...

  16. Radiolabeled Peptide Scaffolds for PET/SPECT - Optical in Vivo Imaging of Carbohydrate-Lectin Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Deutscher, Susan

    2014-09-30

    The objective of this research is to develop phage display-selected peptides into radio- and fluoresecently- labeled scaffolds for the multimodal imaging of carbohydrate-lectin interactions. While numerous protein and receptor systems are being explored for the development of targeted imaging agents, the targeting and analysis of carbohydrate-lectin complexes in vivo remains relatively unexplored. Antibodies, nanoparticles, and peptides are being developed that target carbohydrate-lectin complexes in living systems. However, antibodies and nanoparticles often suffer from slow clearance and toxicity problems. Peptides are attractive alternative vehicles for the specific delivery of radionuclides or fluorophores to sites of interest in vivo, although, because of their size, uptake and retention may be less than antibodies. We have selected high affinity peptides that bind a specific carbohydrate-lectin complex involved in cell-cell adhesion and cross-linking using bacteriophage (phage) display technologies (1,2). These peptides have allowed us to probe the role of these antigens in cell adhesion. Fluorescent versions of the peptides have been developed for optical imaging and radiolabeled versions have been used in single photon emission computed tomography (SPECT) and positron emission tomography (PET) in vivo imaging (3-6). A benefit in employing the radiolabeled peptides in SPECT and PET is that these imaging modalities are widely used in living systems and offer deep tissue sensitivity. Radiolabeled peptides, however, often exhibit poor stability and high kidney uptake in vivo. Conversely, optical imaging is sensitive and offers good spatial resolution, but is not useful for deep tissue penetration and is semi-quantitative. Thus, multimodality imaging that relies on the strengths of both radio- and optical- imaging is a current focus for development of new in vivo imaging agents. We propose a novel means to improve the efficacy of radiolabeled and fluorescently

  17. The Labelling Approach to Deviance.

    Science.gov (United States)

    Rains, Prudence M.; Kitsuse, John L.; Duster, Troy; Freidson, Eliot

    2003-01-01

    This reprint of one chapter from the 1975 text, "Issues in the Classification of Children" by Nicholas Hobbs and others, addresses the theoretical, methodological, and empirical issues involved in the "labeling" approach to the sociology of deviance. It examines the social process of classification, the use of classification in social agencies,…

  18. Psychological effectiveness of carbon labelling

    Science.gov (United States)

    Beattie, Geoffrey

    2012-04-01

    Despite the decision by supermarket-giant Tesco to delay its plan to add carbon-footprint information onto all of its 70,000 products, carbon labelling, if carefully designed, could yet change consumer behaviour. However, it requires a new type of thinking about consumers and much additional work.

  19. On Labeled Traveling Salesman Problems

    DEFF Research Database (Denmark)

    Couetoux, Basile; Gourves, Laurent; Monnot, Jerome;

    2008-01-01

    We consider labeled Traveling Salesman Problems, defined upon a complete graph of n vertices with colored edges. The objective is to find a tour of maximum (or minimum) number of colors. We derive results regarding hardness of approximation, and analyze approximation algorithms for both versions...

  20. Improving the energy labelling scheme

    DEFF Research Database (Denmark)

    Gram-Hanssen, Kirsten; Christensen, Toke Haunstrup

    This report summarises the main results of an EU project on consumer response to energy labels in buildings. This report is mainly directed at Danish policy makers. The main focus is therefore on results that are relevant from a Danish point of view and on how they can be used to further strengthen...