Sample records for peptide labeled fluorescein-5-isothiocyanate

  1. Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.

    PubMed

    Favazza, M; Lerho, M; Houssier, C

    1990-06-01

    Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.

  2. Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples

    NASA Astrophysics Data System (ADS)

    Li, Longhui; Rao, Gong; Lv, Xiaohua; Chen, Ruixi; Cheng, Xiaofeng; Wang, Xiaojun; Zeng, Shaoqun; Liu, Xiuli

    2018-02-01

    Resin embedding is widely used and facilitates microscopic imaging of biological tissues. In contrast, quenching of fluorescence during embedding process hinders the application of resin embedding for imaging of fluorescence-labeled samples. For samples expressing fluorescent proteins, it has been demonstrated that the weakened fluorescence could be recovered by reactivating the fluorophore with alkaline buffer. We extended this idea to immunofluorescence-labeling technology. We showed that the fluorescence of pH-sensitive fluorescein isothiocyanate (FITC) was quenched after resin embedding but reactivated after treating by alkaline buffer. We observed 138.5% fluorescence preservation ratio of reactivated state, sixfold compared with the quenched state in embedding resin, which indicated its application for fluorescence imaging of high signal-to-background ratio. Furthermore, we analyzed the chemical reactivation mechanism of FITC fluorophore. This work would show a way for high-resolution imaging of immunofluorescence-labeled samples embedded in resin.

  3. Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling.

    PubMed

    Hoffmann, Celine; Leroy-Dudal, Johanne; Patel, Salima; Gallet, Olivier; Pauthe, Emmanuel

    2008-01-01

    Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.

  4. A selective reaction of fructose bisphosphate aldolase with fluorescein isothiocyanate in chicken muscle extracts.

    PubMed

    Gehring, Andrew G; Ezzell, John L; Lebherz, Herbert G

    2008-01-01

    The present work describes the selective covalent modification of fructose bisphosphate aldolase in crude extracts of chicken breast muscle by fluorescein 5'-isothiocyanate (5'-FITC) at pH 7.0 and 35 degrees C. The modification was observed after 1 min while no other major soluble protein was labeled even after 30 min. We calculated that ca. one 5'-FITC molecule was incorporated into each aldolase tetramer after a 30 min reaction which resulted in a minimal loss of enzyme activity. The "native" structure of aldolase was required for the selective modification by 5'-FITC since high pH, high temperature, and ionic detergents either inhibited or prevented the reaction of 5'-FITC with aldolase. Certain metabolites (ATP, ADP, CTP, GTP, FBP) and erythrosin B also inhibited the 5'-FITC modification of aldolase. In contrast, F-6-P, AMP, NADH, and NAD(+) as well as free lysine and most importantly, the 6'-isomer of FITC exhibited no competition with 5'-FITC for the labeling of aldolase. Alone, the 6'-isomer of FITC did not exhibit preferential reaction when combined with aldolase. 5'-FITC-labeled and -unlabeled aldolases were not distinguished by their ability to bind to muscle myofibrils (MFs) or by their abilities to refold following reversible denaturation in urea. Structural analysis revealed that 5'-FITC-labeled a tryptic peptide corresponding to residues 112-134 in the primary structure of aldolase, a peptide that does not contain lysine, the amino acid believed to be the primary target of this reagent. Unlike chicken and rabbit muscle aldolases, chicken brain and liver aldolase isoforms along with several other aldolases derived from diverse biological sources did not exhibit this highly selective modification by 5'-FITC. 2008 John Wiley & Sons, Ltd

  5. Fluorescein-labeled stable neurotensin derivatives.

    PubMed

    Maes, Veronique; Hultsch, Christina; Kohl, Suzann; Bergmann, Ralf; Hanke, Thomas; Tourwé, Dirk

    2006-08-01

    Neurotensin(8-13) analogs containing a glycine or 5-aminovaleroyl spacer were labeled with fluorescein through formation of an N-terminal thiourea function. The receptor binding was measured in HT-29 cell cultures and showed a substantial decrease in affinity, especially for the metabolically stabilized [MeArg(9), Tle(11)] analog. Using fluorescence microscopy, the internalization of the fluorescent neurotensin analogs into HT-29 cells was observed. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

  6. Honey-Induced Protein Stabilization as Studied by Fluorescein Isothiocyanate Fluorescence

    PubMed Central

    Abdul Kadir, Habsah; Tayyab, Saad

    2013-01-01

    Protein stabilizing potential of honey was studied on a model protein, bovine serum albumin (BSA), using extrinsic fluorescence of fluorescein isothiocyanate (FITC) as the probe. BSA was labelled with FITC using chemical coupling, and urea and thermal denaturation studies were performed on FITC-labelled BSA (FITC-BSA) both in the absence and presence of 10% and 20% (w/v) honey using FITC fluorescence at 522 nm upon excitation at 495 nm. There was an increase in the FITC fluorescence intensity upon increasing urea concentration or temperature, suggesting protein denaturation. The results from urea and thermal denaturation studies showed increased stability of protein in the presence of honey as reflected from the shift in the transition curve along with the start point and the midpoint of the transition towards higher urea concentration/temperature. Furthermore, the increase in ΔG D H2O and ΔG D 25°C in presence of honey also suggested protein stabilization. PMID:24222758

  7. Fluorescein-labeled β-Glucosidase as a Bacterial Stain

    PubMed Central

    Pital, Abe; Janowitz, Sheldon L.; Hudak, Charles E.; Lewis, Evelyn E.

    1967-01-01

    Fluorescein isothiocyanate-labeled β-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:4169543

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

    PubMed

    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.

  9. Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

    PubMed

    Kessler, Jan H; Mommaas, Bregje; Mutis, Tuna; Huijbers, Ivo; Vissers, Debby; Benckhuijsen, Willemien E; Schreuder, Geziena M Th; Offringa, Rienk; Goulmy, Els; Melief, Cornelis J M; van der Burg, Sjoerd H; Drijfhout, Jan W

    2003-02-01

    We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

  10. DETERMINATION OF ALIPHATIC AMINES IN WATER USING DERIVATIZATION WITH FLUORESCEIN ISOTHIOCYANATE AND CAPILLARY ELECTROPHORESIS/LASER-INDUCED FLUORESCENCE DETECTION.

    EPA Science Inventory

    Detection-oriented derivatization of aliphatic amines and amine functional groups in coumpounds of environmental interest was studied using fluorescein isothiocyanate (FITC) with separation/determination by capillary electrophoresis/laser-induced fluorescence. Determinative level...

  11. Fluorescence turn-on detection of iodide, iodate and total iodine using fluorescein-5-isothiocyanate-modified gold nanoparticles.

    PubMed

    Chen, Yi-Ming; Cheng, Tian-Lu; Tseng, Wei-Lung

    2009-10-01

    Selective turn-on fluorescence detection of I(-) was accomplished using fluorescein isothiocyanate-decorated gold nanoparticles (FITC-AuNPs). FITC molecules, which fluoresce strongly in an alkaline solution, were severely quenched when they were attached to the surface of AuNPs through their isothiocyanate group. Upon the addition of I(-), FITC molecules were detached because of I(-) adsorption on the surface of AuNPs. As a result, released FITC molecules were restored to their original fluorescence intensity. Because I(-) has a higher binding affinity to the surface of Au than do Br(-), Cl(-), or F(-), the FITC-AuNPs obviously have a higher selectivity toward I(-) than toward these other anions. Meanwhile, after IO(3)(-) was reduced to I(-) with ascorbic acid, the detection of IO(3)(-) was successfully achieved using the FITC-AuNPs. Under an optimum pH and AuNP concentration, the lowest detectable concentrations of I(-) and IO(3)(-) using this probe were 10.0 and 50.0 nM, respectively. The FITC-AuNPs provide a number of advantages, including easy preparation, selectivity, sensitivity, and low cost. This unique probe was applied to an analysis of the total iodine in edible salt and seawater.

  12. Exploitation of phosphorescent labelling reagent of fullerol-fluorescein isothiocyanate and new method for the determination of trace alkaline phosphatase as well as forecast of human diseases.

    PubMed

    Liu, Jia-Ming; Huang, Xiao-Mei; Liu, Zhen-Bo; Lin, Shao-Qin; Li, Fei-Ming; Gao, Fei; Li, Zhi-Ming; Zeng, Li-Qing; Li, Lian-Ying; Ouyang, Ying

    2009-08-26

    A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC)(n)-DMA) was developed. The mode of action is based on the reactivity of the active -OH group in F-ol with the -COOH group of FITC to form an F-ol-(FITC)(n)-DMA complex containing several FITC molecules. F-ol-(FITC)(n)-DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC)(n)-DMA (WGA and AP are wheat germ agglutinin and alkaline phosphatase, respectively) which improved the sensitivity using solid substrate room temperature phosphorimetry (SSRTP) detection. The proposed method provided high sensitivity and strong specificity for WGA-AP. The limit of detection (LD) was 0.15 ag AP spot(-1) for F-ol and 0.097 ag AP spot(-1) for FITC in F-ol-(FITC)(n)-DMA, which was lower than the method using single luminescent molecules of F-ol-DMA and FITC-DMA to label WGA (0.20 ag AP spot(-1) for F-ol-DMA and 0.22 ag AP spot(-1) for FITC-DMA). Results for the determination of AP in human serum were in good agreement with those obtained by enzyme-linked immunosorbent assay. The mechanism of F-ol-(FITC)(n)-DMA labelling of WGA was discussed.

  13. Fullerol-fluorescein isothiocyanate-concanavalin agglutinin phosphorescent sensor for the detection of alpha-fetoprotein and forecast of human diseases

    NASA Astrophysics Data System (ADS)

    Liu, Jia-ming; Lin, Li-ping; Jiang, Shu-Lian; Cui, Ma Lin; Jiao, Li; Zhang, Xiao Yang; Zhang, Li-hong; Zheng, Zhi Yong; Lin, Xuan; Lin, Shao-qin

    2013-11-01

    Based on the reaction of the active -OH group in fullerol (F) with the dissociated -COOH group in fluorescein isothiocyanate (FITC) to form an F-FITC and the enhanced effect of N, N-dimethylaniline (DMA) on phosphorescence signal of F-FITC, a new phosphorescent labeling reagent (DMA-F-FITC) was developed. What's more, a phosphorescent sensor for the determination of alpha-fetoprotein variant (AFP-V) has been designed via the coupling technique of the high sensitivity for affinity adsorption-solid substrate-room temperature phosphorimetry (AA-SS-RTP) with the strong specificity reaction between DMA-F-FITC-Con A and AFP-V. The DMA-F-FITC increased the number of luminescent molecules in the biological target which improved the sensitivity of phosphorescent sensor. The proposed sensor was responsive, simple, selective and sensitive, and it has been applied to the determination of trace AFP-V in human serum and the forecast of human diseases using phosphorescence emission wavelength of F or FITC, with the results agreed well with those obtained by enzyme-linked immunoassay (ELISA). Meanwhile, the mechanisms for the labeling reaction and the sensing detection of AFP-V were discussed.

  14. A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA)-chi 2-fluorescein.

    PubMed

    Wiley, J S; Brocklebank, A M; Snook, M B; Jamieson, G P; Sawyer, W H; Craik, J D; Cass, C E; Robins, M J; McAdam, D P; Paterson, A R

    1991-02-01

    The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.

  15. Peptide Reactivity of Isothiocyanates - Implications for Skin Allergy

    NASA Astrophysics Data System (ADS)

    Karlsson, Isabella; Samuelsson, Kristin; Ponting, David J.; Törnqvist, Margareta; Ilag, Leopold L.; Nilsson, Ulrika

    2016-02-01

    Skin allergy is a chronic condition that affects about 20% of the population of the western world. This disease is caused by small reactive compounds, haptens, able to penetrate into the epidermis and modify endogenous proteins, thereby triggering an immunogenic reaction. Phenyl isothiocyanate (PITC) and ethyl isothiocyanate (EITC) have been suggested to be responsible for allergic skin reactions to chloroprene rubber, the main constituent of wetsuits, orthopedic braces, and many types of sports gear. In the present work we have studied the reactivity of the isothiocyanates PITC, EITC, and tetramethylrhodamine-6-isothiocyanate (6-TRITC) toward peptides under aqueous conditions at physiological pH to gain information about the types of immunogenic complexes these compounds may form in the skin. We found that all three compounds reacted quickly with cysteine moieties. For PITC and 6-TRITC the cysteine adducts decomposed over time, while stable adducts with lysine were formed. These experimental findings were verified by DFT calculations. Our results may suggest that the latter are responsible for allergic reactions to isothiocyanates. The initial adduct formation with cysteine residues may still be of great importance as it prevents hydrolysis and facilitates the transport of isothiocyanates into epidermis where they can form stable immunogenic complexes with lysine-containing proteins.

  16. Dual-labeling with 5-aminolevulinic acid and fluorescein for fluorescence-guided resection of high-grade gliomas: technical note.

    PubMed

    Suero Molina, Eric; Wölfer, Johannes; Ewelt, Christian; Ehrhardt, André; Brokinkel, Benjamin; Stummer, Walter

    2018-02-01

    OBJECTIVE Fluorescence guidance with 5-aminolevulinic acid (5-ALA) helps improve resections of malignant gliomas. However, one limitation is the low intensity of blue light for background illumination. Fluorescein has recently been reintroduced into neurosurgery, and novel microscope systems are available for visualizing this fluorochrome, which highlights all perfused tissues but has limited selectivity for tumor detection. Here, the authors investigate a combination of both fluorochromes: 5-ALA for distinguishing tumor and fluorescein for providing tissue fluorescence of adjacent brain tissue. METHODS The authors evaluated 6 patients who harbored cerebral lesions suggestive of high-grade glioma. Patients received 5-ALA (20 mg/kg) orally 4 hours before induction of anesthesia. Low-dose fluorescein (3 mg/kg intravenous) was injected immediately after anesthesia induction. Pentero microscopes (equipped either with Yellow 560 or Blue 400 filters) were used to visualize fluorescence. To simultaneously visualize both fluorochromes, the Yellow 560 module was combined with external blue light illumination (D-light C System). RESULTS Fluorescein-induced fluorescence created a useful background for protoporphyrin IX (PPIX) fluorescence, which appeared orange to red, surrounded by greenly fluorescent normal brain and edematous tissue. Green brain-tissue fluorescence was helpful in augmenting background. Levels of blue illumination that were too strong obscured PPIX fluorescence. Unspecific extravasation of fluorescein was noted at resection margins, which did not interfere with PPIX fluorescence detection. CONCLUSIONS Dual labeling with both PPIX and fluorescein fluorescence is feasible and gives superior background information during fluorescence-guided resections. The authors believe that this technique carries potential as a next step in fluorescence-guided resections if it is completely integrated into the surgical microscope.

  17. In Vitro/In Vivo Evaluation of Radiolabeled [(99m)Tc(CO)3](+)-Hydroxyurea and Fluorescein Isothiocyanate-Hydroxyurea.

    PubMed

    Yilmaz, Baris; Teksoz, Serap; Kilcar, Ayfer Yurt; Ucar, Eser; Ichedef, Cigdem; Medine, Emin Ilker; Ari, Kadir

    2016-02-01

    The aim of current study is to examine hydroxyurea (HU), which is an antineoplastic drug used for the treatment of leukemia, sickle-cell disease, HIV, psoriasis, thrombocythemia, and various neoplastic diseases in two aspects. The active ingredient hydroxyurea was obtained by purification of the capsule form drug, commercially named as HYDREA. Then, [(99m)Tc(CO)3](+)core radiolabeling with HU was performed as first aspect. Quality control studies of (99m)Tc(CO)3-HU complex were performed by thin-layer radiochromatography and high-performance liquid radiochromatography methods. The results demonstrated that the radiolabeling yield was quite high (98.43% ± 2.29%). Also, (99m)Tc(CO)3-HU complex has good stability during the 24-hour period. Biological behavior of (99m)Tc(CO)3-HU complex is evaluated by biodistribution studies on Wistar Albino rats. Fluorescein isothiocyanate (FITC) labeling of HU was performed as second aspect. Fluorometric evaluation of binding efficacy and fluorescence imaging studies on MCF7 and Hela cell lines were carried out. It was thought that the knowledge achieved in this study would contribute to using (99m)Tc(CO)3-HU complex as an imaging agent, which inhibits the DNA synthesis selectively, by inhibiting ribonucleotide reductase enzyme. It was observed that FITC-HU has noteworthy incorporation on both cell lines.

  18. CdS/TiO2-fluorescein isothiocyanate nanoparticles as fluorescence resonance energy transfer probe for the determination of trace alkaline phosphatase based on affinity adsorption assay.

    PubMed

    Liu, Jia-Ming; Lin, Li-ping; Jiao, Li; Cui, Ma-Lin; Wang, Xin-Xing; Zhang, Li-Hong; Zheng, Zhi-Yong

    2012-08-30

    The CdS/TiO(2)-fluorescein isothiocyanate (FITC) luminescent nanoparticles (CdS/TiO(2)-FITC) with the particle size of 20 nm have been synthesized by sol-gel method. CdS/TiO(2)-FITC could emit the fluorescence of both FITC and CdS/TiO(2). The fluorescence resonance energy transfer (FRET) occurred between the donor CdS/TiO(2) and the acceptor FITC in the CdS/TiO(2)-FITC. Taking advantages of the excellent characteristics of FRET, a new CdS/TiO(2)-FITC FRET labeling reagent and a CdS/TiO(2)-FITC-wheat germ agglutinin (CdS/TiO(2)-FITC-WGA) fluorescent probe have been developed. The FRET occurring between the donor CdS/TiO(2) and the acceptor FITC in the labelled product CdS/TiO(2)-FITC-WGA-AP, formed in the affinity adsorption reaction between the WGA in this CdS/TiO(2)-FITC-WGA fluorescent probe and alkaline phosphatase (AP), sharply enhanced the fluorescence signal of FITC and quench the fluorescence signal of CdS/TiO(2). Moreover, the ΔF (the change of the fluorescence signal) of FITC and CdS/TiO(2) were proportional to the content of AP, respectively. Thus, a new method that CdS/TiO(2)-fluorescein isothiocyanate nanoparticles for the determination of trace AP based on FRET-affinity adsorption assay has been established. The limit of quantification (LOQ) of the method was 1.3×10(-17) g AP mL(-1) for CdS/TiO(2) and 1.1×10(-17) g AP mL(-1) for FITC, respectively. This sensitive, rapid, high selective and precise method has been applied to the determination of AP in human serum and the prediction of human disease with the results agreed well with enzyme-linked immunosorbent assay (ELISA) in Zhangzhou Municipal Hospital of Fujian Province. Simultaneously, the reaction mechanism for the determination of AP was also discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. [Evaluation of transfection effectiveness using fluorescein-labelled oligonucleotides and entraster-R siRNA transfection into Plasmodium falciparum].

    PubMed

    Zhou, Hong-Chang; Gao, Yu-Hui; Shao, Sheng-Wen; Zhang, Hui; Zhang, Ting

    2013-12-01

    The cultured Plasmodium falciparum parasites were synchronized twice by 5% sorbitol treatment twice (8-hour window), and then incubated at 37 degrees C for 16 h. Parasites were transfected with fluorescein-labelled oligonucleotides (group A) or fluorescein-labelled oligonucleotides+Entranster-R siRNA transfection reagent (group B). After 5 h a part of parasites was evaluated by fluorescence microscopy and flow cytometry. The rest of parasites were washed with RPMI 1640 medium, and then incubated with 500 microl new medium containing 2% fresh erythrocytes for another 12 h, and detected by flow cytometry. The fluorescein-labelled oligonucleotides were localized in erythrocytes in group B, but nearly no fluorescence was observed for group A. Flow cytometry analysis indicated that the transfection efficiency of group B [(47.40 +/- 3.39)%] was higher than that of group A [(0.60 +/- 0.27)%]. In the second cell cycle, the transfection efficiency in group B was (26.85 +/- 2.90)%, while that of group A was nearly zero. The results indicated that Entranster-R siRNA transfection reagent may increase the oligonucleotides transfection efficiency.

  20. Isothiocyanate-Functionalized Bifunctional Chelates and fac-[M(I)(CO)3](+) (M = Re, (99m)Tc) Complexes for Targeting uPAR in Prostate Cancer.

    PubMed

    Kasten, Benjamin B; Ma, Xiaowei; Cheng, Kai; Bu, Lihong; Slocumb, Winston S; Hayes, Thomas R; Trabue, Steven; Cheng, Zhen; Benny, Paul D

    2016-01-20

    Developing new strategies to rapidly incorporate the fac-[M(I)(CO)3](+) (M = Re, (99m)Tc) core into biological targeting vectors in radiopharmaceuticals continues to expand as molecules become more complex and as efforts to minimize nonspecific binding increase. This work examines a novel isothiocyanate-functionalized bifunctional chelate based on 2,2'-dipicolylamine (DPA) specifically designed for complexing the fac-[M(I)(CO)3](+) core. Two strategies (postlabeling and prelabeling) were explored using the isothiocyanate-functionalized DPA to determine the effectiveness of assembly on the overall yield and purity of the complex with amine containing biomolecules. A model amino acid (lysine) examined (1) amine conjugation of isothiocyanate-functionalized DPA followed by complexation with fac-[M(I)(CO)3](+) (postlabeling) and (2) complexation of fac-[M(I)(CO)3](+) with isothiocyanate-functionalized DPA followed by amine conjugation (prelabeling). Conducted with stable Re and radioactive (99m)Tc analogs, both strategies formed the product in good to excellent yields under macroscopic and radiotracer concentrations. A synthetic peptide (AE105) which targets an emerging biomarker in CaP prognosis, urokinase-type plasminogen activator receptor (uPAR), was also explored using the isothiocyanate-functionalized DPA strategy. In vitro PC-3 (uPAR+) cell uptake assays with the (99m)Tc-labeled peptide (8a) showed 4.2 ± 0.5% uptake at 4 h. In a murine model bearing PC-3 tumor xenografts, in vivo biodistribution of 8a led to favorable tumor uptake (3.7 ± 0.7% ID/g) at 4 h p.i. with relatively low accumulation (<2% ID/g) in normal organs not associated with normal peptide excretion. These results illustrate the promise of the isothiocyanate-functionalized approach for labeling amine containing biological targeting vectors with fac-[M(I)(CO)3](+).

  1. Fluorescein isothiocyanate and rhodamine B dye encapsulated mesoporous SiO2 for applications of blue LED excited white LED

    NASA Astrophysics Data System (ADS)

    Das, Sourav; Manam, J.

    2018-05-01

    In this work, the fluorescein isothiocyanate (FITC) and rhodamine B (RhB) dyes were encapsulated in mesoporous silica nanoparticles (MSNp). The MSNp-FITC-RhB nanohybrids phosphor showed a dichromatic PL emission at green region and orange region when excited at 460 nm. A Forster Resonance Energy Transfer (FRET) was observed from FITC to RhB. The materials were further characterized by XRD, FTIR, TEM, and temperature dependent photoluminescence. The CIE coordinates were tuned from greenish yellow to the orange region and quantum yield was reached 52.04% based on FRET. So by combining the MSNp-FITC-RhB nanohybrids phosphor with the blue LED chip, the white light emission with flexible Color Correlated Temperature and improved Color Rendering Index can be obtained.

  2. Ternary Interactions and Energy Transfer between Fluorescein Isothiocyanate, Adenosine Triphosphate, and Graphene Oxide Nanocarriers.

    PubMed

    Ratajczak, Katarzyna; Stobiecka, Magdalena

    2017-07-20

    The interactions of fluorescent probes and biomolecules with nanocarriers are of key importance to the emerging targeted drug delivery systems. Graphene oxide nanosheets (GONs) as the nanocarriers offer biocompatibility and robust drug binding capacity. The interactions of GONs with fluorophores lead to strong fluorescence quenching, which may interfere with fluorescence bioimaging and biodetection. Herein, we report on the interactions and energy transfers in a model ternary system: GONs-FITC-ATP, where FITC is a model fluorophore (fluorescein isothiocyanate) and ATP is a common biomolecule (adenosine-5'-triphosphate). We have found that FITC fluorescence is considerably quenched by ATP (the quenching constant K SV = 113 ± 22 M -1 ). The temperature coefficient of K SV is positive (α T = 4.15 M -1 deg -1 ). The detailed analysis of a model for internal self-quenching of FITC indicates that the temperature dependence of the net quenching efficiency η for the FITC-ATP pair is dominated by FITC internal self-quenching modes with their contribution estimated at 79%. The quenching of FITC by GONs is much stronger (K SV = 598 ± 29 M -1 ) than that of FITC-ATP and is associated with the formation of supramolecular assemblies bound with hydrogen bonding and π-π stacking interactions. For the analysis of the complex behavior of the ternary system GONs-FITC-ATP, a model of chemisorption of ATP on GONs, with partial blocking of FITC quenching, has been developed. Our results indicate that ATP acts as a moderator for FITC quenching by GONs. The interactions between ATP, FITC, and GONs have been corroborated using molecular dynamics and quantum mechanical calculations.

  3. Measurement of beta-amyloid peptides in specific cells using a photo thin-film transistor

    NASA Astrophysics Data System (ADS)

    Kim, Chang-Beom; Chae, Cheol-Joo; Shin, Hye-Rim; Song, Ki-Bong

    2012-01-01

    The existence of beta-amyloid [Aβ] peptides in the brain has been regarded as the most archetypal biomarker of Alzheimer's disease [AD]. Recently, an early clinical diagnosis has been considered a great importance in identifying people who are at high risk of AD. However, no microscale electronic sensing devices for the detection of Aβ peptides have been developed yet. In this study, we propose an effective method to evaluate a small quantity of Aβ peptides labeled with fluorescein isothiocyanate [FITC] using a photosensitive field-effect transistor [p-FET] with an on-chip single-layer optical filter. To accurately evaluate the quantity of Aβ peptides within the cells cultured on the p-FET device, we measured the photocurrents which resulted from the FITC-conjugated Aβ peptides expressed from the cells and measured the number of photons of the fluorochrome in the cells using a photomultiplier tube. Thus, we evaluated the correlation between the generated photocurrents and the number of emitted photons. We also evaluated the correlation between the number of emitted photons and the amount of FITC by measuring the FITC volume using AFM. Finally, we estimated the quantity of Aβ peptides of the cells placed on the p-FET sensing area on the basis of the binding ratio between FITC molecules and Aβ peptides.

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

    PubMed Central

    Volkmann, Gerrit; Liu, Xiang-Qin

    2009-01-01

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

  5. Protein sorption on polymer surfaces measured by fluorescence labels.

    PubMed

    Brynda, E; Drobník, J; Vacík, J; Kálal, J

    1978-01-01

    Fluorescence labeling can be used in studying protein sorption on various surfaces with a sensitivity of about 10(-8) g/cm2, commensurate with radioactive labeling. Fluorescamine proved to be the most suitable compound for studying protein sorption on hydrophilic gels, because, unlike fluoresceine isothiocyanate and dansylchloride, free fluorochrome does not interfere with measurements. Sorption properties of labeled serum albumin were tested on poly(2-hydroxyethyl methacrylate), on the copolymer of 2-hydroxyethyl methacrylate with methyl methacrylate, and on polyethylene. Labeling does not cause aggregation of the protein, but, as expected, it shifts and somewhat broadens its electrophoretic band while at the same time slightly raising its affinity toward hydrophobic surfaces.

  6. Development of poly-l-lysine-coated calcium-alginate microspheres encapsulating fluorescein-labeled dextrans

    NASA Astrophysics Data System (ADS)

    Charron, Luc; Harmer, Andrea; Lilge, Lothar

    2005-09-01

    A technique to produce fluorescent cell phantom standards based on calcium alginate microspheres with encapsulated fluorescein-labeled dextrans is presented. An electrostatic ionotropic gelation method is used to create the microspheres which are then exposed to an encapsulation method using poly-l-lysine to trap the dextrans inside. Both procedures were examined in detail to find the optimal parameters producing cell phantoms meeting our requirements. Size distributions favoring 10-20 microns microspheres were obtained by varying the high voltage and needle size parameters. Typical size distributions of the samples were centered at 150 μm diameter. Neither the molecular weight nor the charge of the dextrans had a significant effect on their retention in the microspheres, though anionic dextrans were chosen to help in future capillary electrophoresis work. Increasing the exposure time of the microspheres to the poly-l-lysine solution decreased the leakage rates of fluorescein-labeled dextrans.

  7. Determination of Trace Deoxyribonucleic Acid by Using Fluorescein Isothiocyanate-Phenosafranine as a Double-Luminescent Phosphorescence Probe

    PubMed Central

    Huang, Xiao-Mei; Liu, Zhen-Bo; Li, Fei-Ming; Lin, Li-Ping; Wang, Xin-Xing; Lin, Chang-Qing; Huang, Ya-Hong; Li, Zhi-Ming; Lin, Shao-Qin

    2010-01-01

    Using Pb2+ as ion perturber, phenosafranine (PF) and fluorescein isothiocyanate (FITC) could emit strong and stable room temperature phosphorescence (RTP) signal on the filter paper, respectively. When they were mixed, the phenomenon that the RTP signal of PF and FITC enhanced significantly was found. And 1.12 ag DNA spot−1 (sample volume was 0.40 μL, corresponding concentration was 2.8 × 10–15 g mL–1) could cause the RTP signal of both PF and FITC to enhance sharply. The content of DNA was proportional to the ΔIp of PF and FITC in the system at 634 and 659 nm. Thus, a new solid substrate room temperature phosphorimetry (SSRTP) for the determination of trace DNA was established by using FITC-PF as double-luminescent phosphorescence probe. The detection limit (LD) of this method calculated by 3Sb/k was 14 zg DNA spot–1 for PF and 18 zg DNA spot–1 for FITC, respectively, showing high sensitivity. It has been applied to the determination of trace DNA in practical samples and the analysis results were in accordance with those of fluorescence probe. The reaction mechanism of SSRTP for the determination of trace DNA was also discussed. PMID:20665096

  8. Characterization of the swelling-induced alkalinization of endocytotic vesicles in fluorescein isothiocyanate-dextran-loaded rat hepatocytes.

    PubMed Central

    Schreiber, R; Häussinger, D

    1995-01-01

    Short-term cultivated rat hepatocytes were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran and the apparent vesicular pH (pHves) was measured by single-cell fluorescence. After 2 h of exposure to FITC-dextran, the apparent pH in the vesicular compartments accessible to endocytosed FITC-dextran was 6.01 +/- 0.05 (n = 39) in normo-osmotic media. Hypo-osmotic exposure increased, whereas hyper-osmotic exposure decreased apparent pHves. by 0.18 +/- 0.02 (n = 26) and 0.12 +/- 0.01 (n = 23) respectively. Incubation of the cells with unlabelled dextran for 2h before a 2-h FITC-dextran exposure had no effect on apparent pHves and its osmosensitivity. When, however, hepatocytes were exposed to unlabelled dextran for 5 h after a 2 h exposure to FITC-dextran, in order to allow transport of endocytosed FITC-dextran to late endocytotic/lysosomal compartments, apparent pHves. decreased to 5.38 +/- 0.04 (n = 12) and the apparent pH in the vesicular compartment containing the dye was no longer sensitive to aniso-osmotic exposure. These findings indicate that the osomosensitivity of pHves. is apparently restricted to early endocytotic compartments. Aniso-osmotic regulation of apparent pHves. in freshly FITC-loaded hepatocytes was not accompanied by aniso-osmolarity-induced changes of the cytosolic free calcium concentration, and neither vasopressin nor extracellular ATP, which provoked a marked Ca2+ signal, affected apparent pHves. Dibutyryl-cyclic AMP (cAMP) or vanadate (0.5 mmol/l) were without effect on apparent pHves. and its osmosensitivity. However, pertussis toxin-treatment or genistein (but not daidzein) or the erbstatin analogue methyl 2,5-dihydroxycinnamate fully abolished the osmo-sensitivity of apparent pHves., but did not affect apparent pHves. It is concluded that regulation of pHves. by cell volume occurs in early endocytotic compartments, but probably not in lysosomes, and is mediated by a G-protein and tyrosine kinase-dependent, but Ca2+- and

  9. Optimizing Fluorescein Isothiocyanate Dextran Measurement As a Biomarker in a 24-h Feed Restriction Model to Induce Gut Permeability in Broiler Chickens

    PubMed Central

    Baxter, Mikayla F. A.; Merino-Guzman, Ruben; Latorre, Juan D.; Mahaffey, Brittany D.; Yang, Yichao; Teague, Kyle D.; Graham, Lucas E.; Wolfenden, Amanda D.; Hernandez-Velasco, Xochitl; Bielke, Lisa R.; Hargis, Billy M.; Tellez, Guillermo

    2017-01-01

    Fluorescein isothiocyanate dextran (FITC-d) is a 3–5 kDa marker used to measure tight junction permeability. We have previously shown that intestinal barrier function can be adversely affected by stress, poorly digested diets, or feed restriction (FR), resulting in increased intestinal inflammation-associated permeability. However, further optimization adjustments of the current FITC-d methodology are possible to enhance precision and efficacy of results in future. The objective of the present study was to optimize our current model to obtain a larger difference between control and treated groups, by optimizing the FITC-d measurement as a biomarker in a 24-h FR model to induce gut permeability in broiler chickens. One in vitro and four in vivo independent experiments were conducted. The results of the present study suggest that by increasing the dose of FITC-d (8.32 versus 4.16 mg/kg); shortening the collection time of blood samples (1 versus 2.5 h); using a pool of non-FITC-d serum as a blank, compared to previously used PBS; adding a standard curve to set a limit of detection and modifying the software’s optimal sensitivity value, it was possible to obtain more consistent and reliable results. PMID:28470003

  10. A long lifetime chemical sensor: study on fluorescence property of fluorescein isothiocyanate and preparation of pH chemical sensor.

    PubMed

    Ma, Li Ying; Wang, Huai You; Xie, Hui; Xu, Li Xiao

    2004-07-01

    The fluorescence property of fluorescein isothiocyanate (FITC) in acid-alkaline medium was studied by spectrofluorimetry. The characteristic of FITC response to hydrogen ion has been examined in acid-alkaline solution. A novel pH chemical sensor was prepared based on the relationship between the relative fluorescence intensity of FITC and pH. The measurement of relative fluorescence intensity was carried out at 362 nm with excitation at 250 nm. The excellent linear relationship was obtained between relative fluorescence intensity and pH in the range of pH 1-5. The linear regression equation of the calibration graph is F = 66.871 + 6.605 pH (F is relative fluorescence intensity), with a correlation coefficient of linear regression of 0.9995. Effects of temperature, concentration of FITC on the response to hydrogen ion had been examined. It was important that this chemical sensor was long lifetime, and the property of response to hydrogen ion was stable for at least 70 days. This pH sensor can be used for measuring pH value in water solution. The accuracy is 0.01 pH unit. The results obtained by the pH sensor agreed with those by the pH meter. Obviously, this pH sensor is potential for determining pH change real time in biological system.

  11. A long lifetime chemical sensor: study on fluorescence property of fluorescein isothiocyanate and preparation of pH chemical sensor

    NASA Astrophysics Data System (ADS)

    Ma, Li Ying; Wang, Huai You; Xie, Hui; Xu, Li Xiao

    2004-07-01

    The fluorescence property of fluorescein isothiocyanate (FITC) in acid-alkaline medium was studied by spectrofluorimetry. The characteristic of FITC response to hydrogen ion has been examined in acid-alkaline solution. A novel pH chemical sensor was prepared based on the relationship between the relative fluorescence intensity of FITC and pH. The measurement of relative fluorescence intensity was carried out at 362 nm with excitation at 250 nm. The excellent linear relationship was obtained between relative fluorescence intensity and pH in the range of pH 1-5. The linear regression equation of the calibration graph is F=66.871+6.605 pH ( F is relative fluorescence intensity), with a correlation coefficient of linear regression of 0.9995. Effects of temperature, concentration of FITC on the response to hydrogen ion had been examined. It was important that this chemical sensor was long lifetime, and the property of response to hydrogen ion was stable for at least 70 days. This pH sensor can be used for measuring pH value in water solution. The accuracy is 0.01 pH unit. The results obtained by the pH sensor agreed with those by the pH meter. Obviously, this pH sensor is potential for determining pH change real time in biological system.

  12. Preliminary study on the inhibition of nuclear internalization of Tat peptides by conjugation with a receptor-specific peptide and fluorescent dyes

    NASA Astrophysics Data System (ADS)

    Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel

    2006-02-01

    Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

  13. Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris.

    PubMed

    Guo, Yu-Qi; Wu, Qing-Ping; Shao, Xiao-Xia; Shen, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2015-06-01

    Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.

  14. Flow cytometric detection of human immunodeficiency virus type 1 proviral DNA by the polymerase chain reaction incorporating digoxigenin- or fluorescein-labeled dUTP

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Gang; Olson, J.C.; Pu, R.

    1995-10-01

    Serological assays are routinely used in the laboratory diagnosis of human immunodeficiency virus type-1 (HrV-1) infection, but the polymerase chain reaction (PCR) is ultimately the most sensitive and direct method for establishing definitive diagnosis. As an alternative to the conventional radioactive PCR procedure we have developed and evaluated a pair of rapid nonradioisotopic flow cytometric detection methods. Using heminested PCR we directly incorporated fluorescein-12-dUTP (fluo-dUTP) or digoxigenin-11-dUTP (dig-dUTP) into the PCR-amplicons. The labeled amplicons were hybridized with biotinylated antisense and sense probes, followed by capture of the hybrid DNA using streptavidin-coated beads which were finally analyzed in a flow cytometermore » by (1) direct detection of the fluorescence intensity of the amplicons incorporating fluo-dUTP and (2) immunodetection of the amplicons incorporating dig-dUTP by anti-digoxigenin IgG labeled with fluorescein isothiocyanate (FITC). Although both assays were functionally comparable with radiolabeled probe in reliably detecting as low as five copies of HIV-1 proviral DNA sequences, the immunodetection of dig-dUTP consistently yielded higher mean channel fluorescence and gave a stable signal over an extended period of 12-14 weeks. In testing a panel of 20 pedigreed PBMC specimens from blood donors with or without HIV-1 infection, the results of both flow cytometric assays were identical with those of the conventional radioactive procedure. Therefore, we conclude that the dig-dUTP incorporation in amplicons, hybridization with a pair of sense-antisense biotinylated probes and immunodetection of hybrids by flow cytometric analyses is the nonisotopic method of choice for PCR-diagnosis of HIV-1 infection. 21 refs., 2 figs., 4 tabs.« less

  15. Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

    PubMed

    Leng, Jiapeng; Zhu, Dong; Wu, Duojiao; Zhu, Tongyu; Zhao, Ningwei; Guo, Yinlong

    2012-11-15

    Peptidomics analysis of human serum is challenging due to the low abundance of serum peptides and interference from the complex matrix. This study analyzed the differentially expressed (DE) low molecular weight peptides in human serum integrating a DMPITC-based N-terminal isotope labeling technique with nano-liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (nano-LC/MALDI-MS). The workflow introduced a [d(6)]-4,6-dimethoxypyrimidine-2-isothiocyanate (DMPITC)-labeled mixture of aliquots from test samples as the internal standard. The spiked [d(0)]-DMPITC-labeled samples were separated by nano-LC then spotted on the MALDI target. Both quantitative and qualitative studies for serum peptides were achieved based on the isotope-labeled peaks. The DMPITC labeling technique combined with nano-LC/MALDI-MS not only minimized the errors in peptide quantitation, but also allowed convenient recognition of the labeled peptides due to the 6 Da mass difference. The data showed that the entire research procedure as well as the subsequent data analysis method were effective, reproducible, and sensitive for the analysis of DE serum peptides. This study successfully established a research model for DE serum peptides using DMPITC-based N-terminal isotope labeling and nano-LC/MALDI-MS. Application of the DMPITC-based N-terminal labeling technique is expected to provide a promising tool for the investigation of peptides in vivo, especially for the analysis of DE peptides under different biological conditions. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Peptide-membrane Interactions by Spin-labeling EPR

    PubMed Central

    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

  17. Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.

    PubMed

    Ferry, D R; Goll, A; Glossmann, H

    1987-04-01

    The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

  18. Ratiometric, visual, dual-signal fluorescent sensing and imaging of pH/copper ions in real samples based on carbon dots-fluorescein isothiocyanate composites.

    PubMed

    Zhu, Xinxin; Jin, Hui; Gao, Cuili; Gui, Rijun; Wang, Zonghua

    2017-01-01

    In this article, a facile aqueous synthesis of carbon dots (CDs) was developed by using natural kelp as a new carbon source. Through hydrothermal carbonization of kelp juice, fluorescent CDs were prepared and the CDs' surface was modified with polyethylenimine (PEI). The PEI-modified CDs were conjugated with fluorescein isothiocyanate (FITC) to fabricate CDs-FITC composites. To exploit broad applications, the CDs-FITC composites were developed as fluorescent sensing or imaging platforms of pH and Cu 2+ . Analytical performances of the composites-based fluorescence (FL) sensors were evaluated, including visual FL imaging of pH in glass bottle, ratiometric FL sensing of pH in yogurt samples, visual FL latent fingerprint and leaf imaging detection of [Cu 2+ ], dual-signal FL sensing of [Cu 2+ ] in yogurt and human serum samples. Experimental results from ratiometric, visual, dual-signal FL sensing and imaging applications confirmed the high feasibility, accuracy, stabilization and simplicity of CDs-FITC composites-based FL sensors for the detection of pH and Cu 2+ ions in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Acoustic fingerprints of dye-labeled protein submicrosphere photoacoustic contrast agents

    NASA Astrophysics Data System (ADS)

    McDonald, Michael A.; Jankovic, Ladislav; Shahzad, Khalid; Burcher, Michael; Li, King C. P.

    2009-05-01

    Dye-labeled protein microspheres, submicron in size and capable of producing thermoelastically generated ultrasound in response to laser stimulation, are presented as contrast agents for photoacoustic imaging. Incident laser energy absorbed by fluorescein isothiocyanate (FITC)-labeled elastin submicrospheres results in thermoelastically generated sound production. Plotted A-line graphs reveal a distinctive morphology and a greater than two orders of magnitude increase in signal amplitude subsequent to converting FITC elastin into submicrospheres (despite a four orders of magnitude decrease in concentration). Evidence of nonlinearity and enhancement of ultrasound backscatter indicate a potential use in contrast-enhanced harmonic imaging. Photoacoustic and ultrasound imaging of FITC-elastin submicrospheres in a water-filled phantom vessel shows enhanced contrast at low concentration and clear delineation of the phantom vessel wall.

  20. Detection of Peptide-based nanoparticles in blood plasma by ELISA.

    PubMed

    Bode, Gerard H; Pickl, Karin E; Sanchez-Purrà, Maria; Albaiges, Berta; Borrós, Salvador; Pötgens, Andy J G; Schmitz, Christoph; Sinner, Frank M; Losen, Mario; Steinbusch, Harry W M; Frank, Hans-Georg; Martinez-Martinez, Pilar

    2015-01-01

    The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma. A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology. The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS. We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions.

  1. Targeting Phosphatidylserine with a 64Cu-Labeled Peptide for Molecular Imaging of Apoptosis.

    PubMed

    Perreault, Amanda; Richter, Susan; Bergman, Cody; Wuest, Melinda; Wuest, Frank

    2016-10-03

    Molecular imaging of programmed cell death (apoptosis) in vivo is an innovative strategy for early assessment of treatment response and treatment efficacy in cancer patients. Externalization of phosphatidylserine (PS) to the cell membrane surface of dying cells makes this phospholipid an attractive molecular target for the development of apoptosis imaging probes. In this study, we have radiolabeled PS-binding 14-mer peptide FNFRLKAGAKIRFG (PSBP-6) with positron-emitter copper-64 ( 64 Cu) for PET imaging of apoptosis. Peptide PSBP-6 was conjugated with radiometal chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through an aminovaleric acid (Ava) linker for subsequent radiolabeling with 64 Cu to prepare radiotracer 64 Cu-NOTA-Ava-PSBP-6. PS-binding potencies of PSBP-6, NOTA-Ava-PSBP-6, and nat Cu-NOTA-Ava-PSBP-6 were determined in a competitive radiometric PS-binding assay. Radiotracer 64 Cu-NOTA-Ava-PSBP-6 was studied in camptothecin-induced apoptotic EL4 mouse lymphoma cells and in a murine EL4 tumor model of apoptosis using dynamic PET imaging. Peptide PSBP-6 was also conjugated via an Ava linker with fluorescein isothiocyanate (FITC). FITC-Ava-PSBP-6 was evaluated in flow cytometry and fluorescence confocal microscopy experiments. Radiopeptide 64 Cu-NOTA-Ava-PSBP-6 was synthesized in high radiochemical yields of >95%. The IC 50 values for PS-binding potency of PSBP-6, NOTA-Ava-PSBP-6, and nat Cu-NOTA-PSBP-6 were 600 μM, 30 μM, and 23 μM, respectively. A competitive radiometric cell binding assay confirmed binding of 64 Cu-NOTA-Ava-PSBP-6 to camptothecin-induced apoptotic EL4 cells in a Ca 2+ -independent manner. PET imaging studies demonstrated significantly higher uptake of 64 Cu-NOTA-Ava-PSBP-6 in apoptotic EL4 tumors (SUV 5min 0.95 ± 0.04) compared to control tumors (SUV 5min 0.74 ± 0.03). Flow cytometry studies showed significantly higher binding of FITC-Ava-PSBP-6 to EL4 cells treated with camptothecin compared to untreated cells

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

    PubMed

    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.

  3. In Vivo MR Imaging of Glioma Recruitment of Adoptive T-Cells Labeled with NaGdF4 -TAT Nanoprobes.

    PubMed

    Zhang, Hua; Wu, Yue; Wang, Jing; Tang, Zhongmin; Ren, Yan; Ni, Dalong; Gao, Hongbo; Song, Ruixue; Jin, Teng; Li, Qiao; Bu, Wenbo; Yao, Zhenwei

    2018-01-01

    Adoptive T lymphocyte immunotherapy is one of the most promising methods to treat residual lesions after glioma surgery. However, the fate of the adoptively transferred T-cells in vivo is unclear, hampering the understanding of this emerging therapy. Thus, it is highly desirable to develop noninvasive and quantitative in vivo tracking of these T-cells to glioma for better identification of the migratory fate and to provide objective evaluation of outcomes of adoptive T-cell immunotherapy targeting glioma. In this work, ultrasmall T 1 MR-based nanoprobes, NaGdF 4 -TAT, as molecular probes with high longitudinal relaxivity (8.93 mm -1 s -1 ) are designed. By means of HIV-1 transactivator (TAT) peptides, nearly 95% of the adoptive T-cells are labeled with the NaGdF 4 -TAT nanoprobes without any measurable side effects on the labeled T-cells, which is remarkably superior to that of the control fluorescein isothiocyanate-NaGdF 4 concerning labeling efficacy. Labeled adoptive T-cell clusters can be sensitively tracked in an orthotopic GL261-glioma model 24 h after intravenous infusion of 10 7 labeled T-cells by T 1 -weighted MR imaging. Both in vitro and in vivo experiments show that the NaGdF 4 -TAT nanoprobes labeling of T-cells may be a promising method to track adoptive T-cells to improve our understanding of the pathophysiology in adoptive immunotherapy for gliomas. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Detection of Peptide-Based Nanoparticles in Blood Plasma by ELISA

    PubMed Central

    Bode, Gerard H.; Pickl, Karin E.; Sanchez-Purrà, Maria; Albaiges, Berta; Borrós, Salvador; Pötgens, Andy J. G.; Schmitz, Christoph; Sinner, Frank M.; Losen, Mario; Steinbusch, Harry W. M.; Frank, Hans-Georg; Martinez-Martinez, Pilar

    2015-01-01

    Aims The aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma. Materials & Methods A convenient enzyme linked immunosorbent assay (ELISA) was developed for the detection of peptides functionalized with biotin and fluorescein groups. As a proof of principle, polymerized pentafluorophenyl methacrylate nanoparticles linked to biotin-carboxyfluorescein labeled peptides were intravenously injected in Wistar rats. Serial blood plasma samples were analyzed by ELISA and by liquid chromatography mass spectrometry (LC/MS) technology. Results The ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS. Conclusions We detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions. PMID:25996618

  5. Bombesin-like peptides stimulate somatostatin release from rat fundic D cells in primary culture.

    PubMed

    Schaffer, K; Herrmuth, H; Mueller, J; Coy, D H; Wong, H C; Walsh, J H; Classen, M; Schusdziarra, V; Schepp, W

    1997-09-01

    In several species, bombesin-like neuropeptides stimulate somatostatin release in in vitro preparations of gastric mucosa. We sought to determine if this response is due to a direct effect on fundic D cells. Rat fundic mucosal cells were isolated by pronase E (1% D cells). D cells were separated by counterflow elutriation and subsequent density-gradient centrifugation (Nycodenz) (15% D cells) and grown in primary culture for 48 h (46% D cells). Cultured cells were double stained with affinity-purified rabbit-anti-gastrin-releasing peptide (GRP) receptor antibody and mouse monoclonal antibody to human somatostatin. After incubation with rhodamine-labeled anti-rabbit and fluorescein isothiocyanate-labeled anti-mouse antibodies, reactions were visualized by fluorescence microscopy. All cells positive for somatostatin had GRP receptors, whereas all non-D cells showed no expression in this G cell-free culture system. Somatostatin release from cultured cells was stimulated by sulfated cholecystokinin octapeptide (CCK-8; EC50 3 X 10(-10) M) and epinephrine (EC50 4 X 10(-8) M), which are established stimuli for canine fundic D cells. Bombesin (EC50 6 X 10(-11) M), its mammalian analog GRP-27, and neuromedin C (GRP-10) (EC50 1 X 10(-10) M, for both) were almost equally potent stimuli of somatostatin release, eliciting maximal response at 10(-9) M (400-550% above basal). Neuromedin B was less potent and effective (maximal response at 10(-8) M, 230% above basal). [D-Phe6]bombesin-(6-13)-OMe, a specific bombesin receptor antagonist, inhibited bombesin-stimulated somatostatin release in a competitive manner (IC50 9 X 10(-8) M). Potentiating interactions were observed between bombesin and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) or epinephrine, but not between bombesin and CCK-8. We conclude that bombesin-like peptides directly stimulate somatostatin release by interacting with specific receptors on rat fundic D cells. Bombesin-like peptides appear to induce Ca(2

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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 usefulmore » agents for labeling peptides and proteins.« less

  7. Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.

    PubMed

    Manikwar, Prakash; Zimmerman, Tahl; Blanco, Francisco J; Williams, Todd D; Siahaan, Teruna J

    2011-07-20

    Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.

  8. Preparation, characterization and pharmacokinetics of fluorescence labeled propylene glycol alginate sodium sulfate

    NASA Astrophysics Data System (ADS)

    Li, Pengli; Li, Chunxia; Xue, Yiting; Zhang, Yang; Liu, Hongbing; Zhao, Xia; Yu, Guangli; Guan, Huashi

    2014-08-01

    A rapid and sensitive fluorescence labeling method was developed and validated for the microanalysis of a sulfated polysaccharide drug,namely propylene glycol alginate sodium sulfate (PSS), in rat plasma. Fluorescein isothiocyanate (FITC) was selected to label PSS, and 1, 6-diaminohexane was used to link PSS and FITC in order to prepare FITC-labeled PSS (F-PSS) through a reductive amination reaction. F-PSS was identified by UV-Vis, FT-IR and 1H-NMR spectrum. The cell stability and cytotoxicity of F-PSS were tested in Madin-Darby canine kidney (MDCK) cells. The results indicated that the labeling efficiency of F-PSS was 0.522% ± 0.0248% and the absolute bioavailability was 8.39%. F-PSS was stable in MDCK cells without obvious cytotoxicity. The method was sensitive and reliable; it showed a good linearity, precision, recovery and stability. The FITC labeling method can be applied to investigating the absorption and metabolism of PSS and other polysaccharides in biological samples.

  9. A nanodiamond-fluorescein conjugate for cell studies

    NASA Astrophysics Data System (ADS)

    Pedroso-Santana, Seidy; Fleitas-Salazar, Noralvis; Sarabia-Sainz, Andrei; Silva-Campa, Erika; Burgara-Estrella, Alexel; Angulo-Molina, Aracely; Melendrez, Rodrigo; Pedroza-Montero, Martin; Riera, Raul

    2018-03-01

    The use of nanodiamonds in studies with living systems generally involves the modification of their surfaces with functional groups. Fluorescent molecules can be attached to these groups, so that one can know the exact position of the particles in each moment of the interaction with the cells. Here we modify the surface of detonation nanodiamonds and nitrogen-vacancy center nanodiamonds using carboxylation and hydroxylation procedures. Subsequent reactions with silicates and cysteine, before addition of fluorescein allow to obtain fluorescent nano-conjugates. We used confocal microscopy to observe the position of nanodiamonds interacting with HeLa cells. At 3 h post-incubation the green fluorescence is localized in extracellular rounded like-vesicles assemblies while at 24 h the conjugates can be observed inside the cells. The measurement of the fluorescence emitted by both conjugates allowed to find an enhanced emission of fluorescein isothiocyanate (FITC) when the nitrogen-vacancy center is present. We propose the existence of a fluorescence enhancement by electron transference process. The procedure described in this work allows the functionalization of nanodiamonds with FITC and other molecules using functional surface groups and small size mediators. Also, as was proved in our work, the nanodiamond-fluorescein conjugates can be used to track nanoparticles position within the cell. Localization studies are particularly important for drug delivery applications of nanodiamonds.

  10. Peptide and protein quantitation by acid-catalyzed 18O-labeling of carboxyl groups.

    PubMed

    Haaf, Erik; Schlosser, Andreas

    2012-01-03

    We have developed a new method that applies acidic catalysis with hydrochloric acid for (18)O-labeling of peptides at their carboxyl groups. With this method, peptides get labeled at their C-terminus, at Asp and Glu residues, and at carboxymethylated cysteine residues. Oxygen atoms at phosphate groups of phosphopeptide are not exchanged. Our elaborated labeling protocol is easy to perform, fast (5 h and 30 min), and results in 95-97 atom % incorporation of (18)O at carboxyl groups. Undesired side reactions, such as deamidation or peptide hydrolysis, occur only at a very low level under the conditions applied. In addition, data analysis can be performed automatically using common software tools, such as Mascot Distiller. We have demonstrated the capability of this method for the quantitation of peptides as well as for phosphopeptides. © 2011 American Chemical Society

  11. Heterogeneous distribution of dye-labelled biomineralizaiton proteins in calcite crystals

    NASA Astrophysics Data System (ADS)

    Liu, Chuang; Xie, Liping; Zhang, Rongqing

    2015-12-01

    Biominerals are highly ordered crystals mediated by organic matters especially proteins in organisms. However, how specific proteins are distributed inside biominerals are not well understood. In the present study, we use fluorescein isothiocyanate (FITC) to label extracted proteins from the shells of bivalve Pinctada fucata. By confocal laser scanning microscopy (CLSM), we observe a heterogeneous distribution of dye-labelled proteins inside synthetic calcite at the microscale. Proteins from the prismatic calcite layers accumulate at the edge of crystals while proteins from the nacreous aragonite layers accumulate at the center of crystals. Raman and X-ray powder diffraction show that both the proteins cannot alter the crystal phase. Scanning electron microscope demonstrates both proteins are able to affect the crystal morphology. This study may provide a direct approach for the visualization of protein distributions in crystals by small-molecule dye-labelled proteins as the additives in the crystallization process and improve our understanding of intracrystalline proteins distribution in biogenic calcites.

  12. Disposition and pharmacokinetics of phenethyl isothiocyanate and 6-phenylhexyl isothiocyanate in F344 rats.

    PubMed

    Conaway, C C; Jiao, D; Kohri, T; Liebes, L; Chung, F L

    1999-01-01

    Naturally occurring phenethyl isothiocyanate (PEITC) and its synthetic homolog 6-phenylhexyl isothiocyanate (PHITC) are both effective inhibitors of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumor development in A/J mice and F344 rats. To help explain why PHITC is considerably more efficacious than PEITC in chemopreventive potency, comparative disposition and pharmacokinetics data for male F344 rats were obtained after a single gavage dose of 50 micromol/kg (3.71 microCi/micromol) [14C]PEITC or 50 micromol/kg (6.59 microCi/micromol) [14C]PHITC in corn oil. After [14C]PEITC dosing, whole blood 14C peaked at 2.9 h, with an elimination half-life (T1/2e) of 21.7 h; blood 14C from [14C]PHITC-treated rats peaked at 8.9 h, with an T1/2e of 20.5 h. In lungs, the target organ, the T1/2e for [14C]PHITC and its labeled metabolites were more than twice that for [14C]PEITC and its labeled metabolites. The effective dose (area under the concentration-time curve) for 14C from PHITC was greater than 2.5 times the area under the concentration-time curve of 14C from PEITC in liver, lungs, and several other tissues. During 48 h, approximately 16.5% of the administered dose of [14C]PHITC was expired as [14C]CO2, more than 100 times the [14C]CO2 expired by rats treated with [14C]PEITC. In rats given [14C]PEITC, 88.7 +/- 2.2% and 9.9 +/- 1.9% of the dose appeared in the urine and feces, respectively, during 48 h; however, rats given [14C]PHITC excreted 7.2 +/- 0.8% of the dose of 14C in urine and 47.4 +/- 14.0% in the feces. Higher effective doses of PHITC in the lungs and other organs may be the basis, in part, for its greater potency as a chemopreventive agent.

  13. 64Cu-Labeled multifunctional dendrimers for targeted tumor PET imaging.

    PubMed

    Ma, Wenhui; Fu, Fanfan; Zhu, Jingyi; Huang, Rui; Zhu, Yizhou; Liu, Zhenwei; Wang, Jing; Conti, Peter S; Shi, Xiangyang; Chen, Kai

    2018-03-29

    We report the use of multifunctional folic acid (FA)-modified dendrimers as a platform to radiolabel with 64Cu for PET imaging of folate receptor (FR)-expressing tumors. In this study, amine-terminated generation 5 (G5) poly(amidoamine) dendrimers were sequentially modified with fluorescein isothiocyanate (FI), FA, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), followed by acetylation of the remaining dendrimer terminal amines. The as-formed multifunctional DOTA-FA-FI-G5·NHAc dendrimers were then radiolabeled with 64Cu via the DOTA chelation. We show that the FA modification renders the dendrimers with targeting specificity to cancer cells overexpressing FR in vitro. Importantly, the radiolabeled 64Cu-DOTA-FA-FI-G5·NHAc dendrimers can be used as a nanoprobe for specific targeting of FR-overexpressing cancer cells in vitro and targeted microPET imaging of the FR-expressing xenografted tumor model in vivo. The developed 64Cu-labeled multifunctional dendrimeric nanoprobe may hold great promise to be used for targeted PET imaging of different types of FR-expressing cancer.

  14. Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip.

    PubMed

    Wang, Chen; Ouyang, Jun; Ye, De-Kai; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

    2012-08-07

    Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.

  15. The effect of colloidal silica nanoparticles encapsulated fluorescein dye using micelle entrapment method

    NASA Astrophysics Data System (ADS)

    Ahmad, Atiqah; Zakaria, Nor Dyana; Lockman, Zainovia; Razak, Khairunisak Abdul

    2018-05-01

    The advancement of nanoparticle-based approaches such as quantum dots (QDs), metallic (Au and Ag) NPs, silica NPs and other types of nanomaterial have led to a large variety of biomolecular imaging and labelling reagents with controlled size and shaped to overcome the limitation of conventional organic dye. In this study, the yellowish green color of fluorescein dye was encapsulated into colloidal silica nanoparticles by using micelle entrapment approach. Two different size of silica nanoparticles encapsulated fluorescein dye (27.7 ± 5.6 and 46.73 ± 4.3 nm) with spherical and monodispered of nanoparticles were synthesised by varying the volume of co-solvent during the synthesis process. The particles size, particles morphology, absorption spectrum and the photostability of fluorescein dye was measured by using dynamic light scaterring (DLS), Transmission Electron Microscope (TEM) and UV-Vis spectrometer. Furthermore, the effect of photostability of of silica nanoparticles encapsulated fluorescein dye was measured under radiation of 200 W of Halogen lamp for 60 minutes. The silica nanoparticles encapsulated fluorescein dye was more stable compared to bare fluorescein dye after the exposure. In conclusion, the photostability of silica nanoparticles encapsulated fluorescein dye was improved compared to bare fluorescein dye, thus silica nanoparticles encapsulation successfully provides protection from the photobleaching and photodegradation of fluorescein dye.

  16. A method for the 32P labeling of peptides or peptide nucleic acid oligomers

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3'-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5'-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [gamma-32P]ATP.

  17. Lectin Ulex europaeus agglutinin I specifically labels a subset of primary afferent fibers which project selectively to the superficial dorsal horn of the spinal cord.

    PubMed

    Mori, K

    1986-02-19

    To examine differential carbohydrate expression among different subsets of primary afferent fibers, several fluorescein-isothiocyanate conjugated lectins were used in a histochemical study of the dorsal root ganglion (DRG) and spinal cord of the rabbit. The lectin Ulex europaeus agglutinin I specifically labeled a subset of DRG cells and primary afferent fibers which projected to the superficial laminae of the dorsal horn. These results suggest that specific carbohydrates containing L-fucosyl residue is expressed selectively in small diameter primary afferent fibers which subserve nociception or thermoception.

  18. A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

    PubMed

    Magiati, Maria; Sevastou, Areti; Kalogianni, Despina P

    2018-06-04

    A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

  19. Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates.

    PubMed Central

    Walter, N G; Burke, J M

    1997-01-01

    Current methods for evaluating the kinetics of ribozyme-catalyzed reactions rely primarily on the use of radiolabeled RNA substrates, and so require tedious electrophoretic separation and quantitation of reaction products for each data point in any experiment. Here, we report the use of fluorescent substrates for real-time analysis of the time course of reactions of the hairpin ribozyme. Fluorescence of 3' fluorescein-labeled substrates was quenched upon binding to the hairpin ribozyme or its isolated substrate-binding strand (SBS), under conditions of ribozyme or SBS excess. This decrease was accompanied by an increase in anisotropy, and resulted from a base-specific quenching by a guanosine residue added to the 5' end of the SBS, close to fluorescein in the complex. Fluorescence quenching was used to determine rate constants for substrate binding (1.4 x 10(8) M(-1) min(-1)), cleavage (0.15 min(-1)), and substrate dissociation (0.010 min(-1)) by a structurally well-defined ribozyme at 25 degrees C in 50 mM Tris-HCI, pH 7.5, 12 mM MgCl2. These rates are in excellent agreement with those obtained using traditional radioisotopic methods. Measurements of dissociation rates provided physical support for interdomain interactions within the substrate-ribozyme complex. We estimate that 2.1 kcal/mol of additional substrate binding energy is provided by the B domain of the ribozyme. Part of this free energy apparently stems from coaxial stacking of helices in the hinge region between domains, and it is plausible that the remainder might be contributed by direct interactions with loop B. The fluorescence quenching and dequenching methods described here should be readily adaptable to studying a wide variety of RNA interactions and reactions using ribozymes and other model systems. PMID:9085846

  20. Fluorescein thiocarbamyl amino acids as internal standards for migration time correction in capillary sieving electrophoresis

    PubMed Central

    Pugsley, Haley R.; Swearingen, Kristian E.; Dovichi, Norman J.

    2009-01-01

    A number of algorithms have been developed to correct for migration time drift in capillary electrophoresis. Those algorithms require identification of common components in each run. However, not all components may be present or resolved in separations of complex samples, which can confound attempts for alignment. This paper reports the use of fluorescein thiocarbamyl derivatives of amino acids as internal standards for alignment of 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ)-labeled proteins in capillary sieving electrophoresis. The fluorescein thiocarbamyl derivative of aspartic acid migrates before FQ-labeled proteins and the fluorescein thiocarbamyl derivative of arginine migrates after the FQ-labeled proteins. These compounds were used as internal standards to correct for variations in migration time over a two-week period in the separation of a cellular homogenate. The experimental conditions were deliberately manipulated by varying electric field and sample preparation conditions. Three components of the homogenate were used to evaluate the alignment efficiency. Before alignment, the average relative standard deviation in migration time for these components was 13.3%. After alignment, the average relative standard deviation in migration time for these components was reduced to 0.5%. PMID:19249052

  1. Improved 18F Labeling of Peptides with a Fluoride-Aluminum-Chelate Complex

    PubMed Central

    McBride, William J.; D’Souza, Christopher A.; Sharkey, Robert M.; Karacay, Habibe; Rossi, Edmund A.; Chang, Chien-Hsing; Goldenberg, David M.

    2010-01-01

    We reported previously the feasibility to radiolabel peptides with fluorine-18 (18F) using a rapid, one-pot, method that first mixes 18F− with Al3+, and then binds the (Al18F)2+ complex to a NOTA ligand on the peptide. In this report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al18F NOTA complexes were stable in vitro in human serum and those that were tested in vivo also were stable. The radiolabeling reactions were performed at 100°C and the peptides could be labeled in as little as five minutes. The IMP467 peptide could be labeled up to 115 GBq/μmol (3100 Ci/mmol), with a total reaction and purification time of 30 min without chromatographic purification. PMID:20540570

  2. Electron Transfer Dissociation of iTRAQ Labeled Peptide Ions

    PubMed Central

    Han, Hongling; Pappin, Darryl J.; Ross, Philip L; McLuckey, Scott A.

    2009-01-01

    Triply and doubly charged iTRAQ (isobaric tagging for relative and absolute quantitation) labeled peptide cations from a tryptic peptide mixture of bovine carbonic anhydrase II were subjected to electron transfer ion/ion reactions to investigate the effect of charge bearing modifications associated with iTRAQ on the fragmentation pattern. It was noted that electron transfer dissociation (ETD) of triply charged or activated ETD (ETD + supplemental collisional activation of intact electron transfer species) of doubly charged iTRAQ tagged peptide ions yielded extensive sequence information, in analogy with ETD of unmodified peptide ions. That is, addition of the fixed charge iTRAQ tag showed relatively little deleterious effect on the ETD performance of the modified peptides. ETD of the triply charged iTRAQ labeled peptide ions followed by collision-induced dissociation (CID) of the product ion at m/z 162 yielded the reporter ion at m/z 116, which is the reporter ion used for quantitation via CID of the same precursor ions. The reporter ion formed via the two-step activation process is expected to provide quantitative information similar to that directly produced from CID. A 103 Da neutral loss species observed in the ETD spectra of all the triply and doubly charged iTRAQ labeled peptide ions is unique to the 116 Da iTRAQ reagent, which implies that this process also has potential for quantitation of peptides/proteins. Therefore, ETD with or without supplemental collisional activation, depending on the precursor ion charge state, has the potential to directly identify and quantify the peptides/proteins simultaneously using existing iTRAQ reagents. PMID:18646790

  3. STUDIES ON INFLUENZA INFECTION IN FERRETS BY MEANS OF FLUORESCEIN-LABELLED ANTIBODY

    PubMed Central

    Liu, Ch'ien

    1955-01-01

    By means of fluorescein-labelled antibody staining, specific influenza viral antigens were seen in both the cytoplasm and the nucleus of infected ciliated epithelial cells covering the nasal turbinates of infected ferrets. Initially, only a small portion of the nasal epithelium showed fluorescence, and no appreciable abnormality of the cells could be detected by hematoxylin and eosin staining. The fluorescence soon spread to involve the entire epithelium, followed by desquamation coinciding with the onset of manifest illness. Pneumonia was seen in some of the infected ferrets, and in them, viral antigens were found in the bronchial epithelium and in the mediastinal lymph nodes. A rise of viral infectivity titer paralleled the observed spread of viral antigens. Many desquamated nasal epithelial cells and macrophages containing antigen were present in nasal smears. The finding would seem to offer a method for the rapid specific diagnosis of influenza infection. PMID:14367687

  4. Interspecies conservation of retinal guanosine 5'-triphosphatase. Characterization by photoaffinity labelling and tryptic-peptide mapping.

    PubMed Central

    McMurray, M M; Hansen, J S; Haley, B E; Takemoto, D J; Takemoto, L J

    1985-01-01

    Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5'-triphosphatase referred to as 'transducin'. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidoguanosine [gamma-32P]triphosphate demonstrated that the 37000-Mr components of the cow, pig and chick and the 75000-Mr component of the frog were major GTP-binding components. In addition, peptide maps of radioiodinated tryptic peptides indicate that the frog 75000-Mr protein is highly related to the pig transducin. These results demonstrate evolutionary conservation of retinal transducin and the presence of a higher-Mr, but nonetheless highly conserved form, of transducin in the frog. The relationship of this component to the recently reported rod-outer-segment inhibitor protein [Yamazaki, Stein, Chernoff & Bitensky (1983) J. Biol. Chem. 258, 8188-8194] is discussed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2983663

  5. Progression and Metastasis of Mammary Carcinomas: Potential Role of the Muc1 Glycoprotein

    DTIC Science & Technology

    1998-09-01

    polyclonal antibody to the cytoplasmic tail of Mucl or antibody previously blocked with 5mg/ml of synthetic peptide (diluted 1:50) for 1 hr at 25°C...Following three 5 minute washes with PBS pH 7.4, the sections were incubated with fluorescein isothiocyanate-conjugated swine anti-rabbit antibody ...stained with Ab-3 murine monoclonal antibody that recognizes the neu oncogene (Oncogene Science,Uniondale, NY; diluted 1:100), for 1 hr at 25°C

  6. A reduced graphene oxide-based fluorescence resonance energy transfer sensor for highly sensitive detection of matrix metalloproteinase 2

    PubMed Central

    Xi, Gaina; Wang, Xiaoping; Chen, Tongsheng

    2016-01-01

    A novel fluorescence nanoprobe (reduced nano-graphene oxide [nrGO]/fluorescein isothiocyanate-labeled peptide [Pep-FITC]) for ultrasensitive detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the Pep-FITC comprising the specific MMP2 substrate domain (PLGVR) onto the surface of nrGO particles through non-covalent linkage. The nrGO was obtained by water bathing nano-graphene oxide under 90°C for 4 hours. After mixing the nrGO and Pep-FITC for 30 seconds, the fluorescence from Pep-FITC was almost completely quenched due to the fluorescence resonance energy transfer between fluorescein isothiocyanate (FITC) and nrGO. Upon cleavage of the amide bond between Leu and Gly in the Pep-FITC by protease-MMP2, the FITC bound to nrGO was separated from nrGO surface, disrupting the fluorescence resonance energy transfer process and resulting in fluorescence recovery of FITC. Under optimal conditions, the fluorescence recovery of nrGO/Pep-FITC was found to be directly proportional to the concentration of MMP2 within 0.02–0.1 nM. The detection limit of the nrGO/Pep-FITC was determined to be 3 pM, which is approximately tenfold lower than that of the unreduced carboxylated nano-graphene oxide/Pep-FITC probe. PMID:27143876

  7. A reduced graphene oxide-based fluorescence resonance energy transfer sensor for highly sensitive detection of matrix metalloproteinase 2.

    PubMed

    Xi, Gaina; Wang, Xiaoping; Chen, Tongsheng

    2016-01-01

    A novel fluorescence nanoprobe (reduced nano-graphene oxide [nrGO]/fluorescein isothiocyanate-labeled peptide [Pep-FITC]) for ultrasensitive detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the Pep-FITC comprising the specific MMP2 substrate domain (PLGVR) onto the surface of nrGO particles through non-covalent linkage. The nrGO was obtained by water bathing nano-graphene oxide under 90°C for 4 hours. After mixing the nrGO and Pep-FITC for 30 seconds, the fluorescence from Pep-FITC was almost completely quenched due to the fluorescence resonance energy transfer between fluorescein isothiocyanate (FITC) and nrGO. Upon cleavage of the amide bond between Leu and Gly in the Pep-FITC by protease-MMP2, the FITC bound to nrGO was separated from nrGO surface, disrupting the fluorescence resonance energy transfer process and resulting in fluorescence recovery of FITC. Under optimal conditions, the fluorescence recovery of nrGO/Pep-FITC was found to be directly proportional to the concentration of MMP2 within 0.02-0.1 nM. The detection limit of the nrGO/Pep-FITC was determined to be 3 pM, which is approximately tenfold lower than that of the unreduced carboxylated nano-graphene oxide/Pep-FITC probe.

  8. A dual-labeled knottin peptide for PET and near-infrared fluorescence imaging of integrin expression in living subjects

    PubMed Central

    Kimura, Richard H.; Miao, Zheng; Cheng, Zhen; Gambhir, Sanjiv S.; Cochran, Jennifer R.

    2010-01-01

    Previously, we used directed evolution to engineer mutants of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin that bind to αvβ3 and αvβ5 integrin receptors with low nanomolar affinity, and showed that Cy5.5- or 64Cu-DOTA-labeled knottin peptides could be used to image integrin expression in mouse tumor models using near-infrared fluorescence (NIRF) imaging or positron emission tomography (PET). Here, we report the development of a dual-labeled knottin peptide conjugated to both NIRF and PET imaging agents for multimodality imaging in living subjects. We created an orthogonally-protected peptide-based linker for stoichiometric coupling of 64Cu-DOTA and Cy5.5 onto the knottin N-terminus, and confirmed that conjugation did not affect binding to αvβ3 and αvβ5 integrins. NIRF and PET imaging studies in tumor xenograft models showed that Cy5.5 conjugation significantly increased kidney uptake and retention compared to the knottin peptide labeled with 64Cu-DOTA alone. In the tumor, the dual-labeled 64Cu-DOTA/Cy5.5 knottin probe showed decreased wash-out leading to significantly better retention (p < 0.05) compared to the 64Cu-DOTA-labeled knottin probe. Tumor uptake was significantly reduced (p < 0.05) when the dual-labeled probe was co-injected with an excess of unlabeled competitor and when tested in a tumor model with lower levels of integrin expression. Finally, plots of tumor-to-background tissue ratios for Cy5.5 versus 64Cu uptake were well correlated over several time points post injection, demonstrating pharmacokinetic cross validation of imaging labels. This dual-modality NIRF/PET imaging agent is promising for further development in clinical applications where high sensitivity and high-resolution are desired, such as detection of tumors located deep within the body and image-guided surgical resection. PMID:20131753

  9. An investigation of paper based microfluidic devices for size based separation and extraction applications.

    PubMed

    Zhong, Z W; Wu, R G; Wang, Z P; Tan, H L

    2015-09-01

    Conventional microfluidic devices are typically complex and expensive. The devices require the use of pneumatic control systems or highly precise pumps to control the flow in the devices. This work investigates an alternative method using paper based microfluidic devices to replace conventional microfluidic devices. Size based separation and extraction experiments conducted were able to separate free dye from a mixed protein and dye solution. Experimental results showed that pure fluorescein isothiocyanate could be separated from a solution of mixed fluorescein isothiocyanate and fluorescein isothiocyanate labeled bovine serum albumin. The analysis readings obtained from a spectrophotometer clearly show that the extracted tartrazine sample did not contain any amount of Blue-BSA, because its absorbance value was 0.000 measured at a wavelength of 590nm, which correlated to Blue-BSA. These demonstrate that paper based microfluidic devices, which are inexpensive and easy to implement, can potentially replace their conventional counterparts by the use of simple geometry designs and the capillary action. These findings will potentially help in future developments of paper based microfluidic devices. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Combinatorial Labeling Method for Improving Peptide Fragmentation in Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Kuchibhotla, Bhanuramanand; Kola, Sankara Rao; Medicherla, Jagannadham V.; Cherukuvada, Swamy V.; Dhople, Vishnu M.; Nalam, Madhusudhana Rao

    2017-06-01

    Annotation of peptide sequence from tandem mass spectra constitutes the central step of mass spectrometry-based proteomics. Peptide mass spectra are obtained upon gas-phase fragmentation. Identification of the protein from a set of experimental peptide spectral matches is usually referred as protein inference. Occurrence and intensity of these fragment ions in the MS/MS spectra are dependent on many factors such as amino acid composition, peptide basicity, activation mode, protease, etc. Particularly, chemical derivatizations of peptides were known to alter their fragmentation. In this study, the influence of acetylation, guanidinylation, and their combination on peptide fragmentation was assessed initially on a lipase (LipA) from Bacillus subtilis followed by a bovine six protein mix digest. The dual modification resulted in improved fragment ion occurrence and intensity changes, and this resulted in the equivalent representation of b- and y-type fragment ions in an ion trap MS/MS spectrum. The improved representation has allowed us to accurately annotate the peptide sequences de novo. Dual labeling has significantly reduced the false positive protein identifications in standard bovine six peptide digest. Our study suggests that the combinatorial labeling of peptides is a useful method to validate protein identifications for high confidence protein inference. [Figure not available: see fulltext.

  11. Optimizing labelling conditions of 213Bi-DOTATATE for preclinical applications of peptide receptor targeted alpha therapy.

    PubMed

    Chan, Ho Sze; de Blois, Erik; Konijnenberg, Mark W; Morgenstern, Alfred; Bruchertseifer, Frank; Norenberg, Jeffrey P; Verzijlbergen, Fred J; de Jong, Marion; Breeman, Wouter A P

    2017-01-01

    213 Bismuth ( 213 Bi, T 1/2 = 45.6 min) is one of the most frequently used α-emitters in cancer research. High specific activity radioligands are required for peptide receptor radionuclide therapy. The use of generators containing less than 222 MBq 225 Ac (actinium), due to limited availability and the high cost to produce large-scale 225 Ac/ 213 Bi generators, might complicate in vitro and in vivo applications though.Here we present optimized labelling conditions of a DOTA-peptide with an 225 Ac/ 213 Bi generator (< 222 MBq) for preclinical applications using DOTA-Tyr 3 -octreotate (DOTATATE), a somatostatin analogue. The following labelling conditions of DOTATATE with 213 Bi were investigated; peptide mass was varied from 1.7 to 7.0 nmol, concentration of TRIS buffer from 0.15 mol.L -1 to 0.34 mol.L -1 , and ascorbic acid from 0 to 71 mmol.L -1 in 800 μL. All reactions were performed at 95 °C for 5 min. After incubation, DTPA (50 nmol) was added to stop the labelling reaction. Besides optimizing the labelling conditions, incorporation yield was determined by ITLC-SG and radiochemical purity (RCP) was monitored by RP-HPLC up to 120 min after labelling. Dosimetry studies in the reaction vial were performed using Monte Carlo and in vitro clonogenic assay was performed with a rat pancreatic tumour cell line, CA20948. At least 3.5 nmol DOTATATE was required to obtain incorporation ≥ 99 % with 100 MBq 213 Bi (at optimized pH conditions, pH 8.3 with 0.15 mol.L -1 TRIS) in a reaction volume of 800 μL. The cumulative absorbed dose in the reaction vial was 230 Gy/100 MBq in 30 min. A minimal final concentration of 0.9 mmol.L -1 ascorbic acid was required for ~100 MBq (t = 0) to minimize radiation damage of DOTATATE. The osmolarity was decreased to 0.45 Osmol/L.Under optimized labelling conditions, 213 Bi-DOTATATE remained stable up to 2 h after labelling, RCP was ≥ 85 %. In vitro showed a negative correlation between ascorbic acid

  12. Highly sensitive microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer for the detection of neuron-specific enolase.

    PubMed

    Yang, Tingzhen; Vdovenko, Marina; Jin, Xue; Sakharov, Ivan Yu; Zhao, Shulin

    2014-07-01

    A microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer (CRET) was developed for highly sensitive detection of neuron-specific enolase (NSE). The CRET system consisted of horseradish peroxidase (HRP)/luminol as a light donor and fluorescein isothiocyanate as an acceptor. When fluorescein isothiocyanate-labeled antibody binds with HRP-labeled antigen to form immunocomplex, the donor and acceptor are brought close each other and CRET occurs in the immunocomplex. In the MCE, the immunocomplex and excess HRP-NSE were separated, and the chemiluminescense intensity of immunocomplex was used to estimate NSE concentration. The calibration curve showed a linearity in the range of NSE concentrations from 9.0 to 950 pM with a correlation coefficient of 0.9964. Based on a S/N of 3, the detection limit for NSE determination was estimated to be 4.5 pM, which is two-order magnitude lower than that of without CRET detection. This assay was applied for NSE quantification in human serum. The obtained results demonstrated that the proposed immunoassay may serve as an alternative tool for clinical analysis of NSE. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Lysozyme as a recognition element for monitoring of bacterial population.

    PubMed

    Zheng, Laibao; Wan, Yi; Yu, Liangmin; Zhang, Dun

    2016-01-01

    Bacterial infections remain a significant challenge in biomedicine and environment safety. Increasing worldwide demand for point-of-care techniques and increasing concern on their safe development and use, require a simple and sensitive bioanalysis for pathogen detection. However, this goal is not yet achieved. A design for fluorescein isothiocyanate-labeled lysozyme (FITC-LYZ), which provides quantitative binding information for gram-positive bacteria, Micrococcus luteus, and detects pathogen concentration, is presented. The functional lysozyme is used not only as the pathogenic detection platform, but also as a tracking reagent for microbial population in antibacterial tests. A nonlinear relationship between the system response and the logarithm of the bacterial concentration was observed in the range of 1.2×10(2)-1.2×10(5) cfu mL(-1). The system has a potential for further applications and provides a facile and simple method for detection of pathogenic bacteria. Meanwhile, the fluorescein isothiocyanate -labeled lysozyme is also employed as the tracking agent for antibacterial dynamic assay, which show a similar dynamic curve compared with UV-vis test. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Feasibility and availability of ⁶⁸Ga-labelled peptides.

    PubMed

    Decristoforo, Clemens; Pickett, Roger D; Verbruggen, Alfons

    2012-02-01

    (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 (68)Ge/(68)Ga generators, making it independent of cyclotron production. (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 (68)Ga-labelled peptides, including generator technology, (68)Ga generator eluate postprocessing methods, radiolabelling, automation and peptide developments, and also quality assurance and regulatory aspects. (68)Ge/(68)Ga generators based on SnO(2), TiO(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 (68)Ga that could open new ways to utilize this technology. Challenges and limitations in the on-site preparation and use of (68)Ga-labelled peptides outside the marketing authorization track are also discussed.

  15. Dynamic PET and SPECT imaging with radioiodinated, amyloid-reactive peptide p5 in mice: A positive role for peptide dehalogenation

    PubMed Central

    Martin, Emily B.; Kennel, Stephen J.; Richey, Tina; Wooliver, Craig; Osborne, Dustin; Williams, Angela; Stuckey, Alan; Wall, Jonathan S.

    2014-01-01

    Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72 hours post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients. PMID:25102446

  16. Flow cytometry of HEK 293T cells interacting with polyelectrolyte multilayer capsules containing fluorescein-labeled poly(acrylic acid) as a pH sensor.

    PubMed

    Reibetanz, Uta; Halozan, David; Brumen, Milan; Donath, Edwin

    2007-06-01

    Polyelectrolyte multilayer sensor capsules, 5 microm in diameter, which contained fluorescein-labeled poly(acrylic acid) (PAAAF) as pH-sensitive reporter molecules, were fabricated and employed to explore their endocytotic uptake into HEK 293T cells by flow cytometry. The percentage of capsules residing in the endolysosomal compartment was estimated from the fluorescence intensity decrease caused by acidification. Capsules attached to the extracellular surface of the plasma membrane were identified by trypan blue quenching. The number of capsules in the cytoplasm was rather small, being below the detection limit of the method. The advantages of polyelectrolyte multilayer capsules are that the fluorophore is protected from interaction with cellular compartments and that the multilayer can be equipped with additional functions.

  17. Quantitative proteome analysis using isobaric peptide termini labeling (IPTL).

    PubMed

    Arntzen, Magnus O; Koehler, Christian J; Treumann, Achim; Thiede, Bernd

    2011-01-01

    The quantitative comparison of proteome level changes across biological samples has become an essential feature in proteomics that remains challenging. We have recently introduced isobaric peptide termini labeling (IPTL), a novel strategy for isobaric quantification based on the derivatization of peptide termini with complementary isotopically labeled reagents. Unlike non-isobaric quantification methods, sample complexity at the MS level is not increased, providing improved sensitivity and protein coverage. The distinguishing feature of IPTL when comparing it to more established isobaric labeling methods (iTRAQ and TMT) is the presence of quantification signatures in all sequence-determining ions in MS/MS spectra, not only in the low mass reporter ion region. This makes IPTL a quantification method that is accessible to mass spectrometers with limited capabilities in the low mass range. Also, the presence of several quantification points in each MS/MS spectrum increases the robustness of the quantification procedure.

  18. Surface binding properties of aged and fresh (recently excreted) Toxoplasma gondii oocysts

    USDA-ARS?s Scientific Manuscript database

    The surface properties of aged (stored for 10 years) and fresh (recently excreted) oocysts of Toxoplasma gondii were investigated using monoclonal antibody (mAb) and lectin-binding assays. Fresh oocysts bound a wall-specific mAb labeled with fluorescein isothiocyanate while aged oocysts did not. In ...

  19. Isothiocyanates: mechanism of cancer chemopreventive action.

    PubMed

    Thornalley, Paul J

    2002-04-01

    Dietary and synthetic isothiocyanates have cancer chemopreventive activity. Dietary isothiocyanates are formed from glucosinolate precursors of ingested green vegetables. Isothiocyanates are absorbed across intestinal cell membranes by passive diffusion and bind reversibly to plasma protein thiols by thiocarbamoylation. Free isothiocyanate enters cells and is converted to the glutathione conjugate by glutathione S-transferases (GSTs). The glutathione conjugate is exported from cells by multidrug resistance proteins (MRPs), and metabolized in the mercapturic acid pathway to the corresponding mercapturic acid. The isothiocyanate is reformed by fragmentation of mercapturic acid pathway metabolites; it is inactivated by slow hydrolysis to the corresponding amine that is inactive in chemoprevention. Depletion of cellular glutathione and protein thiocarbamoylation activates signal transduction for cancer chemoprevention. Isothiocyanates inhibited and inactivated cytochrome P450 isoforms. They induced increased expression of GST, NADPH: quinone oxidoreductase, aldo-keto reductase and gamma-glutamylcysteine synthetase. These responses were coordinated at the transcription level by nuclear factor-erythroid 2 p45-related factor-2 acting through the antioxidant/electrophile enhancer response element and stimulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1 and c-Jun N-terminal kinase-1 (JNK1) pathway. Isothiocyanates also induced apoptosis of pre-cancerous cells and tumor cells activated by caspase-8 and potentiated by JNK1. The chemopreventive activity of isothiocyanates is influenced by the isothiocyanate bioavailability-as is toxicity, GST polymorphism, protein thiocarbamoylation and probably also by MRP expression. These features of isothiocyanate metabolism and chemoprevention deserve further investigation.

  20. Preparation of fluorescently labeled silica nanoparticles using an amino acid-catalyzed seeds regrowth technique: Application to latent fingerprints detection and hemocompatibility studies.

    PubMed

    Abdelwahab, Walid M; Phillips, Edjohnier; Patonay, Gabor

    2018-02-15

    The efficiency of an amino acid catalyzed seed regrowth technique (ACSRT) in synthesizing twelve fluorescently labeled core-shell silica nanoparticles (FLSNPs) with tunable sizes, tailored hydrophobicity, low polydispersity as well as high labeling efficiency and minimized dye leakage using different combinations of organosilicate monomers and fluorophores have been systematically investigated in this report. The utilization of some of these FLSNPs in some applications that are facilitated by hydrophobicity such as developing and visualizing latent fingerprints (LFPs) on different surfaces was also investigated. The non-specific binding affinity of the developed nanoparticles to human serum albumin (HSA) and immunoglobulin G (IgG) has also been studied. Fluorescein, fluorescein isothiocyanate and its more hydrophilic butenamine derivative (WA6) have been used in this study. Also, the alkoxysilane precursor, tetraethoxyorthosilicate (TEOS) and its binary mixture with phenyltriethoxysilane (PTEOS) or 3-aminopropyl triethoxysilane (APTES) have been used in preparing the FLSNPs with tailored compositions for the core and shell of the nanoparticles. The mean diameters of the PTEOS-coated FLSNPs were between 33.4±5.9 and 42.2±10.8 nm as shown by the SEM measurements. The obtained results highlight the advantages of having a hydrophobic surface along with proper selection of the monomers forming the core to match the properties of the fluorescent reporters for clear detection of LFPs even using dyes of low hydrophobicity such as fluorescein and WA6. Furthermore, some of the developed FLSNPs were compared with bare silica nanoparticles in terms of nonspecific protein adsorption and hemolysis. The obtained results proved that the selected FLSNPs had a superior hemocompatibility in comparison with bare silica nanoparticles. These FLSNPs could also be used in some bio-related and diagnostic applications such as immunoassays and cell imaging purposes. Copyright © 2017

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

    PubMed

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Dynamic PET and SPECT imaging with radioiodinated, amyloid-reactive peptide p5 in mice: a positive role for peptide dehalogenation.

    PubMed

    Martin, Emily B; Kennel, Stephen J; Richey, Tina; Wooliver, Craig; Osborne, Dustin; Williams, Angela; Stuckey, Alan; Wall, Jonathan S

    2014-10-01

    Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72h post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Direct fluorescent labeling for efficient biological assessment of inhalable particles.

    PubMed

    Poudel, Bijay Kumar; Park, Jae Hong; Lim, Jiseok; Byeon, Jeong Hoon

    2017-10-01

    Labeling of aerosol particles with a radioactive, magnetic, or optical tracer has been employed to confirm particle localization in cell compartments, which has provided useful evidence for correlating toxic effects of inhaled particles. However, labeling requires several physicochemical steps to identify functionalities of the inner or outer surfaces of particles, and moreover, these steps can cause changes in size, surface charge, and bioactivity of the particles, resulting in misinterpretations regarding their toxic effects. This study addresses this challenging issue with a goal of introducing an efficient strategy for constantly supplying labeled aerosol particles in a single-pass configuration without any pre- or post-physicochemical batch treatments of aerosol particles. Carbon black (CB, simulating combustion-generated soot) or calcium carbonate (CC, simulating brake-wear fragments) particles were constantly produced via spark ablation or bubble bursting, respectively. These minute particles were incorporated with fluorescein isothiocyanate-poly(ethylene glycol) 2-aminoethyl ether acetic acid solution at the orifice of a collison atomizer to fabricate hybrid droplets. The droplets successively entered a diffusion dryer containing 254-nm UV irradiation; therefore, the droplets were dynamically stiffened by UV to form fluorescent probes on particles during solvent extraction in the dryer. Particle size distributions, morphologies, and surface charges before and after labeling were measured to confirm fluorescence labeling without significant changes in the properties. In vitro assays, including confocal imaging, were conducted to confirm the feasibility of the labeling approach without inducing significant differences in bioactivity compared with untreated CB or CC particles.

  4. Fluorescent Staining of Tea Pathogenic Fungi in Tea Leaves Using Fluorescein-labeled Lectin

    NASA Astrophysics Data System (ADS)

    Yamada, Kengo; Yoshida, Katsuyuki; Sonoda, Ryoichi

    Fluorochrome-labeled lectin, fluorescein conjugated wheat germ agglutinin (F-WGA) was applied to stain tea pathogenic fungi in tea leaf tissue. Infected leaves were fixed and decolorized with a mixture of ethanol and acetic acid, and cleared with 10% KOH for whole mount before staining with F-WGA. Hyphae of Pestalotiopsis longiseta, Pseudocercospora ocellata, Botrytis cinerea and Colletotrichum theae-sinensis fluoresced brightly in whole mount and sectioned samples of infected leaf tissue. In browned tissue, hyphae did not fluoresce frequently in whole mount sample. Autofluorescence of leaf tissue was strong in browned tissue of sections, it was removed by 10% KOH treatment before staining. Penetration hyphae of C. theae-sinensis in cell wall of trichome and hyphae in basal part of trichome did not fluoresced frequently. In whole mount samples of tea leaf infected with Exobasidium vexans and E. reticulatum, hymenia appeared on leaf surface fluoresced, but hyphae in leaf tissue did not fluoresce. In sectioned samples, hyphae fluoresced brightly when sections were treated with 10% KOH before staining.

  5. Stable, water extractable isothiocyanates from Moringa oleifera leaves attenuate inflammation in vitro

    PubMed Central

    Waterman, Carrie; Cheng, Diana M.; Rojas-Silva, Patricio; Poulev, Alexander; Dreifus, Julia; Ann Lila, Mary; Raskin, Ilya

    2014-01-01

    Moringa (Moringa oleifera Lam.) is an edible plant used as food and medicine throughout the tropics. A moringa concentrate (MC) made by extracting fresh leaves with water utilized naturally occurring myrosinase to convert four moringa glucosinolates (1–4) into moringa isothiocyanates (5–8). Optimum conditions maximizing MC yield, compound 5 (4-[(α-L-rhamnosyloxy)benzyl]isothiocyanate), and compound 8 (4-[(4’-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate) content were established (1:5 fresh leaf weight to water ratio at room temperature). The optimized MC contained 1.66% isothiocyanates and 3.82% total polyphenols. Compound 8 exhibited 80% stability at 37 °C for 30 days. MC, 5, and 8 significantly decreased gene expression and production of inflammatory markers in RAW macrophages. Specifically, 5 and 8 attenuated expression of iNOS and IL-1β and production of nitric oxide and TNFβ at 1 and 5 µM. Our results suggest a potential for stable and concentrated moringa isothiocyanates (5–8), delivered in MC as a food-grade product, to alleviate low-grade inflammation associated with chronic diseases. PMID:24731259

  6. RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells.

    PubMed

    He, Xuedan; Alves, Carla S; Oliveira, Nilsa; Rodrigues, João; Zhu, Jingyi; Bányai, István; Tomás, Helena; Shi, Xiangyang

    2015-01-01

    Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvβ3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvβ3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvβ3 integrin-overexpressing cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Stable, water extractable isothiocyanates from Moringa oleifera leaves attenuate inflammation in vitro.

    PubMed

    Waterman, Carrie; Cheng, Diana M; Rojas-Silva, Patricio; Poulev, Alexander; Dreifus, Julia; Lila, Mary Ann; Raskin, Ilya

    2014-07-01

    Moringa (Moringa oleifera Lam.) is an edible plant used as both a food and medicine throughout the tropics. A moringa concentrate (MC), made by extracting fresh leaves with water, utilized naturally occurring myrosinase to convert four moringa glucosinolates into moringa isothiocyanates. Optimum conditions maximizing MC yield, 4-[(α-L-rhamnosyloxy)benzyl]isothiocyanate, and 4-[(4'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate content were established (1:5 fresh leaf weight to water ratio at room temperature). The optimized MC contained 1.66% isothiocyanates and 3.82% total polyphenols. 4-[(4'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate exhibited 80% stability at 37°C for 30 days. MC, and both of the isothiocyanates described above significantly decreased gene expression and production of inflammatory markers in RAW macrophages. Specifically, both attenuated expression of iNOS and IL-1β and production of nitric oxide and TNFα at 1 and 5 μM. These results suggest a potential for stable and concentrated moringa isothiocyanates, delivered in MC as a food-grade product, to alleviate low-grade inflammation associated with chronic diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging.

    PubMed

    Mendive-Tapia, Lorena; Subiros-Funosas, Ramon; Zhao, Can; Albericio, Fernando; Read, Nick D; Lavilla, Rodolfo; Vendrell, Marc

    2017-08-01

    Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C 2 -BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).

  9. Characterization of labelling and de-labelling reagents for detection and recovery of tyrosine residue in peptide.

    PubMed

    Toyo'oka, Toshimasa; Mantani, Tomomi; Kato, Masaru

    2003-01-01

    This paper characterized the labelling and de-labelling reagents for reversible labelling of tyrosine (Tyr)-containing peptide, which involves detection and recovery. The phenolic hydroxyl group (-OH) in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), and 1-fluoro-2,4-dinitrobenzene (DNFB) under mild conditions at room temperature at pH 9.3. The labels in the resulting derivatives were removed with the treatment of nucleophiles, such as thiols (cysteine, N-acetyl-L-cysteine and dithiothreitol) and amines (dimethylamine, methylamine, diethylamine, ethylamine and pyrrolidine). The de-labelling reactions of NBD-labelled N-acetyl-L-tyrosine (N-AcTyr) with the nucleophiles produced N-AcTyr, accompanied by NBD-nucleophile. Although DBD-F and DNFB also successfully labeled the -OH group in N-AcTyr, the efficiency of Cbond;O bond cleavage and recovery of N-AcTyr by the nucleophiles was relatively low compared with NBD-label. Among the de-labelling reagents, N-acetyl-L-cysteine and dimethylamine were recommended for the elimination of NBD moiety, with respect to the reaction rate, the side reaction, and the yield of recovery. The proposed procedure, which includes the labelling with NBD-F and the removal of NBD moiety by the nucleophiles, was successfully applied to the reversible labelling of N-terminal amine-blocked peptides, i.e. N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Copyright 2003 John Wiley & Sons, Ltd.

  10. Neutrophil phagocytosis following inoculation of Salmonella choleraesuis into swine.

    PubMed

    Stabel, T J; Fedorka-Cray, P J; Gray, J T

    2002-02-01

    Neutrophils are an important mediator of host defence, especially in early stages of infection. A major function of neutrophils is the uptake and killing of invading microbes. Little is known about the effect of neutrophil activity on the pathogenesis and development of the carrier state in swine following infection with Salmonella choleraesuis. A human whole-blood microassay using flow cytometry was modified to measure the effect of S. choleraesuis infection in vivo on the rate of ingestion, or rate of uptake, of homologous bacteria by porcine neutrophils. Pigs were inoculated intranasally with 5-8 x 10(8) CFU S. choleraesuis and blood was collected in heparinized tubes at -5, 0, 1, 2, 3 and 4 days post inoculation (PI). Heat-killed S. choleraesuis were labelled with fluorescein isothiocyanate and incubated for various times with diluted whole blood. Red blood cells were lysed, external non-phagocytized bacteria were quenched with a commercially available lysing solution, and fluorescence from internalized bacteria labelled with fluorescein isothiocyanate was detected by flow cytometry. The rate of uptake by neutrophils did not increase until 2 days PI and then remained elevated to 4 days PI. The minimal uptake of S. choleraesuis early after exposure to these organisms may provide an opportunity for the pathogen to colonize and/or replicate to levels that facilitate establishment of a carrier state or clinical infection in swine.

  11. TRIBUTYLTIN ISOTHIOCYANATE AND ISOCYANIDE,

    DTIC Science & Technology

    Infrared evidence was used to demonstrate that tributyltin isothiocyanate is formed when certain tributyltin compounds are reacted with inorganic...thiocyanates or thiourea. Molecular refraction data supporting this contention are also reported. The preparation of tributyltin isothiocyanate by several different methods and of tributyltin succinimide is described. (Author)

  12. Novel Route to Transition Metal Isothiocyanate Complexes Using Metal Powders and Thiourea

    NASA Technical Reports Server (NTRS)

    Harris, Jerry D.; Eckles, William E.; Hepp, Aloysius F.; Duraj, Stan A.; Hehemann, David G.; Fanwick, Phillip E.; Richardson, John

    2003-01-01

    A new synthetic route to isothiocyanate-containing materials is presented. Eight isothiocyanate- 4-methylpyridine (y-picoline) compounds were prepared by refluxing metal powders (Mn, Fe, Co, Ni, and Cu) with thiourea in y-picoline. With the exception of compound 5,prepared with Co, the isothiocyanate ligand was generated in situ by the isomerization of thiourea to NH4+SCN- at reflux temperatures. The complexes were characterized by x-ray crystallography. Compounds 1,2, and 8 are the first isothiocyanate- 4-methylpyridine anionic compounds ever prepared and structurally characterized. Compounds 1 and 2 are isostructural with four equatorially bound isothiocyanate ligands and two axially bound y-picoline molecules. Compound 8 is a five-coordinate copper(II) molecule with a distorted square-pyramidal geometry. Coordinated picoline and two isothiocyanates form the basal plane and the remaining isothiocyanate is bound at the apex. Structural data are presented for all compounds.

  13. Immunochemical detection of food-derived isothiocyanate as a lysine conjugate.

    PubMed

    Nakamura, Toshiyuki; Kitamoto, Noritoshi; Osawa, Toshihiko; Kato, Yoji

    2010-01-01

    In a previous study we prepared monoclonal antibody against allyl isothiocyanate (AITC)-modified lysine (Lys), and found that AITC reacted with Lys under physiological conditions in vitro (T. Nakamura et al., Chem. Res. Toxicol., 22, 536-542 (2009)). In the present study, antibodies against benzyl isothiocyanate (ITC), 6-methylsulfinylhexyl ITC and phenethyl ITC modified protein were prepared, and the respective monoclonal antibodies, B6C9, 6MS3D10, and PE3A10 were obtained. These antibodies were applied to ITC detection in food using shredded Wasabia japonica (wasabi) and ground Carica papaya (papaya) seed by trapping ITC with biotin-labeled bovine serum albumin. ITC formation from the wasabi and papaya seed samples was confirmed using the antibodies in a dose-dependent manner. These antibodies might be applicable in identifying food-derived ITC.

  14. Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

    PubMed

    Katz, B M; Lundquist, L J; Walsh, D A; Glass, D B

    1989-06-01

    PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

  15. 44Sc for labeling of DOTA- and NODAGA-functionalized peptides: preclinical in vitro and in vivo investigations.

    PubMed

    Domnanich, Katharina A; Müller, Cristina; Farkas, Renata; Schmid, Raffaella M; Ponsard, Bernard; Schibli, Roger; Türler, Andreas; van der Meulen, Nicholas P

    2017-01-01

    Recently, 44 Sc (T 1/2  = 3.97 h, Eβ + av  = 632 keV, I = 94.3 %) has emerged as an attractive radiometal candidate for PET imaging using DOTA-functionalized biomolecules. The aim of this study was to investigate the potential of using NODAGA for the coordination of 44 Sc. Two pairs of DOTA/NODAGA-derivatized peptides were investigated in vitro and in vivo and the results obtained with 44 Sc compared with its 68 Ga-labeled counterparts.DOTA-RGD and NODAGA-RGD, as well as DOTA-NOC and NODAGA-NOC, were labeled with 44 Sc and 68 Ga, respectively. The radiopeptides were investigated with regard to their stability in buffer solution and under metal challenge conditions using Fe 3+ and Cu 2+ . Time-dependent biodistribution studies and PET/CT imaging were performed in U87MG and AR42J tumor-bearing mice. Both RGD- and NOC-based peptides with a DOTA chelator were readily labeled with 44 Sc and 68 Ga, respectively, and remained stable over at least 4 half-lives of the corresponding radionuclide. In contrast, the labeling of NODAGA-functionalized peptides with 44 Sc was more challenging and the resulting radiopeptides were clearly less stable than the DOTA-derivatized matches. 44 Sc-NODAGA peptides were clearly more susceptible to metal challenge than 44 Sc-DOTA peptides under the same conditions. Instability of 68 Ga-labeled peptides was only observed if they were coordinated with a DOTA in the presence of excess Cu 2+ . Biodistribution data of the 44 Sc-labeled peptides were largely comparable with the data obtained with the 68 Ga-labeled counterparts. It was only in the liver tissue that the uptake of 68 Ga-labeled DOTA compounds was markedly higher than for the 44 Sc-labeled version and this was also visible on PET/CT images. The 44 Sc-labeled NODAGA-peptides showed a similar tissue distribution to those of the DOTA peptides without any obvious signs of in vivo instability. Although DOTA revealed to be the preferred chelator for stable coordination of 44

  16. Italian multicentre study on intrathecal fluorescein for craniosinusal fistulae.

    PubMed

    Felisati, G; Bianchi, A; Lozza, P; Portaleone, S

    2008-08-01

    Cerebrospinal fluid leak (CSF), clinical sign of a dural lesion of the skull base, is a relatively rare event that can present with a variety of symptoms. Every craniosinus fistula should be considered a serious, potentially life-threatening situation (even those cases with hidden CSF leak). Reports of experience concerning diagnosis and treatment of craniosinus fistulae have appeared in the Literature. In the last few years, the endoscopic nasal approach is proving effective as it makes diagnosis much easier and is the least invasive surgical approach, with the greatest percentage of success. Various classifications are being proposed to improve clinical evaluation of CSF leaks and to simplify the diagnostic and therapeutic approach. The most common parameters of classification are: aetiology (traumatic, iatrogenic, non-traumatic, etc.) site, type of flow (high or low pressure) and, as far as concerns treatment, the type of graft used, all of which have contributed to various diagnostic and therapeutic algorithms being proposed. Therefore, the subject seems to be widely schematized and the therapeutic attitude widely agreed. However, one of the diagnostic and therapeutic approaches is now being questioned. For some, it is the heart of the clinical approach, while for others, it is a useful tool yet too dangerous to be used on account of potential side effects: namely, the fluorescein test. This procedure, consisting of intrathecal injection of a colorant (fluorescein), is well known by the Food and Drug Administration (FDA) which neither explicitly prohibits it, nor allows it, intrathecal administration is, therefore, an off label use. As far as the Authors know, authorization of this procedure has not been forthcoming anywhere in the world although the procedure itself is widely employed. As far as concerns the use of intrathecal fluorescein, many scientific papers have been written, clearly supporting its clinical usefulness. One limit to the use of fluorescein

  17. Investigation of phosphatidylcholine enhancing FITC-insulin across buccal mucosa by confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Tian, Weiqun; Su, Li; Zeng, Shaoqun; Luo, Qingming; Gao, Qiuhua; Xu, Huibi

    2002-04-01

    The aim was to characterize the transport of fluorescein isothiocyanate (FITC)-labeled dextran and insulin with different resoluble compounds for peptides and proteins through buccal mucosa. The penetration rate of insulin molecules through porcine buccal mucosa (a nonkeratinized epithelium, comparable to human buccal mucosa) was investigated by measuring transbuccal fluxes and by analyzing the distribution of the fluorescent probe in the rabbit buccal mucosa epithelium, using confocal laser scanning microscopy for visualizing permeation pathways. The confocal images of the distribution pattern of FITC-dextran and FITC-insulin showed that the paracellular route is the major pathway of FITC-dextran through buccal mucosa epithelium, the intra-cellular route is the major pathway of FITC-insulin through buccal mucosa epithelium. The permeation rate can be increased by co-administration of soybean phosphatidylcholine (SPC).

  18. Efficient 18F-Labeling of Large 37-Amino Acid pHLIP Peptide Analogues and their Biological Evaluation

    PubMed Central

    Daumar, Pierre; Wanger-Baumann, Cindy A.; Pillarsetty, NagaVaraKishore; Fabrizio, Laura; Carlin, Sean D.; Andreev, Oleg A.; Reshetnyak, Yana K.; Lewis, Jason S.

    2012-01-01

    Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP®) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pHe<7). Labeling of peptides with [18F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known “click” methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC “click chemistry” for the simple and efficient 18F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and a L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[18F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥ 98%. The subsequent CuI catalyzed “click” reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [18F]-D-WT-pHLIP and [18F]-L-K-pHLIP were obtained with total radiochemical yields of 5–20% after HPLC purification. The total reaction time was only 85 min including formulation. In vitro stability tests revealed high stability of the [18F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65 and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [18F]-D-WT-pHLIP and the negative

  19. A new strategy for the preparation of peptide-targeted technetium and rhenium radiopharmaceuticals. The automated solid-phase synthesis, characterization, labeling, and screening of a peptide-ligand library targeted at the formyl peptide receptor.

    PubMed

    Stephenson, Karin A; Banerjee, Sangeeta Ray; Sogbein, Oyebola O; Levadala, Murali K; McFarlane, Nicole; Boreham, Douglas R; Maresca, Kevin P; Babich, John W; Zubieta, Jon; Valliant, John F

    2005-01-01

    A new solid-phase synthetic methodology was developed that enables libraries of peptide-based Tc(I)/Re(I) radiopharmaceuticals to be prepared using a conventional automated peptide synthesizer. Through the use of a tridentate ligand derived from N-alpha-Fmoc-l-lysine, which we refer to as a single amino acid chelate (SAAC), a series of 12 novel bioconjugates [R-NH(CO)ZLF(SAAC)G, R = ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, benzyl; Z = Met, Nle] that are designed to target the formyl peptide receptor (FPR) were prepared. Construction of the library was carried out in a multiwell format on an Advanced ChemTech 348 peptide synthesizer where multi-milligram quantities of each peptide were isolated in high purity without HPLC purification. After characterization, the library components were screened for their affinity for the FPR receptor using flow cytometry where the K(d) values were found to be in the low micromolar range (0.5-3.0 microM). Compound 5j was subsequently labeled with (99m)Tc(I) and the product isolated in high radiochemical yield using a simple Sep-Pak purification procedure. The retention time of the labeled compound matched that of the fully characterized Re-analogue which was prepared through the use of the same solid-phase synthesis methodology that was used to construct the library. The work reported here is a rare example of a method by which libraries of peptide-ligand conjugates and their rhenium complexes can be prepared.

  20. 77 FR 23269 - Determination That FUNDUSCEIN-25 (fluorescein sodium injection), 25%, and AK-FLUOR (fluorescein...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-18

    ...] Determination That FUNDUSCEIN-25 (fluorescein sodium injection), 25%, and AK-FLUOR (fluorescein sodium injection... FUNDUSCEIN-25 (fluorescein sodium injection), 25%, and AK-FLUOR (fluorescein sodium injection), 25%, were not... abbreviated new drug applications (ANDAs) for fluorescein sodium injection, 25%, if all other legal and...

  1. Novel Fitc-Labeled Igy Antibody: Fluorescence Imaging Toxoplasma Gondii In Vitro.

    PubMed

    Sert, Mehtap; Cakir Koc, Rabia; Budama Kilinc, Yasemin

    2017-04-12

    Toxoplasmosis is caused by T. gondii and can create serious health problems in humans and also worldwide economic harm. Because of the clinical and veterinary importance of toxoplasmosis, its timely and accurate diagnosis has a major impact on disease-fighting strategies. T. gondii surface antigen 1 (SAG1), an immunodominant-specific antigen, is often used as a diagnostic tool. Therefore, the aim of this study was the optimization of novel fluorescein isothiocyanate (FITC) labeling of the SAG1-specific IgY antibody to show the potential for immunofluorescence imaging of T. gondii in vitro. The specificity of IgY antibodies was controlled by an enzyme-linked immunosorbent assay (ELISA), and the concentration of the IgY antibody was detected using a spectrophotometer. The optimum incubation time and FITC concentration were determined with a fluorescence spectrometer. The obtained FITC-labeled IgY was used for marking T. gondii tachyzoites, which were cultured in vitro and viewed using light microscopy. The interaction of the fluorescence-labeled antibody and the T. gondii tachyzoites was examined with a fluorescence microscope. In this study, for the first time, a FITC-labeled anti-SAG1 IgY antibody was developed according to ELISA, fluorescence spectrometer, and fluorescence imaging of cell culture. In the future, the obtained FITC-labeled T. gondii tachyzoites' specific IgY antibodies may be used as diagnostic tools for the detection of T. gondii infections in different samples.

  2. Spin-labelled diketopiperazines and peptide-peptoid chimera by Ugi-multi-component-reactions.

    PubMed

    Sultani, Haider N; Haeri, Haleh H; Hinderberger, Dariush; Westermann, Bernhard

    2016-12-28

    For the first time, spin-labelled coumpounds have been obtained by isonitrile-based multi component reactions (IMCRs). The typical IMCR Ugi-protocols offer a simple experimental setup allowing structural variety by which labelled diketopiperazines (DKPs) and peptide-peptoid chimera have been synthesized. The reaction keeps the paramagnetic spin label intact and offers a simple and versatile route to a large variety of new and chemically diverse spin labels.

  3. Type and location of fluorescent probes incorporated into the potent mu-opioid peptide [Dmt]DALDA affect potency, receptor selectivity and intrinsic efficacy.

    PubMed

    Schiller, P W; Berezowska, I; Weltrowska, G; Chen, H; Lemieux, C; Chung, N N

    2005-06-01

    The dermorphin-derived tetrapeptide H-Dmt-d-Arg-Phe-Lys-NH(2) (Dmt = 2',6'-dimethyltyrosine) ([Dmt(1)]DALDA) is a highly potent and selective mu-opioid agonist capable of crossing the blood-brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt(1)]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6-dimethylamino-2'-naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea-pig ileum and mouse vas deferens assays, and in mu-, delta- and kappa-opioid receptor-binding assays. The analogues showed various degrees of mu receptor-binding selectivity, but all of them were less mu-selective than the [Dmt(1)]DALDA parent peptide. Most analogues retained potent, full mu-agonist activity, except for one with fluorescein attached at the C-terminus (3a) (partial mu-agonist) and one containing beta-(6'-dimethylamino-2'-naphthoyl)alanine (aladan) in place of Phe(3) (4) (mu- and kappa-antagonist). The obtained data indicate that the receptor-binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence-labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group (2a) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy.

  4. Proximity-Induced Covalent Labeling of Proteins with a Reactive Fluorophore-Binding Peptide Tag.

    PubMed

    Sunbul, Murat; Nacheva, Lora; Jäschke, Andres

    2015-08-19

    Labeling of proteins with fluorescent dyes in live cells enables the investigation of their roles in biological systems by fluorescence microscopy. Because the labeling procedure should not disturb the native function of the protein of interest, it is of high importance to find the optimum labeling method for the problem to be studied. Here, we developed a rapid one-step method to covalently and site-specifically label proteins with a TexasRed fluorophore in vitro and in live bacteria. To this end, a genetically encodable TexasRed fluorophore-binding peptide (TR512) was converted into a reactive tag (ReacTR) by adjoining a cysteine residue which rapidly reacts with N-α-chloroacetamide-conjugated TexasRed fluorophore owing to the proximity effect; ReacTR tag first binds to the TexasRed fluorophore and this interaction brings the nucleophilic cysteine and the electrophilic N-α-chloroacetamide groups in close proximity. Our method has several advantages over existing methods: (i) it utilizes a peptide tag much smaller than fluorescent proteins, the SNAP, CLIP, or HaLo tags; (ii) it allows for labeling of proteins with a small, photostable, red-emitting TexasRed fluorophore; (iii) the probe used is very easy to synthesize; (iv) no enzyme is required to transfer the fluorophore to the peptide tag; and (v) labeling yields a stable covalent product in a very fast reaction.

  5. Staining intraoperative topical solutions with fluorescein: enhancing the safety of sinus surgery.

    PubMed

    Yao, William C; Regone, Rachel M; Takashima, Masayoshi

    2015-09-01

    Oxymetazoline HCl 0.05%, 1:100,000 lidocaine with epinephrine (lido+epi), and 1:1000 epinephrine are all colorless solutions employed in sinus surgery. Because lido+epi is injected whereas others are not, care must be taken to label all solutions to avoid inadvertent injection of oxymetazoline or concentrated epinephrine because of life-threatening complications. Dyes have been used to color solutions for visual identification, but efficacy and cost have never been compared. We sought to determine the effectiveness of surgical marking pen (gentian violet) and fluorescein strips as coloring agents in mediums commonly used in sinus surgery. In this specialty techniques study, 4 liquid mediums (normal saline [NS], 0.05% oxymetazoline, 1:1,000 epinephrine, and lido+epi) and 2 coloring agents (fluorescein strip and surgical marking pen) were combined separately. Photos and absorbance of each solution were obtained at 15-minute intervals over 2 hours using a spectrophotometer. Peak absorbance remained stable for all solutions with fluorescein. Absorbance also remained constant for NS (0.37 absorbance units [AU] → 0.37 AU) and oxymetazoline (2.8 AU → 2.8 AU) mixed with marking pen. Absorbance decreased over 2 hours when marking pen was mixed with 1:1000 epinephrine (0.82 AU → 0.32 AU) and lido+epi (1.19 AU → 0.33 AU). Furthermore, the majority of color visibility and absorbance decreased in the first 15 minutes for concentrated epinephrine (0.82 → 0.33) and lido+epi (1.19 → 0.51). Cost of fluorescein strips ($0.14 to $0.20/strip) was less than marking pens ($13 to 46/pen). Compared to marking pen dye, solutions dyed with fluorescein retained their color and did not decay over time. We recommend fluorescein strips to label colorless materials in the operating room because of their lack of color decay and cost advantages. © 2015 ARS-AAOA, LLC.

  6. Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses

    PubMed Central

    Hernandez, Hilda; Niehauser, Sarah; Boltz, Stacey A.; Gawandi, Vijay; Phillips, Robert S.; Amster, I. Jonathan

    2006-01-01

    A method for improving the identification of peptides in a shotgun proteome analysis using accurate mass measurement has been developed. The improvement is based upon the derivatization of cysteine residues with a novel reagent, 2,4-dibromo-(2′-iodo)acetanilide. The derivitization changes the mass defect of cysteine-containing proteolytic peptides in a manner that increases their identification specificity. Peptide masses were measured using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron mass spectrometry. Reactions with protein standards show that the derivatization of cysteine is rapid and quantitative, and the data suggest that the derivatized peptides are more easily ionized or detected than unlabeled cysteine-containing peptides. The reagent was tested on a 15N-metabolically labeled proteome from M. maripaludis. Proteins were identified by their accurate mass values and from their nitrogen stoichiometry. A total of 47% of the labeled peptides are identified versus 27% for the unlabeled peptides. This procedure permits the identification of proteins from the M. maripaludis proteome that are not usually observed by the standard protocol and shows that better protein coverage is obtained with this methodology. PMID:16689545

  7. Simultaneous monitoring of insulin and islet amyloid polypeptide secretion from islets of Langerhans on a microfluidic device.

    PubMed

    Lomasney, Anna R; Yi, Lian; Roper, Michael G

    2013-08-20

    A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature enabled changes in the perfusion media within 5 s, allowing rapid exchange of the glucose concentrations delivered to groups of islets. The perfusate was continuously sampled by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15 cm mixing channel maintained at 37 °C. The immunoassay mixture was injected for 0.3 s onto a 1.5 cm separation channel at 11.75 s intervals and immunoassay reagents detected using 488 and 635 nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2 to 7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4-10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 to 11 or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis.

  8. Improved 68 Ga-labeling method using ethanol addition: Application to the α-helical peptide DOTA-FAMP.

    PubMed

    Hasegawa, Koki; Kawachi, Emi; Uehara, Yoshinari; Yoshida, Tsuyoshi; Imaizumi, Satoshi; Ogawa, Masahiro; Miura, Shin-Ichiro; Saku, Keijiro

    2017-01-01

    We examined the 68 Ga labeling of the α-helical peptide, DOTA-FAMP, and evaluated conformational changes during radiolabeling. 68 Ga-DOTA-FAMP is a positron emission tomography probe candidate for atherosclerotic plaques. The labeling yield achieved by Zhernosekov's method (using acetone for 68 Ga purification) was compared with that achieved by the original and 2 modified Mueller's methods (using NaCl solution). Modified method I involves desalting the 68 Ga prior to labeling, and modified method II involves the inclusion of ethanol in the labeling solution. The labeling yield using Zhernosekov's method was 62% ± 5.4%. In comparison, Mueller's original method gave 8.9% ± 1.7%. Modified method I gave a slight improvement of 32% ± 2.1%. Modified method II further increased the yield to 66% ± 3.4%. Conformational changes were determined by circular dichroism spectroscopy, revealing that these differences could be attributed to conformational changes. Heat treatment affects peptide conformation, which leads to aggregation and decreases the labeling yield. Mueller's method is simpler, but harsh conditions preclude its application to biomolecules. To suppress aggregation, we included a desalting process and added ethanol in the labeling solution. These changes significantly improved the labeling yield. Before use for imaging, conformational changes of biomolecules during radiolabeling should be evaluated by circular dichroism spectroscopy to ensure the homogeneity of the labeled product. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Fluorescein-related extensive jaundice.

    PubMed

    Kalkan, Asim; Turedi, Suleyman; Aydin, Ibrahim

    2015-03-01

    Fluorescein is a chemical dye frequently used in eye diseases to assess blood flow in the retina, choroid tissue, and iris. Although it has many known adverse effects, it has not previously been reported to lead to jaundice. The purpose of this case report was to emphasize that for patients presenting at the emergency department with jaundice symptoms, it should not be forgotten by emergency physicians that jaundice can develop after fluorescein angiography. Case: A 65-year-old woman presented at the emergency department with extensive jaundice that had developed on her entire body a few hours after fluorescein angiography applied because of vision impairment. The test results for all the diseases considered to cause jaundice were normal,and fluorescein-related jaundice was diagnosed. Conclusion: A detailed anamnesis should be taken when jaundice is seen in patients who have undergone fluorescein angiography, and it should not be forgotten that fluorescein dye is a rare cause of jaundice.

  10. Pharmacokinetics and microbiodistribution of 64Cu-labeled collagen binding peptides in chronic myocardial infarction

    PubMed Central

    Kim, Heejung; Lee, Sung-Jin; Kim, Jin Su; Davies-Venn, Cynthia; Cho, Hong-Jun; Won, Samuel Jaeyoon; Dejene, Eden; Yao, Zhengsheng; Kim, Insook; Paik, Chang H.; Bluemke, David A.

    2016-01-01

    Objectives To evaluate the pharmacokinetics and microbiodistribution of 64Cu-labeled collagen binding peptides. Method The affinity constant (KD), association (ka) and dissociation rate constant (kd) for the peptide collagelin or its analogue (named CRPA) binding to collagen were measured by bio-layer interferometric analysis. Rats (n = 4–5) with myocardial infarction or normal were injected IV with the 64Cu-labeled peptides or 64Cu-DOTA as a control. Dynamic PET imaging was performed for 60 min at 7- to 8-week post-infarct. [18F]FDG PET imaging was performed to identify the viable myocardium. To validate the PET images, slices of heart samples from the base to the apex were analyzed using autoradiography and histology. Result The peptides bound to collagen with KD of ~ 0.9 μM. The 64Cu-peptides and 64Cu-DOTA accumulated in the infarct area (confirmed by autoradiography and histology images) within 1 minute of injection and were excreted rapidly via the renal system. The blood clearance curves were bi-phasic with the elimination half-lives, 21.9 ± 2.4, 26.2 ± 4.6 and 21.2 ± 2.1 min for 64Cu-CRPA, 64Cu-collagelin and the control 64Cu-DOTA, respectively. The clearance half-lives from the focal fibrotic tissue (24.1 ± 1.5, 25.6 ± 8.0 and 21.4 ± 1.3 min, respectively) and remote myocardium (20.8 ± 0.7, 21.0 ± 5.5 and 19.1 ± 2.4 min, respectively) were not significantly different. The uptake ratios of infarct-to-remote myocardium (1.93 ± 0.18, 2.15 ± 0.38 and 1.88 ± 0.08, respectively) for 64Cu-CRPA, 64Cu-collagelin and 64Cu-DOTA remained stable for the time period between 10 to 60 min. Conclusion The distribution of the 64Cu-collagelin probes corresponds to the heterogeneous distribution of expanded extracellular space in the setting of myocardial infarction. The overall washout rate from the fibrous tissue was determined by the slow washout rate (t1/2, ≥ 20 min) of the peptides from the extracellular space to the vasculature, not by the dissociation

  11. Transient receptor potential ankyrin 1 activation enhances hapten sensitization in a T-helper type 2-driven fluorescein isothiocyanate-induced contact hypersensitivity mouse model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shiba, Takahiro; Tamai, Takuma; Sahara, Yurina

    2012-11-01

    Some chemicals contribute to the development of allergies by increasing the immunogenicity of other allergens. We have demonstrated that several phthalate esters, including dibutyl phthalate (DBP), enhance skin sensitization to fluorescein isothiocyanate (FITC) in a mouse contact hypersensitivity model, in which the T-helper type 2 (Th2) response is essential. On the other hand, some phthalate esters were found to activate transient receptor potential ankyrin 1 (TRPA1) cation channels on sensory neurons. We then found a positive correlation between the enhancing effects of several types of phthalate esters on skin sensitization to FITC and their ability to activate TRPA1. Here wemore » examined the involvement of TRPA1 in sensitization to FITC by using TRPA1 agonists other than phthalate esters. During skin sensitization to FITC, the TRPA1 agonists (menthol, carvacrol, cinnamaldehyde and DBP) augmented the ear-swelling response as well as trafficking of FITC-presenting dendritic cells to draining lymph nodes. We confirmed that these TRPA1 agonists induced calcium influx into TRPA1-expressing Chinese hamster ovary (CHO) cells. We also found that TRPA1 antagonist HC-030031 inhibited DBP-induced calcium influx into TRPA1-expressing CHO cells. After pretreatment with this antagonist upon skin sensitization to FITC, the enhancing effect of DBP on sensitization was suppressed. These results suggest that TRPA1 activation will become a useful marker to find chemicals that facilitate sensitization in combination with other immunogenic haptens. -- Highlights: ► Role of TRPA1 activation was revealed in a mouse model of skin sensitization to FITC. ► TRPA1 agonists enhanced skin sensitization as well as dendritic cell trafficking. ► Dibutyl phthalate (DBP) has been shown to enhance skin sensitization to FITC. ► TRPA1 activation by DBP was inhibited by a selective antagonist, HC-030031. ► HC-030031 inhibited the enhancing effect of DBP on skin sensitization to FITC.« less

  12. Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.

    PubMed

    Altai, Mohamed; Honarvar, Hadis; Wållberg, Helena; Strand, Joanna; Varasteh, Zohreh; Rosestedt, Maria; Orlova, Anna; Dunås, Finn; Sandström, Mattias; Löfblom, John; Tolmachev, Vladimir; Ståhl, Stefan

    2014-11-24

    Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs. Copyright © 2014 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

  13. Secondary structure propensity and chirality of the amyloidophilic peptide p5 and its analogues impacts ligand binding - In vitro characterization

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wall, Jonathan S.; Williams, Angela; Wooliver, Craig

    Here, polybasic helical peptides, such as peptide p5, bind human amyloid extracts and synthetic amyloid fibrils. When radio labeled, peptide p5 has been shown to specifically bind amyloid in vivo thereby allowing imaging of the disease. Structural requirements for heparin and amyloid binding have been studied using analogues of p5 that modify helicity and chirality.

  14. Secondary structure propensity and chirality of the amyloidophilic peptide p5 and its analogues impacts ligand binding - In vitro characterization

    DOE PAGES

    Wall, Jonathan S.; Williams, Angela; Wooliver, Craig; ...

    2016-08-11

    Here, polybasic helical peptides, such as peptide p5, bind human amyloid extracts and synthetic amyloid fibrils. When radio labeled, peptide p5 has been shown to specifically bind amyloid in vivo thereby allowing imaging of the disease. Structural requirements for heparin and amyloid binding have been studied using analogues of p5 that modify helicity and chirality.

  15. Endoscopic detection of murine colonic dysplasia using a novel fluorescence-labeled peptide

    NASA Astrophysics Data System (ADS)

    Miller, Sharon J.; Joshi, Bishnu P.; Gaustad, Adam; Fearon, Eric R.; Wang, Thomas D.

    2011-03-01

    Current endoscopic screening does not detect all pre-malignant (dysplastic) colorectal mucosa, thus requiring the development of more sensitive, targeted techniques to improve detection. The presented work utilizes phage display to identify a novel peptide binder to colorectal dysplasia in a CPC;Apc mouse model. A wide-field, small animal endoscope capable of fluorescence excitation (450-475 nm) identified polyps via white light and also collected fluorescence images (510 nm barrier filter) of peptide binding. The peptide bound ~2-fold greater to the colonic adenomas when compared to the control peptide. We have imaged fluorescence-labeled peptide binding in vivo that is specific towards distal colonic adenomas.

  16. Photoaffinity Labeling of Ras Converting Enzyme using Peptide Substrates that Incorporate Benzoylphenylalanine (Bpa) Residues: Improved Labeling and Structural Implications

    PubMed Central

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

    2012-01-01

    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. PMID:22079863

  17. SaxA-Mediated Isothiocyanate Metabolism in Phytopathogenic Pectobacteria

    PubMed Central

    Rosengarten, Jamila F.; de Graaf, Rob M.; Jetten, Mike S. M.

    2016-01-01

    Pectobacteria are devastating plant pathogens that infect a large variety of crops, including members of the family Brassicaceae. To infect cabbage crops, these plant pathogens need to overcome the plant's antibacterial defense mechanisms, where isothiocyanates are liberated by hydrolysis of glucosinolates. Here, we found that a Pectobacterium isolate from the gut of cabbage root fly larvae was particularly resistant to isothiocyanate and even seemed to benefit from the abundant Brassica root metabolite 2-phenylethyl isothiocyanate as a nitrogen source in an ecosystem where nitrogen is scarce. The Pectobacterium isolate harbored a naturally occurring mobile plasmid that contained a sax operon. We hypothesized that SaxA was the enzyme responsible for the breakdown of 2-phenylethyl isothiocyanate. Subsequently, we heterologously produced and purified the SaxA protein and characterized the recombinant enzyme. It hydrolyzed 2-phenylethyl isothiocyanate to yield the products carbonyl sulfide and phenylethylamine. It was also active toward another aromatic isothiocyanate but hardly toward aliphatic isothiocyanates. It belongs to the class B metal-dependent beta-lactamase fold protein family but was not, however, able to hydrolyze beta-lactam antibiotics. We discovered that several copies of the saxA gene are widespread in full and draft Pectobacterium genomes and therefore hypothesize that SaxA might be a new pathogenicity factor of the genus Pectobacterium, possibly compromising food preservation strategies using isothiocyanates. PMID:26873319

  18. Glycogen synthase kinase-3β inhibition of 6-(methylsulfinyl)hexyl isothiocyanate derived from wasabi (Wasabia japonica Matsum).

    PubMed

    Yoshida, Jun; Nomura, Satomi; Nishizawa, Naoyuki; Ito, Yoshiaki; Kimura, Ken-ichi

    2011-01-01

    A new biological activity of 6-(methylsulfinyl)hexyl isothiocyanate derived from Wasabia japonica was discovered as an inhibitor of glycogen synthase kinase-3β. The most potent isothiocyanate, 9-(methylsulfinyl)hexyl isothiocyanate, inhibited glycogen synthase kinase-3β at a K(i) value of 10.5 µM and showed ATP competitive inhibition. The structure-activity relationship revealed an inhibitory potency of methylsulfinyl isothiocyanate dependent on the alkyl chain length and the sulfoxide, sulfone, and/or the isothiocyanate moiety.

  19. Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage.

    PubMed

    Hamaguchi, Y; Toriyama, M; Sakai, H; Hiramoto, Y

    1985-04-01

    Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.

  20. Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage

    PubMed Central

    1985-01-01

    Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC- labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin. PMID:3920225

  1. Generation of Small 32P-Labeled Peptides as a Potential Approach to Colorectal Cancer Therapy

    PubMed Central

    Abraham, John M.; Cheng, Yulan; Hamilton, James P.; Paun, Bogdan; Jin, Zhe; Agarwal, Rachana; Kan, Takatsugu; David, Stefan; Olaru, Alexandru; Yang, Jian; Ito, Tetsuo; Selaru, Florin M.; Mori, Yuriko; Meltzer, Stephen J.

    2008-01-01

    Cancers have been revealed to be extremely heterogenous in terms of the frequency and types of mutations present in cells from different malignant tumors. Thus, it is likely that uniform clinical treatment is not optimal for all patients, and that the development of individualized therapeutic regimens may be beneficial. We describe the generation of multiple, unique small peptides nine to thirty-four amino acids in length which, when labeled with the radioisotope 32P, bind with vastly differing efficiencies to cell lines derived from different colon adenocarcinomas. In addition, the most effective of these peptides permanently transfers the 32P radioisotope to colorectal cancer cellular proteins within two hours at a rate that is more than 150 times higher than in cell lines derived from other cancers or from the normal tissues tested. Currently, the only two FDA-approved radioimmunotherapeutic agents in use both employ antibodies directed against the B cell marker CD20 for the treatment of non-Hodgkin's lymphoma. By using the method described herein, large numbers of different 32P-labeled peptides can be readily produced and assayed against a broad spectrum of cancer types. This report proposes the development and use of 32P-labeled peptides as potential individualized peptide-binding therapies for the treatment of colon adenocarcinoma patients. PMID:18575578

  2. Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.

    PubMed

    LeBlanc, André; Michaud, Sarah A; Percy, Andrew J; Hardie, Darryl B; Yang, Juncong; Sinclair, Nicholas J; Proudfoot, Jillaine I; Pistawka, Adam; Smith, Derek S; Borchers, Christoph H

    2017-07-07

    When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

  3. Temperature-responsive peptide-mimetic coating based on poly(N-methacryloyl-l-leucine): properties, protein adsorption and cell growth.

    PubMed

    Raczkowska, Joanna; Ohar, Mariya; Stetsyshyn, Yurij; Zemła, Joanna; Awsiuk, Kamil; Rysz, Jakub; Fornal, Katarzyna; Bernasik, Andrzej; Ohar, Halyna; Fedorova, Svitlana; Shtapenko, Oksana; Polovkovych, Svyatoslav; Novikov, Volodymyr; Budkowski, Andrzej

    2014-06-01

    Poly(N-methacryloyl-l-leucine) (PNML) coatings were successfully fabricated via polymerization from peroxide initiator grafted to premodified glass substrate. Chemical composition and thickness of PNML coatings were determined using time of flight-secondary ion mass spectrometry (TOF- SIMS) and ellipsometry, respectively. PNML coatings exhibit thermal response of the wettability, between 4 and 28°C, which indicates a transition between hydrated loose coils and hydrophobic collapsed chains. Morphology of the PNML coating was observed with the AFM, transforming with increasing temperature from initially relatively smooth surface to rough and more structured surface. Protein adsorption observed by fluorescence microscopy for model proteins (bovine serum albumin and lentil lectin labeled with fluorescein isothiocyanate) at transition from 5 to 25°C, showed high affinity of PNML coating to proteins at all investigated temperatures and pH. Thus, PNML coating have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). The high proliferation growth of the human embryonic kidney cell (HEK 293) onto PNML coating was demonstrated, indicating its excellent cytocompatibility. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Synthesis, Characterization, and Initial Biological Evaluation of [99m Tc]Tc-Tricarbonyl-labeled DPA-α-MSH Peptide Derivatives for Potential Melanoma Imaging.

    PubMed

    Gao, Feng; Sihver, Wiebke; Bergmann, Ralf; Belter, Birgit; Bolzati, Cristina; Salvarese, Nicola; Steinbach, Jörg; Pietzsch, Jens; Pietzsch, Hans-Jürgen

    2018-06-06

    α-Melanocyte stimulating hormone (α-MSH) derivatives target the melanocortin-1 receptor (MC1R) specifically and selectively. In this study, the α-MSH-derived peptide NAP-NS1 (Nle-Asp-His-d-Phe-Arg-Trp-Gly-NH 2 ) with and without linkers was conjugated with 5-(bis(pyridin-2-ylmethyl)amino)pentanoic acid (DPA-COOH) and labeled with [ 99m Tc]Tc-tricarbonyl by two methods. With the one-pot method the labeling was faster than with the two-pot method, while obtaining similarly high yields. Negligible trans-chelation and high stability in physiological solutions was determined for the [ 99m Tc]Tc-tricarbonyl-peptide conjugates. Coupling an ethylene glycol (EG)-based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [ 99m Tc]Tc-tricarbonyl-peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the 99m Tc-labeled peptides for in vivo imaging. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Targeted radionuclide therapy for lung cancer with iodine-131-labeled peptide in a nude-mouse model.

    PubMed

    Chen, Zhenzhu; Gao, Hongyi; Li, Man; Fang, Shun; Li, Guiping; Guo, Linlang

    2017-06-01

    Integrin α3β1 has been shown to be a novel candidate target for the imaging and specific therapy of non-small-cell lung cancer. We have previously reported on a peptide containing a novel motif of NGXG that specifically binds to the integrin α3 receptor on lung cancer cells using a one-bead one-peptide combinatorial library. In this study, we developed the peptide cNGEGQQc-based therapeutic agent labeling with radionuclide iodine-131 (I) and evaluated its characteristics including stability, biodistribution, antitumor activity, and safety. The results showed that I-cNGEGQQc was stable in serum. Furthermore, the biodistribution of I-cNGEGQQc was determined in normal mice and rabbits. In-vivo biodistribution studies showed that radiolabeled peptide in the kidney was significantly higher than that in other organs. Nude mice bearing lung cancer cell xenografts (H1975 and L78) were used as an in-vivo model for tumor-inhibition efficacy studies with I-cNGEGQQc. The tumor growth decreased significantly in mice receiving I-labeled peptide compared with the controls and the effect of I-labeled peptide can be blocked by unlabeled cNGEGQQc. Safety studies showed that I-cNGEGQQc was relatively safe for animals without significant toxicity. Our data suggest that I-cNGEGQQc has potential as a targeted radiotherapeutic agent for non-small-cell lung cancer.

  6. PET imaging of αvβ3 integrin expression in tumours with 68Ga-labelled mono-, di- and tetrameric RGD peptides

    PubMed Central

    Yim, Cheng-Bin; Franssen, Gerben M.; Schuit, Robert C.; Luurtsema, Gert; Liu, Shuang; Oyen, Wim J. G.; Boerman, Otto C.

    2010-01-01

    Purpose Due to the restricted expression of αvβ3 in tumours, αvβ3 is considered a suitable receptor for tumour targeting. In this study the αvβ3-binding characteristics of 68Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their 111In-labelled counterparts. Methods A monomeric (E-c(RGDfK)), a dimeric (E-[c(RGDfK)]2) and a tetrameric (E{E[c(RGDfK)]2}2) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with 68Ga. In vitro αvβ3-binding characteristics were determined in a competitive binding assay. In vivo αvβ3-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. Results The IC50 values for the Ga(III)-labelled DOTA-E-c(RGDfK), DOTA-E-[c(RGDfK)]2 and DOTA-E{E[c(RGDfK)]2}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 68Ga-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 111In-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. Conclusion The integrin affinity and the tumour uptake followed the order of DOTA-tetramer > DOTA-dimer > DOTA-monomer. The 68Ga-labelled tetrameric RGD peptide has excellent characteristics for imaging of αvβ3 expression with PET. Electronic supplementary material The online version of this article (doi:10.1007/s00259-010-1615-x) contains supplementary material, which is available to authorized users. PMID:20857099

  7. STUDIES ON FLUORESCENT ANTIBODY STAINING

    PubMed Central

    Goldstein, Gerald; Slizys, Irene S.; Chase, Merrill W.

    1961-01-01

    1. A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2. In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3. Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: (a) conjugating only gamma globulins with fluorescein isothiocyanate; (b) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; (c) use of crystalline preparations of fluorescein isothiocyanate. 4. Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5. The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6. A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules. PMID:13706641

  8. Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein.

    PubMed

    Tabachnick, M; Perret, V

    1987-08-01

    [125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.

  9. In vitro cell studies of technetium-99m labeled RGD-HYNIC peptide, a comparison of tricine and EDDA as co-ligands.

    PubMed

    Su, Zi-Fen; He, Jiang; Rusckowski, Mary; Hnatowich, Donald J

    2003-02-01

    The level of alpha(V)beta(3) integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to alpha(V)beta(3) integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with (99m)Tc. The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the (99m)Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of alpha(V)beta(3) integrin proteins are expressed on the cells. Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved

  10. A tumor-penetrating peptide enhances circulation-independent targeting of peritoneal carcinomatosis

    PubMed Central

    Sugahara, Kazuki N.; Scodeller, Pablo; Braun, Gary B.; de Mendoza, Tatiana Hurtado; Yamazaki, Chisato M.; Kluger, Michael D.; Kitayama, Joji; Alvarez, Edwin; Howell, Stephen B.; Teesalu, Tambet; Ruoslahti, Erkki; Lowy, Andrew M.

    2015-01-01

    Peritoneal carcinomatosis is a major source of morbidity and mortality in patients with advanced abdominal neoplasms. Intraperitoneal chemotherapy (IPC) is an area of intense interest given its efficacy in ovarian cancer. However, IPC suffers from poor drug penetration into peritoneal tumors. As such, extensive cytoreductive surgery is required prior to IPC. Here, we explore the utility of iRGD, a tumor-penetrating peptide, for improved tumor-specific penetration of intraperitoneal compounds and enhanced IPC in mice. Intraperitoneally administered iRGD significantly enhanced penetration of an attached fluorescein into disseminated peritoneal tumor nodules. The penetration was tumor-specific, circulation-independent, and mediated by the neuropilin-binding RXXK tissue-penetration peptide motif of iRGD. Q-iRGD, which fluoresces upon cleavage, including the one that leads to RXXK activation, specifically labeled peritoneal metastases displaying different growth patterns in mice. Importantly, iRGD enhanced intratumoral entry of intraperitoneally co-injected dextran to approximately 300% and doxorubicin to 250%. Intraperitoneal iRGD/doxorubicin combination therapy inhibited the growth of bulky peritoneal tumors and reduced systemic drug toxicity. iRGD delivered attached fluorescein and co-applied nanoparticles deep into fresh human peritoneal metastasis explants. These results indicate that intraperitoneal iRGD co-administration serves as a simple and effective strategy to facilitate tumor detection and improve the therapeutic index of IPC for peritoneal carcinomatosis. PMID:26071630

  11. Biocatalytic response of multi-layer assembled collagen/hyaluronic acid nanoengineered capsules.

    PubMed

    Sousa, Fernanda; Kreft, Oliver; Sukhorukov, Gleb B; Möhwald, Helmuth; Kokol, Vanja

    2014-01-01

    Biodegradable hollow capsules filled with fluorescently labelled bovine serum albumin (BSA) as a model drug were prepared via layer-by-layer (LbL) self-assembly of type-I collagen (COL) and hyaluronic acid (HA) using calcium carbonate micro-particles and co-precipitation method. Capsules loaded with fluorescein isothiocyanate (FITC)-BSA, tetramethylrhodamin isothiocyanate (TRITC)-BSA or Alex-Fluor-488-BSA, respectively, were characterised before and after core removal using Confocal Laser Scanning Microscopy (CLSM), whilst the morphologies of individual hollow capsules were assessed using Atomic Force Microscopy (AFM). The sustained release of the encapsulated FITC-BSA protein was attained using enzymatic degradation of the capsule shells by collagenase. The released profile of the fluorescently-labelled BSA indicated that it could be successfully controlled by modulating the number of layers and/or by collagen crosslinking either before or after the capsule's assembly.

  12. Allergic contact dermatitis from allyl isothiocyanate in a Danish cohort of 259 selected patients.

    PubMed

    Lerbaek, Anne; Rastogi, Suresh Chandra; Menné, Torkil

    2004-08-01

    Allyl isothiocyanate is present in many plants. Allergic contact dermatitis from allyl isothiocyanate is well known but infrequently reported. The aim of this study was to investigate the prevalence of contact allergy to allyl isothiocyanate in patients with suspected contact dermatitis from vegetables and food. 259 such patients were tested at the Department of Dermatology, Gentofte Hospital, Denmark, from 1994 to 2003. Only 2 patients (0.8%) had a positive reaction (+) to allyl isothiocyanate and 43 patients (16.6%) had a ?+ reaction. One of the patients with a positive reaction provided samples of margarine, salad cream, oil and mayonnaise. These were analysed with high-performance liquid chromatography, and a moderate concentration of allyl isothiocyanate (2.5 ppm) was detected in the sample of margarine. This patient was a professional sandwich maker presenting with fingertip dermatitis mimicking 'tulip fingers' or allergic contact dermatitis from garlic and onions. In conclusion, allergic contact dermatitis from allyl isothiocyanate occurs in only a limited number of cases, despite frequent exposure. The large number of ?+ reactions raises the question as to whether the recommended patch test concentration is too low.

  13. Functional reconstitution of the human serotonin receptor 5-HT(6) using synthetic transmembrane peptides.

    PubMed

    Lee, Won-Kyu; Han, Jason J; Jin, Bong-Suk; Boo, Doo Wan; Yu, Yeon Gyu

    2009-12-18

    Seven transmembrane (7TM) synthetic peptides mimicking the alpha-helical TM domains of the human serotonin receptor subtype-6 (5-HT(6)) were autonomously reconstituted in detergent micelle and liposome environments. The degree of assembly of the 7TM peptides was characterized by monitoring the fluorescence resonance energy transfer (FRET) between donor and acceptor probes labeled at the amino termini of the second and fourth TM-peptides, respectively. The FRET efficiency of these peptides significantly increased when the 7TM peptides were reconstituted in liposome compare to detergent micelles. Furthermore, the 7TM peptides reconstituted in liposomes selectively bound to free serotonin and serotonin-conjugated magnetic beads, yielding a dissociation constant of 0.84 microM. These results show that the seven individual TM domains of 5-HT(6) can spontaneously assemble into liposomes in a conformation that mimics a native structure, and further demonstrate that specific interactions between TM helices play a critical role in the folding and stabilizing of GPCRs. The autonomous assembly of 7TM-peptides can be applied to the screening of agonists for GPCRs that are difficult to manipulate.

  14. Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5).

    PubMed

    Zannetti, Antonella; Del Vecchio, Silvana; Iommelli, Francesca; Del Gatto, Annarita; De Luca, Stefania; Zaccaro, Laura; Papaccioli, Angela; Sommella, Jvana; Panico, Mariarosaria; Speranza, Antonio; Grieco, Paolo; Novellino, Ettore; Saviano, Michele; Pedone, Carlo; Salvatore, Marco

    2009-08-15

    To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging. The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

  15. A novel approach to infection imaging using a synthetic Tc-99m-labeled leukotactic peptide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Som, P.; Oster, Z.H.; Sharma, S.

    1996-05-01

    RMT1, a synthetic peptide binding to PMN and macrophage receptors was labeled with Tc-99m and investigated as a potential imaging agent for abscess and inflammation. Experimental abscesses were induced in rabbits and dogs by turpentine and E.coli injection. After injection of Tc-99m-RMT1 two and twelve day old abscesses were visualized within 20 min. In initial studies, a dose of 30 {mu}g of peptide/3 mCi was used. This amount was subsequently reduced to 1.5 {mu}g peptide with same amount of Tc-99m yielding similar imaging results. Technetium-99m-IgG and Tc-99m-MAG-3 were used as positive and negative controls, respectively. After injection of IgG abscessesmore » were visualized but activity in blood was always higher than in abscess. When using Tc-99m RMT1 rapid abscess visualization and faster blood clearance was observed. The accumulation of RMT1 was monophasic, i.e., following the initial visualization, activity continued to build up continuously for 1{1/2} hr. Tc-99m-MAG3 accumulated initially in abscess, but activity washed out. In dogs, RMT1 blood clearance showed three components: a fast component with t{1/2}=1.9 min, 73%, intermediate t{1/2}=22 min, 24.5% and slow component, t{1/2}=115, 9.5% with 3 hours cumulative urine excretion of 40-51%. RMT1 appears to be more advantageous than currently available methods because of rapidity of imaging, simpler preparation before injection and will probably be less expensive and time consuming compared to labeled WBC. These results indicate that clinical experiments are warranted.« less

  16. Cellular uptake mechanism and clearance kinetics of fluorescence-labeled glycyrrhetinic acid and glycyrrhetinic acid-modified liposome in hepatocellular carcinoma cells.

    PubMed

    Sun, Yuqi; Lu, Jinghua; Yan, Dongxue; Shen, Liping; Hu, Haiyang; Chen, Dawei

    2017-07-01

    Glycyrrhetinic acid (GA) is a natural pentacyclic triterpene derivative that exerts significant effects in the suppression of liver cancer. The receptors of GA on liver cells and hepatocellular carcinoma (HCC) cells have drawn broad attention. The effects of GA might depend on its transport into and out of cells. However, the question has not been previously addressed despite its obvious and fundamental importance. In this paper, GA and GA-modified liposome (GA-Lip) were labeled with fluorescein isothiocyanate (FITC) or coumarin 6 (Cou6) using chemical or pharmaceutical techniques. The transport courses of FITC-GA and GA-Cou6-Lip were studied in HepG2 cells in vitro. We found that the fluorescence labeled GA and GA-Lip uptake and clearance were time-dependent. FITC-GA uptake involved passive diffusion and active transport, and the receptors were in the cytomembrane proteins. GA-Cou6-Lip uptake was mediated by caveolae-dependent endocytosis. In addition, FITC-GA and GA-Cou6-Lip clearance of the HCC cells fitted exponential decay and second-order processes, respectively. These findings provide new insights into the anti-HCC actions of GA. Copyright © 2017. Published by Elsevier B.V.

  17. NI-16INTRA-OPERATIVE USE OF FLUORESCEIN FOR MALIGNANT GLIOMA RESECTION DIFFERENTIATES TUMOR FROM NORMAL BRAIN TISSUE BASED ON HISTOPATHOLOGIC ANALYSIS

    PubMed Central

    Decker, Matthew; Kresak, Jesse; Yachnis, Anthony; Bova, Frank; Rahman, Maryam

    2014-01-01

    OBJECTIVES: To determine whether the use of IV fluorescein during surgery for malignant glioma can reliably be used to differentiate between infiltrative tumor and normal brain tissue. BACKGROUND: Fluorescein sodium is a molecular compound with fluorescent capabilities between light wavelengths of 520-530nm, appearing yellow-green (1). Neurosurgical application of fluorescein has been studied primarily for increasing intra-operative visibility of malignant gliomas (1). The mechanism of action has been hypothesized to involve disruption of the blood brain barrier (BBB) (2). Cells in areas with disrupted BBB take up fluorescein with a sensitivity of 94% and specificity of 89% for high-grade gliomas (2). We performed histopathologic analysis on tissue obtained during fluorescein-guided tumor resections to evaluate the differences between fluorescent and non-fluorescent tissue. METHODS: Two adult patients with suspected high-grade gliomas underwent surgical resection. Prior to opening of the dura 3mg/kg of IV fluorescein was given. A Zeiss OPMI Pentero microscope (Carl Zeiss Meditech Inc.) with a yellow 560nm filter was used to visualize the tumor. At the tumor margins, tissue was identified as "bright" and "dark" and sent as separate specimens for histopathological analysis. RESULTS: Histological sections of specimens labeled "bright" contained infiltrating glioma with focal microvascular proliferation. Histological sections of specimens labeled "dark" contained gray matter and focal subcortical white matter with no high-grade glioma identified. Final grading for both patients was WHO Grade IV, glioblastoma. CONCLUSION: Intra-operative use of fluorescein in surgical resection of malignant gliomas can help to distinguish between infiltrating tumor and normal brain tissue based on histopathological analysis. Further evaluation of the utility of flurorescein during high and low-grade glioma surgery is necessary.

  18. Design of bactericidal peptides against Escherichia coli O157:H7, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus.

    PubMed

    Cruz, Jenniffer; Rondon, Paola; Torres, Rodrigo; Urquiza, Mauricio; Guzman, Fanny; Alvarez, Claudio; Abengozar, Maria Angeles; Sierra, Daniel A; Rivas, Luis; Fernandez-Lafuente, Roberto; Ortiz, Claudia

    2018-05-08

    Antimicrobial peptides are on the first line of defense against pathogenic microorganisms of many living beings. These compounds are considered natural antibiotics that can overcome bacterial resistance to conventional antibiotics. Due to this characteristic, new peptides with improved properties are quite appealing for designing new strategies for fighting pathogenic bacteria Methods: Sixteen designed peptides were synthesized using Fmoc chemistry; five of them are new cationic antimicrobial peptides (CAMPs) designed using a genetic algorithm that optimizes the antibacterial activity based on selected physicochemical descriptors and 11 analog peptides derived from these five peptides were designed and constructed by single amino acid substitutions. These 16 peptides were structurally characterized and their biological activity was determined against Escherichia coli O157:H7 (E. coli O157:H7), and methicillin-resistant strains of Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa) were determined Results: These 16 peptides were folded into an α-helix structure in membrane-mimicking environment. Among these 16 peptides, GIBIM-P5S9K (ATKKCGLFKILKGVGKI) showed the highest antimicrobial activity against E. coli O157:H7 (MIC=10µM), methicillin resistant Staphylococcus aureus (MRSA) (MIC=25µM) and Pseudomonas aeruginosa (MIC=10 µM). Peptide GIBIM-P5S9K caused permeabilization of the bacterial membrane at 25 µM as determined by the Sytox Green uptake assay and the labelling of these bacteria by using the fluoresceinated peptide. GIBIM-P5S9K seems to be specific for these bacteria because at 50 µM provoked lower than 40% of erythrocyte hemolysis. New CAMPs have been designed using a genetic algorithm based on selected physicochemical descriptors and single amino acid substitution. These CAMPs interacted quite specifically with the bacterial cell membrane, GIBIM-P5S9K exhibiting high antibacterial activity on Escherichia coli O157:H7, methicillin

  19. Brain-Targeted Delivery of Trans-Activating Transcriptor-Conjugated Magnetic PLGA/Lipid Nanoparticles

    PubMed Central

    Zhang, Yifang; Sun, Tingting; Zhang, Fang; Wu, Jian; Fu, Yanyan; Du, Yang; Zhang, Lei; Sun, Ying; Liu, YongHai; Ma, Kai; Liu, Hongzhi; Song, Yuanjian

    2014-01-01

    Magnetic poly (D,L-lactide-co-glycolide) (PLGA)/lipid nanoparticles (MPLs) were fabricated from PLGA, L-α-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino (polyethylene glycol) (DSPE-PEG-NH2), and magnetic nanoparticles (NPs), and then conjugated to trans-activating transcriptor (TAT) peptide. The TAT-MPLs were designed to target the brain by magnetic guidance and TAT conjugation. The drugs hesperidin (HES), naringin (NAR), and glutathione (GSH) were encapsulated in MPLs with drug loading capacity (>10%) and drug encapsulation efficiency (>90%). The therapeutic efficacy of the drug-loaded TAT-MPLs in bEnd.3 cells was compared with that of drug-loaded MPLs. The cells accumulated higher levels of TAT-MPLs than MPLs. In addition, the accumulation of QD-loaded fluorescein isothiocyanate (FITC)-labeled TAT-MPLs in bEnd.3 cells was dose and time dependent. Our results show that TAT-conjugated MPLs may function as an effective drug delivery system that crosses the blood brain barrier to the brain. PMID:25187980

  20. Brain-targeted delivery of trans-activating transcriptor-conjugated magnetic PLGA/lipid nanoparticles.

    PubMed

    Wen, Xiangru; Wang, Kai; Zhao, Ziming; Zhang, Yifang; Sun, Tingting; Zhang, Fang; Wu, Jian; Fu, Yanyan; Du, Yang; Zhang, Lei; Sun, Ying; Liu, YongHai; Ma, Kai; Liu, Hongzhi; Song, Yuanjian

    2014-01-01

    Magnetic poly (D,L-lactide-co-glycolide) (PLGA)/lipid nanoparticles (MPLs) were fabricated from PLGA, L-α-phosphatidylethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino (polyethylene glycol) (DSPE-PEG-NH2), and magnetic nanoparticles (NPs), and then conjugated to trans-activating transcriptor (TAT) peptide. The TAT-MPLs were designed to target the brain by magnetic guidance and TAT conjugation. The drugs hesperidin (HES), naringin (NAR), and glutathione (GSH) were encapsulated in MPLs with drug loading capacity (>10%) and drug encapsulation efficiency (>90%). The therapeutic efficacy of the drug-loaded TAT-MPLs in bEnd.3 cells was compared with that of drug-loaded MPLs. The cells accumulated higher levels of TAT-MPLs than MPLs. In addition, the accumulation of QD-loaded fluorescein isothiocyanate (FITC)-labeled TAT-MPLs in bEnd.3 cells was dose and time dependent. Our results show that TAT-conjugated MPLs may function as an effective drug delivery system that crosses the blood brain barrier to the brain.

  1. A Sol-gel Integrated Dual-readout Microarray Platform for Quantification and Identification of Prostate-specific Antigen.

    PubMed

    Lee, SangWook; Lee, Jong Hyun; Kwon, Hyuck Gi; Laurell, Thomas; Jeong, Ok Chan; Kim, Soyoun

    2018-01-01

    Here, we report a sol-gel integrated affinity microarray for on-chip matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) that enables capture and identification of prostate?specific antigen (PSA) in samples. An anti-PSA antibody (H117) was mixed with a sol?gel, and the mixture was spotted onto a porous silicon (pSi) surface without additional surface modifications. The antibody easily penetrates the sol-gel macropore fluidic network structure, making possible high affinities. To assess the capture affinity of the platform, we performed a direct assay using fluorescein isothiocyanate-labeled PSA. Pure PSA was subjected to on-chip MALDI-TOF-MS analysis, yielding three clear mass peptide peaks (m/z = 1272, 1407, and 1872). The sol-gel microarray platform enables dual readout of PSA both fluorometric and MALDI-TOF MS analysis in biological samples. Here we report a useful method for a means for discovery of biomarkers in complex body fluids.

  2. A novel prosthetic group for site-selective labeling of peptides for positron emission tomography.

    PubMed

    Olberg, Dag Erlend; Hjelstuen, Ole Kristian; Solbakken, Magne; Arukwe, Joseph; Karlsen, Hege; Cuthbertson, Alan

    2008-06-01

    Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.

  3. CW dipolar broadening EPR spectroscopy and mechanically aligned bilayers used to measure distance and relative orientation between two TOAC spin labels on an antimicrobial peptide

    NASA Astrophysics Data System (ADS)

    Sahu, Indra D.; Hustedt, Eric J.; Ghimire, Harishchandra; Inbaraj, Johnson J.; McCarrick, Robert M.; Lorigan, Gary A.

    2014-12-01

    An EPR membrane alignment technique was applied to measure distance and relative orientations between two spin labels on a protein oriented along the surface of the membrane. Previously we demonstrated an EPR membrane alignment technique for measuring distances and relative orientations between two spin labels using a dual TOAC-labeled integral transmembrane peptide (M2δ segment of Acetylcholine receptor) as a test system. In this study we further utilized this technique and successfully measured the distance and relative orientations between two spin labels on a membrane peripheral peptide (antimicrobial peptide magainin-2). The TOAC-labeled magainin-2 peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 15.3 Å from a dual TOAC-labeled magainin-2 peptide at positions 8 and 14 that closely matches with the 13.3 Å distance obtained from a model of the labeled magainin peptide. In addition, the angles determining the relative orientations of the two nitroxides have been determined, and the results compare favorably with molecular modeling. This study demonstrates the utility of the technique for proteins oriented along the surface of the membrane in addition to the previous results for proteins situated within the membrane bilayer.

  4. Preprocessing Significantly Improves the Peptide/Protein Identification Sensitivity of High-resolution Isobarically Labeled Tandem Mass Spectrometry Data*

    PubMed Central

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

    2015-01-01

    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. PMID:25435543

  5. Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site.

    PubMed

    Bause, E; Wesemann, M; Bartoschek, A; Breuer, W

    1997-02-15

    Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethylglycine in the Asn-Xaa-Thr glycosylation triplet. Incubation of the enzyme in the presence of Dol-PP-linked [14C]oligosaccharides and the N-3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both of which are involved in the catalytic activity. Labelling of both subunits was blocked competitively by the acceptor peptide N-benzoyl-Asu-Gly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-alpha,gamma-diaminobutyric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy-inhibitor by replacing asparagine with glutamine. Our data clearly show that double-labelling is an active-site-directed modification, involving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Double-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the course of which OST catalyses its own 'suicide' inactivation, is more likely, as suggested by indirect experimental evidence. The syn-catalytic mechanism corresponds with our current view of the functional role of the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation.

  6. Laboratory Measured Emission Losses of Methyl Isothiocyanate at Pacific Northwest Soil Surface Fumigation Temperatures.

    PubMed

    Lu, Zhou; Hebert, Vincent R; Miller, Glenn C

    2017-02-01

    Temperature is a major environmental factor influencing land surface volatilization at the time of agricultural field fumigation. Cooler fumigation soil temperatures relevant to Pacific Northwest (PNW) application practices with metam sodium/potassium should result in appreciably reduced methyl isothiocyanate (MITC) emission rates, thus minimizing off target movement and bystander inhalation exposure. Herein, a series of laboratory controlled flow-through soil column assessments were performed evaluating MITC emissions over the range of cooler temperatures (2-13°C). Assessments were also conducted at the maximum allowed label application temperature of 32°C. All assessments were conducted at registration label-specified field moisture capacity, and no more than 50% cumulative MITC loss was observed over the 2-day post-fumigation timeframe. Three-fold reductions in MITC peak fluxes at cooler PNW application temperatures were observed compared to the label maximum temperature. This study supports current EPA metam sodium/potassium label language that indicates surface fumigations during warmer soil conditions should be discouraged.

  7. Caseins from bovine colostrum and milk strongly bind piscidin-1, an antimicrobial peptide from fish.

    PubMed

    Kütt, Mary-Liis; Stagsted, Jan

    2014-09-01

    A model system of bovine colostrum and piscidin, a fish-derived antimicrobial peptide, was developed to study potential interactions of antimicrobial peptides in colostrum. We did not detect any antimicrobial activity of colostrum using the radial plate diffusion assay; in fact colostrum completely abrogated activity of added piscidin. This could not be explained by degradation of piscidin by colostrum, which was less than ten percent. We found that colostrum even protected piscidin against degradation by added proteases. We further observed that colostrum and milk rapidly quenched the fluorescence of fluorescein-piscidin but not that of fluorescein. This effect was not seen with BSA and the specific quenching of fluorescein-piscidin by colostrum was saturably inhibited with unlabeled piscidin. Size exclusion chromatography indicated that fluorescein-piscidin bound to casein micelles with no apparent binding to IgG or whey proteins. Further, addition of pure caseins was able to quench fluorescence of fluorescein-piscidin and to inhibit the antimicrobial activity of piscidin. The interaction between caseins and piscidin could be dissociated by guanidine hydrochloride and recovered piscidin had antimicrobial activity against bacteria. Based on our results we propose that caseins could be carriers for antimicrobial peptides in colostrum and milk. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Effects of phalloidin microinjection and localization of fluorescein-labeled phalloidin in living sand dollar eggs.

    PubMed

    Hamaguchi, Y; Mabuchi, I

    1982-01-01

    Effects of microinjection of phalloidin on fertilization and cleavage of sand dollar (Clypeaster japonicus and Scaphechinus mirabilis) eggs were studied. The drug, previously injected into unfertilized eggs, showed no effect on the elevation of the fertilization membrane upon insemination up to an intracellular concentration of 50 microM. However, the movement of the egg pronucleus to the sperm pronucleus was inhibited and the fusion of pronuclei did not occur. The subsequent development no longer took place. When phalloidin was injected into fertilized eggs, the thickness of the cortical layer increased and the microvilli became conspicuous. Both nuclear division and cleavage were inhibited at the intracellular concentration of more than 20 microM, though the latter seemed to be more sensitive to phalloidin than the former. Fluorescein-labeled phalloidin (FL-phalloidin) was injected into eggs in order to investigate F-actin localization by fluorescence microscopy. In both unfertilized and fertilized eggs, FL-phalloidin was localized in the cortical layer within 1 min after injection. It was also localized in the cortical layer as radially oriented rod-like structures when injected into fertilized eggs before the disappearance of the nuclear membrane. No distinct fluorescence was detected in the mitotic apparatus or in the cleavage furrow. FL-phalloidin redistributed gradually into egg cytoplasm. In unfertilized eggs, fluorescent rods were found especially in the egg pronucleus 30 min after injection.

  9. Turn-on fluorescence sensor based on single-walled-carbon-nanohorn-peptide complex for the detection of thrombin.

    PubMed

    Zhu, Shuyun; Liu, Zhongyuan; Hu, Lianzhe; Yuan, Yali; Xu, Guobao

    2012-12-14

    Proteases play a central role in several widespread diseases. Thus, there is a great need for the fast and sensitive detection of various proteolytic enzymes. Herein, we have developed a carbon nanotube (CNT)-based protease biosensing platform that uses peptides as a fluorescence probe for the first time. Single-walled carbon nanohorns (SWCNHs) and thrombin were used to demonstrate this detection strategy. SWCNHs can adsorb a fluorescein-based dye (FAM)-labeled peptide (FAM-pep) and quench the fluorescence of FAM. In contrast, thrombin can cleave FAM-pep on SWCNHs and recover the fluorescence of FAM, which allows the sensitive detection of thrombin. This biosensor has a high sensitivity and selectivity toward thrombin, with a detection limit of 100 pM. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Selective Photoaffinity Labeling Identifies the Signal Peptide Binding Domain on SecA

    PubMed Central

    Musial-Siwek, Monika; Rusch, Sharyn L.; Kendall, Debra A.

    2007-01-01

    SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the Signal Peptide Binding Groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53-residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acids 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA. PMID:17084862

  11. Antibody-mediated fluorescence enhancement based on shifting the intramolecular dimer<-->monomer equilibrium of fluorescent dyes.

    PubMed

    Wei, A P; Blumenthal, D K; Herron, J N

    1994-05-01

    A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer<-->dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.

  12. Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

    PubMed Central

    Nelson, P S; Kent, M; Muthini, S

    1992-01-01

    Novel CE-phosphoramidite (7a-e) and CPG (8a, c, d, e) reagents have been prepared from a unique 2-aminobutyl-1,3-propanediol backbone. The reagents have been used to directly label oligonucleotides with fluorescein, acridine, and biotin via automated DNA synthesis. The versatile 2-aminobutyl-1,3-propanediol backbone allows for labeling at any position (5', internal, and 3') during solid phase oligonucleotide synthesis. Multiple labels can be achieved by repetitive coupling cycles. Furthermore, the 3-carbon atom internucleotide phosphate distance is retained when inserted internally. Using this method, individual oligonucleotides possessing two and three different reporter molecules have been prepared. PMID:1475185

  13. Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

    PubMed Central

    Stender, Henrik; Kurtzman, Cletus; Hyldig-Nielsen, Jens J.; Sørensen, Ditte; Broomer, Adam; Oliveira, Kenneth; Perry-O'Keefe, Heather; Sage, Andrew; Young, Barbara; Coull, James

    2001-01-01

    A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity. PMID:11157265

  14. Isothiocyanates: cholinesterase inhibiting, antioxidant, and anti-inflammatory activity.

    PubMed

    Burčul, Franko; Generalić Mekinić, Ivana; Radan, Mila; Rollin, Patrick; Blažević, Ivica

    2018-12-01

    Finding a new type of cholinesterase inhibitor that would overcome the brain availability and pharmacokinetic parameters or hepatotoxic liability has been a focus of investigations dealing with the treatment of Alzheimer's disease. Isothiocyanates have not been previously investigated as potential cholinesterase inhibitors. These compounds can be naturally produced from their glucosinolate precursors, secondary metabolites widely distributed in our daily Brassica vegetables. Among 11 tested compounds, phenyl isothiocyanate and its derivatives showed the most promising inhibitory activity. 2-Methoxyphenyl ITC showed best inhibition on acetylcholinesterase with IC 50 of 0.57 mM, while 3-methoxyphenyl ITC showed the best inhibition on butyrylcholinesterase having 49.2% at 1.14 mM. Assessment of the antioxidant efficacy using different methods led to a similar conclusion. The anti-inflammatory activity was also tested using human COX-2 enzyme, ranking phenyl isothiocyanate, and 3-methoxyphenyl isothiocyanate as most active, with ∼99% inhibition at 50 μM.

  15. Human Alveolar Macrophages May Not Be Susceptible to Direct Infection by a Human Influenza Virus.

    PubMed

    Ettensohn, David B; Frampton, Mark W; Nichols, Joan E; Roberts, Norbert J

    2016-12-01

    The current studies were undertaken to determine the susceptibility of human alveolar macrophages (AMs) to influenza A virus (IAV) infection in comparison with autologous peripheral blood-derived monocytes-macrophages (PBMs). AMs and PBMs were exposed to IAV in vitro and examined for their ability to bind and internalize IAV, and synthesize viral proteins and RNA. PBMs but not AMs demonstrated binding and internalization of the virus, synthesizing viral proteins and RNA. Exposure of AMs in the presence of a sialidase inhibitor or anti-IAV antibody resulted in viral protein synthesis by the cells. Exposure of AMs to fluorescein isothiocyanate-labeled IAV in the presence of anti-fluorescein isothiocyanate antibody also resulted in viral protein synthesis. Thus, human AMs are apparently not susceptible to direct infection by a human IAV but are likely to be infected indirectly in the setting of exposure in the presence of antibody that binds the challenging strain of IAV. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  16. A silicon-based peptide biosensor for label-free detection of cancer cells

    NASA Astrophysics Data System (ADS)

    Martucci, Nicola M.; Rea, Ilaria; Ruggiero, Immacolata; Terracciano, Monica; De Stefano, Luca; Migliaccio, Nunzia; Dardano, Principia; Arcari, Paolo; Rendina, Ivo; Lamberti, Annalisa

    2015-05-01

    Sensitive and accurate detection of cancer cells plays a crucial role in diagnosis of cancer and minimal residual disease, so being one of the most hopeful approaches to reduce cancer death rates. In this paper, a strategy for highly selective and sensitive detection of lymphoma cells on planar silicon-based biosensor has been evaluated. In this setting an Idiotype peptide, able to specifically bind the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, has been covalently linked to the sensor active surface and used as molecular probe. The biochip here presented showed a coverage efficiency of 85% with a detection efficiency of 8.5×10-3 cells/μm2. The results obtained suggested an efficient way for specific label-free cell detection by using a silicon-based peptide biosensor. In addition, the present recognition strategy, besides being useful for the development of sensing devices capable of monitoring minimal residual disease, could be used to find and characterize new specific receptor-ligand interactions through the screening of a recombinant phage library.

  17. Microbeads display of proteins using emulsion PCR and cell-free protein synthesis.

    PubMed

    Gan, Rui; Yamanaka, Yumiko; Kojima, Takaaki; Nakano, Hideo

    2008-01-01

    We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell-free protein synthesis in emulsion. A PCR mixture containing streptavidin-coated microbeads was compartmentalized by water-in-oil (w/o) emulsion with estimated 0.5 template molecules per droplet. The template molecules were amplified and immobilized on beads via bead-linked reverse primers and biotinylated forward primers. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti-glutathione S-transferase (GST) antibody. The pool of beads was then subjected to cell-free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We mixed two types of DNA templates of Histidine6 tag (His6)-fused and FLAG tag-fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. After incubation with fluorescein isothiocyanate (FITC)-labeled anti-His6 (C-term) antibody, the beads with the His6 gene were enriched 917-fold in a single-round screening by using flow cytometry. A library with a theoretical diversity of 10(6) was constructed by randomizing the middle four residues of the His6 tag. After a two-round screening, the randomized sequences were substantially converged to peptide-encoding sequences recognized by the anti-His6 antibody.

  18. Net Fluorescein Flux Across Corneal Endothelium Strongly Suggests Fluid Transport is due to Electro-osmosis.

    PubMed

    Sanchez, J M; Cacace, V; Kusnier, C F; Nelson, R; Rubashkin, A A; Iserovich, P; Fischbarg, J

    2016-08-01

    We have presented prior evidence suggesting that fluid transport results from electro-osmosis at the intercellular junctions of the corneal endothelium. Such phenomenon ought to drag other extracellular solutes. We have investigated this using fluorescein-Na2 as an extracellular marker. We measured unidirectional fluxes across layers of cultured human corneal endothelial (HCE) cells. SV-40-transformed HCE layers were grown to confluence on permeable membrane inserts. The medium was DMEM with high glucose and no phenol red. Fluorescein-labeled medium was placed either on the basolateral or the apical side of the inserts; the other side carried unlabeled medium. The inserts were held in a CO2 incubator for 1 h (at 37 °C), after which the entire volume of the unlabeled side was collected. After that, label was placed on the opposite side, and the corresponding paired sample was collected after another hour. Fluorescein counts were determined with a (Photon Technology) DeltaScan fluorometer (excitation 380 nm; emission 550 nm; 2 nm bwth). Samples were read for 60 s. The cells utilized are known to transport fluid from the basolateral to the apical side, just as they do in vivo in several species. We used 4 inserts for influx and efflux (total: 20 1-h periods). We found a net flux of fluorescein from the basolateral to the apical side. The flux ratio was 1.104 ± 0.056. That difference was statistically significant (p = 0.00006, t test, paired samples). The endothelium has a definite restriction at the junctions. Hence, an asymmetry in unidirectional fluxes cannot arise from osmosis, and can only point instead to paracellular solvent drag. We suggest, once more, that such drag is due to electro-osmotic coupling at the paracellular junctions.

  19. Absorption-enhancing effects of gemini surfactant on the intestinal absorption of poorly absorbed hydrophilic drugs including peptide and protein drugs in rats.

    PubMed

    Alama, Tammam; Kusamori, Kosuke; Katsumi, Hidemasa; Sakane, Toshiyasu; Yamamoto, Akira

    2016-02-29

    In general, the intestinal absorption of small hydrophilic molecules and macromolecules like peptides, after oral administration is very poor. Absorption enhancers are considered to be one of the most promising agents to enhance the intestinal absorption of drugs. In this research, we focused on a gemini surfactant, a new type of absorption enhancer. The intestinal absorption of drugs, with or without sodium dilauramidoglutamide lysine (SLG-30), a gemini surfactant, was examined by an in situ closed-loop method in rats. The intestinal absorption of 5(6)-carboxyfluorescein (CF) and fluorescein isothiocyanate-dextrans (FDs) was significantly enhanced in the presence of SLG-30, such effect being reversible. Furthermore, the calcium levels in the plasma significantly decreased when calcitonin was co-administered with SLG-30, suggestive of the increased intestinal absorption of calcitonin. In addition, no significant increase in the of lactate dehydrogenase (LDH) activity or in protein release from the intestinal epithelium was observed in the presence of SLG-30, suggestive of the safety of this compound. These findings indicate that SLG-30 is an effective absorption-enhancer for improving the intestinal absorption of poorly absorbed drugs, without causing serious damage to the intestinal epithelium. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Snake spectacle vessel permeability to sodium fluorescein.

    PubMed

    Bellhorn, Roy W; Strom, Ann R; Motta, Monica J; Doval, John; Hawkins, Michelle G; Paul-Murphy, Joanne

    2018-03-01

    Assess vascular permeability of the snake spectacle to sodium fluorescein during resting and shedding phases of the ecdysis cycle. Ball python (Python regius). The snake was anesthetized, and spectral domain optic coherence tomography was performed prior to angiographic procedures. An electronically controlled digital single-lens reflex camera with a dual-head flash equipped with filters suitable for fluorescein angiography was used to make images. Sodium fluorescein (10%) solution was administered by intracardiac injection. Angiographic images were made as fluorescein traversed the vasculature of the iris and spectacle. Individually acquired photographic frames were assessed and sequenced into pseudovideo image streams for further evaluation CONCLUSIONS: Fluorescein angiograms of the snake spectacle were readily obtained. Vascular permeability varied with the phase of ecdysis. Copious leakage of fluorescein occurred during the shedding phase. This angiographic method may provide diverse opportunities to investigate vascular aspects of snake spectacle ecdysis, dysecdysis, and the integument in general. © 2017 American College of Veterinary Ophthalmologists.

  1. Site-Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne-Azide Cycloaddition Reactions.

    PubMed

    Perols, Anna; Arcos Famme, Melina; Eriksson Karlström, Amelie

    2015-11-01

    Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine-reactive probes that target surface-exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site-specifically labeled product. Z32 BPA was synthesized by solid-phase peptide synthesis and further functionalized to give alkyne-Z32 BPA and azide-Z32 BPA for Cu(I) -catalyzed cycloaddition, as well as DBCO-Z32 BPA for Cu-free strain-promoted cycloaddition. The Z32 BPA variants were conjugated to the human IgG1 antibody trastuzumab and site-specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2-expressing cells in immunofluorescence microscopy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Rash, fever, and chills after intravenous fluorescein angiography.

    PubMed

    Johnson, R N; McDonald, H R; Schatz, H

    1998-12-01

    To report a previously unreported complication associated with intravenous injection of fluorescein dye. Case report. A 75-year-old man developed a unique complication after intravenous injection of fluorescein dye for angiography. Two hours after receiving an intravenous injection of fluorescein for angiography, the patient developed a fever, rash, and chills. Admission to a hospital and careful systemic evaluation determined that this reaction was a noninfectious allergic response to intravenous fluorescein dye injection. A delayed allergic response to intravenous fluorescein dye injection can occur.

  3. Use of a Phage-Display Method to Identify Peptides that Bind to a Tin Oxide Nanosheets.

    PubMed

    Nakazawa, Hikaru; Seta, Yasuko; Hirose, Tatsuya; Masuda, Yoshitake; Umetsu, Mitsuo

    2018-01-01

    Nanosheets of SnO2 which an n-type semiconductor with a rutile-type crystalline structure are predominantly used as gas sensors. SnO2 nanosheets have a tetragonal crystal structure where growth along the c-axis is suppressed to form a sheet. The major exposed facets of SnO2 nanosheets have {110}, {101} and {211} crystal planes along the a-axis, with the reduced {110} surface having a particularly high surface energy. Identifying peptides that bind to specific crystal planes by using peptide phage-display approach will increase the potential applications of metal oxide nanomaterials by fusing proteins with desirable active sites to peptides that adsorb at high density on the major exposed crystal plane of nanosheets. It may be possible to construct highly sensitive biosensors. The main objective of the present study is to identify peptides that adsorb preferentially to a SnO2 nanosheet by using peptide-phage display approach. Four milligrams of SnO2 nanosheet were mixed with 1011 plaque-forming units of Ph.D.-12 Phage Display Peptide Library. Phage-bound nanosheet particles were washed 10 times with 1 mL of phosphatebuffered saline containing 0.5% Tween 20. Phages bound to the nanosheet were eluted with three different buffers: (1) high-salt buffer containing 2 M NaCl (pH 7.5); (2) acidic buffer containing 200 mM Gly-HCl (pH 2.2); and (3) high-phosphate-ion buffer containing 500 mM NaH2PO4 (pH 7.5). The eluted phages were subjected to four or five rounds of biopanning. At each round, individual plaques were picked from the plates, and the amino acid sequences of the peptides were identified by DNA sequencing. The identified SnO2-binding peptides labeled with fluorescein isothiocyanate were synthesized. Adsorption isotherms were constructed at peptide concentrations ranging from 0.25 to 2.0 µM with 4mg of nanomaterials. We were determined the sequences of 11 clones with the high-salt buffer, 7 with the high-phosphateion buffers, and 6 with the acidic buffer and

  4. Single molecule resolution of the antimicrobial action of quantum dot-labeled sushi peptide on live bacteria.

    PubMed

    Leptihn, Sebastian; Har, Jia Yi; Chen, Jianzhu; Ho, Bow; Wohland, Thorsten; Ding, Jeak Ling

    2009-05-11

    Antimicrobial peptides are found in all kingdoms of life. During the evolution of multicellular organisms, antimicrobial peptides were established as key elements of innate immunity. Most antimicrobial peptides are thought to work by disrupting the integrity of cell membranes, causing pathogen death. As antimicrobial peptides target the membrane structure, pathogens can only acquire resistance by a fundamental change in membrane composition. Hence, the evolution of pathogen resistance has been a slow process. Therefore antimicrobial peptides are valuable alternatives to classical antibiotics against which multiple drug-resistant bacteria have emerged. For potential therapeutic applications as antibiotics a thorough knowledge of their mechanism of action is essential. Despite the increasingly comprehensive understanding of the biochemical properties of these peptides, the actual mechanism by which antimicrobial peptides lyse microbes is controversial. Here we investigate how Sushi 1, an antimicrobial peptide derived from the horseshoe crab (Carcinoscorpius rotundicauda), induces lysis of Gram-negative bacteria. To follow the entire process of antimicrobial action, we performed a variety of experiments including transmission electron microscopy and fluorescence correlation spectroscopy as well as single molecule tracking of quantum dot-labeled antimicrobial peptides on live bacteria. Since in vitro measurements do not necessarily correlate with the in vivo action of a peptide we developed a novel fluorescent live bacteria lysis assay. Using fully functional nanoparticle-labeled Sushi 1, we observed the process of antimicrobial action at the single-molecule level. Recently the hypothesis that many antimicrobial peptides act on internal targets to kill the bacterium has been discussed. Here, we demonstrate that the target sites of Sushi 1 are outer and inner membranes and are not cytosolic. Further, our findings suggest four successive steps of the bactericidal process

  5. Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis.

    PubMed

    Masterson, Larry R; Bortone, Nadia; Yu, Tao; Ha, Kim N; Gaffarogullari, Ece C; Nguyen, Oanh; Veglia, Gianluigi

    2009-04-01

    Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI(5-24)). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His(6) tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.

  6. ABC Transporters and Isothiocyanates: Potential for Pharmacokinetic Diet–Drug Interactions

    PubMed Central

    Telang, Urvi; Ji, Yan; Morris, Marilyn E.

    2013-01-01

    Isothiocyanates, a class of anti-cancer agents, are derived from cruciferous vegetables such as broccoli, cabbage and watercress, and have demonstrated chemopreventive activity in a number of cancer models and epidemiologic studies. Due to public interest in cancer prevention and alternative therapies in cancer, the consumption of herbal supplements and vegetables containing these compounds is widespread and increasing. Isothiocyanates interact with ATP-binding cassette (ABC) efflux transporters such as P-glycoprotein, MRP1, MRP2 and BCRP, and may influence the pharmacokinetics of substrates of these transporters. This review discusses the pharmacokinetic properties of isothiocyanates, their interactions with ABC transporters, and presents some data describing the potential for isothiocyanate-mediated diet–drug interactions. PMID:19623673

  7. Biotin and fluorescent labeling of RNA using T4 RNA ligase.

    PubMed Central

    Richardson, R W; Gumport, R I

    1983-01-01

    Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5'-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNAPhe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100%. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochloride, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3'-terminus of RNA molecules. Images PMID:6194506

  8. Protective effects of poly (butyl) cyanoacrylate nanoparticles containing vasoactive intestinal peptide against 6-hydroxydopamine-induced neurotoxicity in vitro.

    PubMed

    Xu, Zhi-Ran; Wang, Wu-Fang; Liang, Xin-Fang; Liu, Ze-Hua; Liu, Yu; Lin, Liang; Zhu, Xuan

    2015-04-01

    The present study investigated brain delivery system of vasoactive intestinal peptide (VIP) adsorbed on poly (butyl cyanoacrylate) nanoparticles coated with polysorbate 80 (P80-poly (butyl) cyanoacrylate (PBCA)-nanoparticles (NPs)) and the neuroprotective effects on the formulation in the model of 6-hydroxydopamine (6-OHDA)-induced Parkinsonian dysfunction in the human neuroblastoma cell line SH-SY5Y. Drug-loaded nanoparticles were prepared by emulsion polymerization method using VIP and PBCA and then stirring with polysorbate 80. The resulting nanoparticles possessed high entrapment efficiency and favorable stability against CaCl2 or fetal bovine serum (FBS)-induced aggregation. Use of fluorescein isothiocyanate (FITC)-conjugated polysorbate 80-PBCA nanoparticles in confocal microscopy revealed that nanoparticles are located inside, while the FITC solution could not penetrate into the cells. The blank nanoparticles showed no significant effects on cell viability, indicating that they had no role in protection; however, polysorbate 80-modified VIP-loading PBCA nanoparticles showed enhanced cell viability compared to free VIP in 6-OHDA-mimic cellular model of Parkinson's disease. In addition, the nanoparticles strikingly increased the anti-apoptosis activity and restored the loss of mitochondrial membrane potential (MMP) significantly after the treatment of 6-OHDA. These results demonstrated that the activity of VIP was enhanced by polysorbate 80-PBCA nanoparticles compared to control solutions, suggesting that PBCA nanoparticles coated with polysorbate 80 could be an effective carrier system for VIP.

  9. Contact and fumigant toxicity of Armoracia rusticana essential oil, allyl isothiocyanate and related compounds to Dermatophagoides farinae.

    PubMed

    Yun, Yeon-Kyeong; Kim, Hyun-Kyung; Kim, Jun-Ran; Hwang, Kumnara; Ahn, Young-Joon

    2012-05-01

    The toxicity to adult Dermatophagoides farinae of allyl isothiocyanate identified in horseradish, Armoracia rusticana, oil and another 27 organic isothiocyanates was evaluated using contact + fumigant and vapour-phase mortality bioassays. Results were compared with those of two conventional acaricides, benzyl benzoate and dibutyl phthalate. Horseradish oil (24 h LC(50), 1.54 µg cm(-2)) and allyl isothiocyanate (2.52 µg cm(-2)) were highly toxic. Benzyl isothiocyanate (LC(50) , 0.62 µg cm(-2)) was the most toxic compound, followed by 4-chlorophenyl, 3-bromophenyl, 3,5-bis(trifluoromethyl)phenyl, cyclohexyl, 2-chlorophenyl, 4-bromophenyl and 2-bromophenyl isothiocyanates (0.93-1.41 µg cm(-2)). All were more effective than either benzyl benzoate (LC(50) , 4.58 µg cm(-2)) or dibutyl phthalate (24.49 µg cm(-2)). The structure-activity relationship indicates that types of functional group and chemical structure appear to play a role in determining the isothiocyanate toxicities to adult D. farinae. In the vapour-phase mortality bioassay, these isothiocyanates were consistently more toxic in closed versus open containers, indicating that their mode of delivery was, in part, a result of vapour action. In the light of global efforts to reduce the level of highly toxic synthetic acaricides in indoor environments, the horseradish oil-derived compounds and the isothiocyanates described herein merit further study as potential acaricides for the control of house dust mite populations as fumigants with contact action. Copyright © 2011 Society of Chemical Industry.

  10. Identification of Quorum-Sensing Inhibitors Disrupting Signaling between Rgg and Short Hydrophobic Peptides in Streptococci

    PubMed Central

    Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Lee, Hyun; Chlipala, George E.; Ratia, Kiira

    2015-01-01

    ABSTRACT Bacteria coordinate a variety of social behaviors, important for both environmental and pathogenic bacteria, through a process of intercellular chemical signaling known as quorum sensing (QS). As microbial resistance to antibiotics grows more common, a critical need has emerged to develop novel anti-infective therapies, such as an ability to attenuate bacterial pathogens by means of QS interference. Rgg quorum-sensing pathways, widespread in the phylum Firmicutes, employ cytoplasmic pheromone receptors (Rgg transcription factors) that directly bind and elicit gene expression responses to imported peptide signals. In the human-restricted pathogen Streptococcus pyogenes, the Rgg2/Rgg3 regulatory circuit controls biofilm development in response to the short hydrophobic peptides SHP2 and SHP3. Using Rgg-SHP as a model receptor-ligand target, we sought to identify chemical compounds that could specifically inhibit Rgg quorum-sensing circuits. Individual compounds from a diverse library of known drugs and drug-like molecules were screened for their ability to disrupt complexes of Rgg and FITC (fluorescein isothiocyanate)-conjugated SHP using a fluorescence polarization (FP) assay. The best hits were found to bind Rgg3 in vitro with submicromolar affinities, to specifically abolish transcription of Rgg2/3-controlled genes, and to prevent biofilm development in S. pyogenes without affecting bacterial growth. Furthermore, the top hit, cyclosporine A, as well as its nonimmunosuppressive analog, valspodar, inhibited Rgg-SHP pathways in multiple species of Streptococcus. The Rgg-FITC-peptide-based screen provides a platform to identify inhibitors specific for each Rgg type. Discovery of Rgg inhibitors constitutes a step toward the goal of manipulating bacterial behavior for purposes of improving health. PMID:25968646

  11. Secondary nuclear targeting of mesoporous silica nano-particles for cancer-specific drug delivery based on charge inversion

    PubMed Central

    Wang, Xiyong; Fan, Xiaobo; Wu, Guoqiu

    2016-01-01

    A novel multifunctional nano-drug delivery system based on reversal of peptide charge was successfully developed for anticancer drug delivery and imaging. Mesoporous silica nano-particles (MSN) ~50 nm in diameter were chosen as the drug reservoirs, and their surfaces were modified with HIV-1 transactivator peptide-fluorescein isothiocyanate (TAT-FITC) and YSA-BHQ1. The short TAT peptide labeled with FITC was used to facilitate intranuclear delivery, while the YSA peptide tagged with the BHQ1 quencher group was used to specifically bind to the tumor EphA2 membrane receptor. Citraconic anhydride (Cit) was used to invert the charge of the TAT peptide in neutral or weak alkaline conditions so that the positively charged YSA peptide could combine with the TAT peptide through electrostatic attraction. The FITC fluorescence was quenched by the spatial approach of BHQ1 after the two peptides bound to each other. However, the Cit-amino bond was unstable in the acidic atmosphere, so the positive charge of the TAT peptide was restored and the positively charged YSA moiety was repelled. The FITC fluorescence was recovered after the YSA-BHQ1 moiety was removed, and the TAT peptide led the nano-particles into the nucleolus. This nano-drug delivery system was stable at physiological pH, rapidly released the drug in acidic buffer, and was easily taken up by MCF-7 cells. Compared with free doxorubicin hydrochloride at an equal concentration, this modified MSN loaded with doxorubicin molecules had an equivalent inhibitory effect on MCF-7 cells. This nano-drug delivery system is thus a promising method for simultaneous cancer diagnosis and therapy. PMID:27661121

  12. Secondary nuclear targeting of mesoporous silica nano-particles for cancer-specific drug delivery based on charge inversion.

    PubMed

    Zhao, Jianwen; Zhao, Fengfeng; Wang, Xiyong; Fan, Xiaobo; Wu, Guoqiu

    2016-10-25

    A novel multifunctional nano-drug delivery system based on reversal of peptide charge was successfully developed for anticancer drug delivery and imaging. Mesoporous silica nano-particles (MSN) ~50 nm in diameter were chosen as the drug reservoirs, and their surfaces were modified with HIV-1 transactivator peptide-fluorescein isothiocyanate (TAT-FITC) and YSA-BHQ1. The short TAT peptide labeled with FITC was used to facilitate intranuclear delivery, while the YSA peptide tagged with the BHQ1 quencher group was used to specifically bind to the tumor EphA2 membrane receptor. Citraconic anhydride (Cit) was used to invert the charge of the TAT peptide in neutral or weak alkaline conditions so that the positively charged YSA peptide could combine with the TAT peptide through electrostatic attraction. The FITC fluorescence was quenched by the spatial approach of BHQ1 after the two peptides bound to each other. However, the Cit-amino bond was unstable in the acidic atmosphere, so the positive charge of the TAT peptide was restored and the positively charged YSA moiety was repelled. The FITC fluorescence was recovered after the YSA-BHQ1 moiety was removed, and the TAT peptide led the nano-particles into the nucleolus. This nano-drug delivery system was stable at physiological pH, rapidly released the drug in acidic buffer, and was easily taken up by MCF-7 cells. Compared with free doxorubicin hydrochloride at an equal concentration, this modified MSN loaded with doxorubicin molecules had an equivalent inhibitory effect on MCF-7 cells. This nano-drug delivery system is thus a promising method for simultaneous cancer diagnosis and therapy.

  13. THE EFFECT OF PRESSURE ON THE OXIDATION STATE OF IRON, IV. THIOCYANATE AND ISOTHIOCYANATE LIGANDS*

    PubMed Central

    Fung, S. C.; Drickamer, H. G.

    1969-01-01

    The effect of pressure on the Mössbauer resonance spectra of Fe(III) with thiocyanate (M-SCN) and isothiocyanate (M-NCS) ligands has been studied. Fe(NCS)3·6H2O, which has the isothiocyanate structure, reduces with increasing pressure, reversibly, and with a pressure dependence for the conversion very similar to that shown by a wide variety of ionic ferric compounds. K3Fe(SCN)6 has the thiocyanate structure. At low pressures, it exhibits a significantly larger reduction than the Fe(NCS)3. With increasing pressure the thiocyanate complexes isomerize, each complex apparently exhibiting about the same degree of conversion at a given pressure. At 150 kb the isomerization is essentially complete. The reduction of the Fe(III) to Fe(II) is reversible but the isomerization is not, and the sample, when powdered and reloaded in the high-pressure cell, exhibits the isomer shift, quadrupole splitting, and Fe(III) to Fe(II) conversion characteristic of an isothiocyanate. Heating the thiocyanate to 110°C at 5 kb yields a mixture of thiocyanate and isothiocyanate that converts with pressure completely to the isothiocyanate. PMID:16591728

  14. Severe blood-brain barrier disruption and surrounding tissue injury.

    PubMed

    Chen, Bo; Friedman, Beth; Cheng, Qun; Tsai, Phil; Schim, Erica; Kleinfeld, David; Lyden, Patrick D

    2009-12-01

    Blood-brain barrier opening during ischemia follows a biphasic time course, may be partially reversible, and allows plasma constituents to enter brain and possibly damage cells. In contrast, severe vascular disruption after ischemia is unlikely to be reversible and allows even further extravasation of potentially harmful plasma constituents. We sought to use simple fluorescent tracers to allow wide-scale visualization of severely damaged vessels and determine whether such vascular disruption colocalized with regions of severe parenchymal injury. Severe vascular disruption and ischemic injury was produced in adult Sprague Dawley rats by transient occlusion of the middle cerebral artery for 1, 2, 4, or 8 hours, followed by 30 minutes of reperfusion. Fluorescein isothiocyanate-dextran (2 MDa) was injected intravenously before occlusion. After perfusion-fixation, brain sections were processed for ultrastructure or fluorescence imaging. We identified early evidence of tissue damage with Fluoro-Jade staining of dying cells. With increasing ischemia duration, greater quantities of high molecular weight dextran-fluorescein isothiocyanate invaded and marked ischemic regions in a characteristic pattern, appearing first in the medial striatum, spreading to the lateral striatum, and finally involving cortex; maximal injury was seen in the mid-parietal areas, consistent with the known ischemic zone in this model. The regional distribution of the severe vascular disruption correlated with the distribution of 24-hour 2,3,5-triphenyltetrazolium chloride pallor (r=0.75; P<0.05) and the cell death marker Fluoro-Jade (r=0.86; P<0.05). Ultrastructural examination showed significantly increased areas of swollen astrocytic foot process and swollen mitochondria in regions of high compared to low leakage, and compared to contralateral homologous regions (ANOVA P<0.01). Dextran extravasation into the basement membrane and surrounding tissue increased significantly from 2 to 8 hours of

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

    PubMed

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

    2004-04-15

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

  16. Convergent synthesis of 13N-labelled Peptidic structures using aqueous [13N]NH3.

    PubMed

    Blower, Julia E; Cousin, Samuel F; Gee, Antony D

    2017-01-01

    Nitrogen-13 has a 10-min half-life which places time constraints on the complexity of viable synthetic methods for its incorporation into PET imaging agents. In exploring ways to overcome this limitation, we have used the Ugi reaction to develop a rapid one-pot method for radiolabelling peptidic molecules using [ 13 N]NH 3 as a synthetic precursor. Carrier-added [ 13 N]NH 3 (50 μL) was added to a solution of carboxylic acid, aldehyde, and isocyanide in 2,2,2-TFE (200 μL). The mixture was heated in a microwave synthesiser at 120 °C for 10 min. Reactions were analysed by radio-HPLC and radio-LCMS. Isolation of the target 13 N-labelled peptidic Ugi compound was achieved via semi-preparative radio-HPLC as demonstrated for Ugi 1. Radio-HPLC analysis of each reaction confirmed the formation of radioactive products co-eluting with their respective reference standards with radiochemical yields of the crude products ranging from 11% to 23%. Two cyclic γ-lactam structures were also achieved via intra-molecular reactions. Additional radioactive by-products observed in the radio-chromatogram were identified as 13 N-labelled di-imines formed from the reaction of [ 13 N]NH 3 with two isocyanide molecules. The desired 13 N-labelled Ugi product was isolated using semi-preparative HPLC. We have developed a one-pot method that opens up new routes to radiolabel complex, peptidic molecules with 13 N using aqueous [ 13 N]NH 3 as a synthetic precursor.

  17. Antimicrobial Activities of Isothiocyanates Against Campylobacter jejuni Isolates

    PubMed Central

    Dufour, Virginie; Alazzam, Bachar; Ermel, Gwennola; Thepaut, Marion; Rossero, Albert; Tresse, Odile; Baysse, Christine

    2012-01-01

    Food-borne human infection with Campylobacter jejuni is a medical concern in both industrialized and developing countries. Efficient eradication of C. jejuni reservoirs within live animals and processed foods is limited by the development of antimicrobial resistances and by practical problems related to the use of conventional antibiotics in food processes. We have investigated the bacteriostatic and bactericidal activities of two phytochemicals, allyl-isothiocyanate (AITC), and benzyl isothiocyanate (BITC), against 24 C. jejuni isolates from chicken feces, human infections, and contaminated foods, as well as two reference strains NCTC11168 and 81-176. AITC and BITC displayed a potent antibacterial activity against C. jejuni. BITC showed a higher overall antibacterial effect (MIC of 1.25–5 μg mL−1) compared to AITC (MIC of 50–200 μg mL−1). Both compounds are bactericidal rather than bacteriostatic. The sensitivity levels of C. jejuni isolates against isothiocyanates were neither correlated with the presence of a GGT (γ-Glutamyl Transpeptidase) encoding gene in the genome, with antibiotic resistance nor with the origin of the biological sample. However the ggt mutant of C. jejuni 81-176 displayed a decreased survival rate compared to wild-type when exposed to ITC. This work determined the MIC of two ITC against a panel of C. jejuni isolates, showed that both compounds are bactericidal rather than bacteriostatic, and highlighted the role of GGT enzyme in the survival rate of C. jejuni exposed to ITC. PMID:22919644

  18. Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides

    PubMed Central

    Schuchardt, Christiane; Kulkarni, Harshad R.; Shahinfar, Mostafa; Singh, Aviral; Glatting, Gerhard; Baum, Richard P.; Beer, Ambros J.

    2016-01-01

    In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25−29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1–3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the

  19. Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides.

    PubMed

    Kletting, Peter; Schuchardt, Christiane; Kulkarni, Harshad R; Shahinfar, Mostafa; Singh, Aviral; Glatting, Gerhard; Baum, Richard P; Beer, Ambros J

    2016-01-01

    In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25-29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1-3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the peptide

  20. 111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues for melanoma imaging.

    PubMed

    Miao, Yubin; Gallazzi, Fabio; Guo, Haixun; Quinn, Thomas P

    2008-02-01

    The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized alpha-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with (111)In. The internalization and efflux of (111)In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics, and SPECT/CT imaging of (111)In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH were 9.53+/-1.41% injected dose/gram (% ID/g) and 10.40+/-1.40% ID/g at 2 h postinjection, respectively. Flank melanoma tumors were clearly visualized with (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h postinjection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h postinjection. There was low radioactivity (<0.8% ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h postinjection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using (111)In-labeled lactam bridge-cyclized alpha-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.

  1. Synthesis and characterization of a novel inhibitor of C-reactive protein-mediated proinflammatory effects.

    PubMed

    Kumaresan, Pappanaicken R; Devaraj, Sridevi; Huang, Wenzhe; Lau, Edmond Y; Liu, Ruiwu; Lam, Kit S; Jialal, Ishwarlal

    2013-06-01

    Numerous studies have shown that high C-reactive protein (CRP) levels predict cardiovascular disease and augur a poor prognosis in patients with acute coronary syndromes. Much in vitro and in vivo data support of a role for CRP in atherogenesis. There is an urgent need to develop inhibitors that specifically block the biological effects of CRP in vivo. The one-bead-one-compound (OBOC) combinatorial library method has been used to discover ligands against several biological targets. In this study, we use a novel fluorescence-based screening method to screen an OBOC combinatorial library for the discovery of peptides against human CRP. Human CRP was labeled with fluorescein isothiocyanate (FITC) and human serum albumin (HuSA) was labeled with phycoerythrin (PE) and used for screening. The OBOC library LWH-01 was synthesized on TentaGel resin beads using a standard solid-phase "split/mix" approach. By subtraction screening, eight peptides that bind specifically to CRP and not to HuSA were identified. In human aortic endothelial cells (HAECs) incubated with CRP, inhibitors CRPi-2, CRPi-3, and CRPi-6 significantly inhibited CRP-induced superoxide, cytokine release, and nuclear factor-κB (NFκB) activity. Molecular docking studies demonstrate that CRPi-2 interacts with the two Ca(2+) ions in the single subunit of CRP. The binding of CRPi-2 is reminiscent of choline binding. Future studies will examine the utility of this inhibitor in animal models and clinical trials.

  2. Analysis of Intrinsic Peptide Detectability via Integrated Label-Free and SRM-Based Absolute Quantitative Proteomics.

    PubMed

    Jarnuczak, Andrew F; Lee, Dave C H; Lawless, Craig; Holman, Stephen W; Eyers, Claire E; Hubbard, Simon J

    2016-09-02

    Quantitative mass spectrometry-based proteomics of complex biological samples remains challenging in part due to the variability and charge competition arising during electrospray ionization (ESI) of peptides and the subsequent transfer and detection of ions. These issues preclude direct quantification from signal intensity alone in the absence of a standard. A deeper understanding of the governing principles of peptide ionization and exploitation of the inherent ionization and detection parameters of individual peptides is thus of great value. Here, using the yeast proteome as a model system, we establish the concept of peptide F-factor as a measure of detectability, closely related to ionization efficiency. F-factor is calculated by normalizing peptide precursor ion intensity by absolute abundance of the parent protein. We investigated F-factor characteristics in different shotgun proteomics experiments, including across multiple ESI-based LC-MS platforms. We show that F-factors mirror previously observed physicochemical predictors as peptide detectability but demonstrate a nonlinear relationship between hydrophobicity and peptide detectability. Similarly, we use F-factors to show how peptide ion coelution adversely affects detectability and ionization. We suggest that F-factors have great utility for understanding peptide detectability and gas-phase ion chemistry in complex peptide mixtures, selection of surrogate peptides in targeted MS studies, and for calibration of peptide ion signal in label-free workflows. Data are available via ProteomeXchange with identifier PXD003472.

  3. Low cost labeling with highlighter ink efficiently visualizes developing blood vessels in avian and mouse embryos.

    PubMed

    Takase, Yuta; Tadokoro, Ryosuke; Takahashi, Yoshiko

    2013-12-01

    To understand how blood vessels form to establish the intricate network during vertebrate development, it is helpful if one can visualize the vasculature in embryos. We here describe a novel labeling method using highlighter ink, easily obtained in stationery stores with a low cost, to visualize embryo-wide vasculatures in avian and mice. We tested 50 different highlighters for fluorescent microscopy with filter sets equipped in a standard fluorescent microscope. The yellow and violet inks yielded fluorescent signals specifically detected by the filters used for green fluorescent protein (GFP) and red fluorescent protein (RFP) detections, respectively. When the ink solution was infused into chicken/quail and mouse embryos, vasculatures including large vessels and capillaries were labeled both in living and fixed embryos. Ink-infused embryos were further subjected to histological sections, and double stained with antibodies including QH-1 (quail), α smooth muscle actin (αSMA), and PECAM-1 (mouse), revealing that the endothelial cells were specifically labeled by the infused highlighter ink. Highlighter-labeled signals were detected with a resolution comparable to or higher than signals of fluorescein isothiocyanate (FITC)-lectin and Rhodamine-dextran, conventionally used for angiography. Furthermore, macroconfocal microscopic analyses with ink-infused embryos visualized fine vascular structures of both embryo proper and extra-embryonic plexus in a Z-stack image of 2400 μm thick with a markedly high resolution. Together, the low cost highlighter ink serves as an alternative reagent useful for visualization of blood vessels in developing avian and mouse embryos and possibly in other animals. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  4. PSMA-11-Derived Dual-Labeled PSMA Inhibitors for Preoperative PET Imaging and Precise Fluorescence-Guided Surgery of Prostate Cancer.

    PubMed

    Baranski, Ann-Christin; Schäfer, Martin; Bauder-Wüst, Ulrike; Roscher, Mareike; Schmidt, Jana; Stenau, Esther; Simpfendörfer, Tobias; Teber, Dogu; Maier-Hein, Lena; Hadaschik, Boris; Haberkorn, Uwe; Eder, Matthias; Kopka, Klaus

    2018-04-01

    Resection of tumors using targeted dual-modality probes combining preoperative imaging with intraoperative guidance is of high clinical relevance and might considerably affect the outcome of prostate cancer therapy. This work aimed at the development of dual-labeled prostate-specific membrane antigen (PSMA) inhibitors derived from the established N,N' -bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine- N,N' -diacetic acid (HBED-CC)-based PET tracer 68 Ga-Glu-urea-Lys(Ahx)-HBED-CC ( 68 Ga-PSMA-11) to allow accurate intraoperative detection of PSMA-positive tumors. Methods: A series of novel PSMA-targeting fluorescent dye conjugates of Glu-urea-Lys-HBED-CC was synthesized, and their biologic properties were determined in cell-based assays and confocal microscopy. As a preclinical proof of concept, specific tumor uptake, pharmacokinetics, and feasibility for intraoperative fluorescence guidance were investigated in tumor-bearing mice and healthy pigs. Results: The designed dual-labeled PSMA inhibitors exhibited high binding affinity and PSMA-specific effective internalization. Conjugation of fluorescein isothiocyanate (10.86 ± 0.94 percentage injected dose [%ID]/g), IRDye800CW (13.66 ± 3.73 %ID/g), and DyLight800 (15.62 ± 5.52 %ID/g) resulted in a significantly increased specific tumor uptake, whereas 68 Ga-Glu-urea-Lys-HBED-CC-AlexaFluor488 (9.12 ± 5.47 %ID/g) revealed a tumor uptake similar to that of 68 Ga-PSMA-11 (4.89 ± 1.34 %ID/g). The first proof-of-concept studies with the clinically relevant candidate 68 Ga-Glu-urea-Lys-HBED-CC-IRDye800CW reinforced a fast, specific enrichment in PSMA-positive tumors, with rapid background clearance. With regard to intraoperative navigation, a specific fluorescence signal was detected in PSMA-expressing tissue. Conclusion: This study demonstrated that PSMA-11-derived dual-labeled dye conjugates are feasible for providing PSMA-specific pre-, intra-, and postoperative detection of prostate cancer lesions and have high

  5. Accidental intra-arterial injection of fluorescein dye.

    PubMed

    Bovino, J A; Marcus, D F

    1984-12-01

    During fluorescein angiography, sodium fluorescein dye intended for intravenous use was inadvertently injected into an artery in the antecubital fossa. An immediate and dramatic orange discoloration of the skin distal to the injection combined with intense burning pain of the right forearm and hand were noted. The patient was treated with ice packs and analgesics. The fluorescein angiogram showed a delayed arm to eye circulation time, but was of normal quality. There were no long-term complications.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matzke, Melissa M.; Brown, Joseph N.; Gritsenko, Marina A.

    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 frommore » 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.« less

  7. Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells.

    PubMed

    Garland, A M; Grady, E F; Payan, D G; Vigna, S R; Bunnett, N W

    1994-10-01

    Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P.

  8. Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes.

    PubMed

    Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke; Saini, Sunil Kumar; Ramskov, Sofie; Donia, Marco; Such, Lina; Furness, Andrew J S; McGranahan, Nicholas; Rosenthal, Rachel; Straten, Per Thor; Szallasi, Zoltan; Svane, Inge Marie; Swanton, Charles; Quezada, Sergio A; Jakobsen, Søren Nyboe; Eklund, Aron Charles; Hadrup, Sine Reker

    2016-10-01

    Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10-100 distinct T-cell specificities in one sample. Here we use peptide-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.

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

    PubMed

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

    2014-11-20

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

  10. Fluorescence probe techniques to monitor protein adsorption-induced conformation changes on biodegradable polymers.

    PubMed

    Benesch, Johan; Hungerford, Graham; Suhling, Klaus; Tregidgo, Carolyn; Mano, João F; Reis, Rui L

    2007-08-15

    The study of protein adsorption and any associated conformational changes on interaction with biomaterials is of great importance in the area of implants and tissue constructs. This study aimed to evaluate some fluorescent techniques to probe protein conformation on a selection of biodegradable polymers currently under investigation for biomedical applications. Because of the fluorescence emanating from the polymers, the use of monitoring intrinsic protein fluorescence was precluded. A highly solvatochromic fluorescent dye, Nile red, and a well-known protein label, fluorescein isothiocyanate, were employed to study the adsorption of serum albumin to polycaprolactone and to some extent also to two starch-containing polymer blends (SPCL and SEVA-C). A variety of fluorescence techniques, steady state, time resolved, and imaging were employed. Nile red was found to leach from the protein, while fluorescein isothiocyanate proved useful in elucidating a conformational change in the protein and the observation of protein aggregates adsorbed to the polymer surface. These effects were seen by making use of the phenomenon of energy migration between the fluorescent tags to monitor interprobe distance and the use of fluorescence lifetime imaging to ascertain the surface packing of the protein on polymer.

  11. UNiquant, a program for quantitative proteomics analysis using stable isotope labeling.

    PubMed

    Huang, Xin; Tolmachev, Aleksey V; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A; Smith, Richard D; Chan, Wing C; Hinrichs, Steven H; Fu, Kai; Ding, Shi-Jian

    2011-03-04

    Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for postmeasurement normalization of peptide ratios, which is required by the other programs.

  12. UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling

    PubMed Central

    Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven H.; Fu, Kai; Ding, Shi-Jian

    2011-01-01

    Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for post-measurement normalization of peptide ratios, which is required by the other programs. PMID:21158445

  13. EVALUATION OF CRYPTOSPORIDIUM OOCYST RECOVERY IN WATER BY EPA METHOD 1623 WITH A MODIFIED IMS DISSOCIATION PROCEDURE.

    EPA Science Inventory

    EPA Methods 1622 and 1623 are the benchmarks for detection of Cryptosporidium spp. oocysts in water. 5-7 These methods consist of filtration, elution, purification by immunomagnetic separation (IMS), and microscopic analysis after staining with a fluorescein isothiocyanate conju...

  14. 49 CFR 583.5 - Label requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 7 2013-10-01 2013-10-01 false Label requirements. 583.5 Section 583.5 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AUTOMOBILE PARTS CONTENT LABELING § 583.5 Label...

  15. 49 CFR 583.5 - Label requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 7 2010-10-01 2010-10-01 false Label requirements. 583.5 Section 583.5 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AUTOMOBILE PARTS CONTENT LABELING § 583.5 Label...

  16. 49 CFR 583.5 - Label requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 7 2011-10-01 2011-10-01 false Label requirements. 583.5 Section 583.5 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AUTOMOBILE PARTS CONTENT LABELING § 583.5 Label...

  17. 49 CFR 583.5 - Label requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 7 2012-10-01 2012-10-01 false Label requirements. 583.5 Section 583.5 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AUTOMOBILE PARTS CONTENT LABELING § 583.5 Label...

  18. 49 CFR 583.5 - Label requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 7 2014-10-01 2014-10-01 false Label requirements. 583.5 Section 583.5 Transportation Other Regulations Relating to Transportation (Continued) NATIONAL HIGHWAY TRAFFIC SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AUTOMOBILE PARTS CONTENT LABELING § 583.5 Label...

  19. Heat Shock Protein 27-Targeted Heptapeptide of the PKC{Delta} Catalytic V5 Region Sensitizes Tumors With Radio- and Chemoresistance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Hae-June; Kim, Eun-Ho; School of Life Sciences and Biotechnology, Korea University, Seoul

    Purpose: Previous data suggest that the PKC{delta} catalytic V5 (PKC{delta}-V5) heptapeptide (HEPT) (FEQFLDI) binds HSP27 and blocks HSP27-mediated radio- or chemoresistance. Here we investigated further the in vivo function of the PKC{delta}-V5 HEPT. Methods and Materials: Labeling of HEPT with Cy5.5 or fluorescein isothiocyanate was performed to evaluate in vitro or in vivo distribution of HEPT. A clonogenic survival assay, flow cytometry, and Western blotting of cleaved caspase-3 were performed to determine in vitro sensitization effects of HEPT plus ionizing radiation (IR) versus IR alone or those of HEPT plus cisplatin(Cis) versus Cis alone. A nude mouse xenografting system wasmore » also applied to detect in vivo sensitizing effects of HEPT. Results: HEPT efficiently bound to HSP27 and showed sensitization after combined treatment with IR versus treatment with Cis alone in NCI-H1299 lung carcinoma cells, with higher HSP27 expression, which was similar to that of combined treatment with IR or with Cis alone in NCI-H460 lung carcinoma cells with lower HSP27 expression. In vivo image analysis using Cy5.5-labeled HEPT showed that HEPT was retained in HSP27-overexpressing cancer cells after xenografting to nude mice. Combined treatment of HEPT with IR versus that with Cis alone in xenografted mice showed that HEPT increased radio- or chemosensitization in NCI-H1299 cells compared to that in mice xenografted with NCI-H460 cells. Conclusions: The novel PKC{delta}-V5 HEPT may help overcome HSP27-mediated radio- or chemoresistance.« less

  20. Comparative assessment of a 99mTc labeled H1299.2-HYNIC peptide bearing two different co-ligands for tumor-targeted imaging.

    PubMed

    Torabizadeh, Seyedeh Atekeh; Abedi, Seyed Mohammad; Noaparast, Zohreh; Hosseinimehr, Seyed Jalal

    2017-05-01

    Peptides are a class of targeting agents that bind to cancer-specific cell surfaces. Since they specifically target cancer cells, they could be used as molecular imaging tools. In this study, the 15-mer peptide Ac-H1299.2 (YAAWPASGAWTGTAP) was conjugated with HYNIC via lysine amino acid on C-terminus and labeled with 99m Tc using tricine and EDDA/tricine as the co-ligands. These radiotracers were evaluated for potential utilization in diagnostic imaging of ovarian cancer cells (SKOV-3). The cell-specificity of these radiolabeled peptides was determined based on their binding on an ovarian cancer cell line (SKOV-3), and displaying a low affinity for lung adenocarcinoma cell line (A549) and breast cancer cell line (MCF7). Biodistribution studies were conducted in normal mice as well as in nude mice bearing SKOV-3 ovarian cancer xenografts. HYNIC-peptide was labeled with 99m Tc with more than 99% efficiency and showed high stability in buffer and serum. We observed nanomolar binding affinities for both radiolabeled peptides. The tumor uptakes were 3.27%±0.46% and 1.55%±0.20% for tricine and 2.34±1.1% and 1.09%±0.18% for EDDA/tricine at 1 and 4h after injection, respectively. A higher tumor to background ratio and lower radioactivity in the blood were observed for EDDA/tricine co-ligands, leading to clear tumor visualization in imaging with injection of this peptide. This new 99m Tc-labeled peptide selectively targeted ovarian cancer and introduction of a (EDDA/tricine) as a co-ligand improved the pharmacokinetics of 99m Tc-labeled H1299.2 for tumor imaging in animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Transdermal Protein Delivery Using Choline and Geranate (CAGE) Deep Eutectic Solvent.

    PubMed

    Banerjee, Amrita; Ibsen, Kelly; Iwao, Yasunori; Zakrewsky, Michael; Mitragotri, Samir

    2017-08-01

    Transdermal delivery of peptides and other biological macromolecules is limited due to skin's inherent low permeability. Here, the authors report the use of a deep eutectic solvent, choline and geranate (CAGE), to enhance topical delivery of proteins such as bovine serum albumin (BSA, molecular weight: ≈66 kDa), ovalbumin (OVA, molecular weight: ≈45 kDa) and insulin (INS, molecular weight: 5.8 kDa). CAGE enhances permeation of BSA, OVA, and insulin into porcine skin ex vivo, penetrating deep into the epidermis and dermis. Studies using tritium-labeled BSA and fluorescein isothiocyanate labeled insulin show significantly enhanced delivery of proteins into and across porcine skin, penetrating the skin in a time-dependent manner. Fourier transform IR spectra of porcine stratum corneum (SC) samples before and after incubation in CAGE show a reduction in peak area attributed to SC lipid content, suggesting lipid extraction from the SC. Circular dichroism confirms that CAGE does not affect insulin's secondary conformation. In vivo studies in rats show that topical application of 10 U insulin dispersed in CAGE (25 U kg -1 insulin dose) leads to a highly significant 40% drop in blood glucose levels in 4 h that is relatively sustained for 12 h. Taken together, these studies demonstrate that CAGE is a promising vehicle for transdermal delivery of therapeutic proteins; specifically, as a noninvasive delivery alternative to injectable insulin for the treatment of diabetes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Fluorescein isothiocyanate (FITC)-Dextran Extravasation as a Measure of Blood-Brain Barrier Permeability

    PubMed Central

    Natarajan, Reka; Northrop, Nicole

    2017-01-01

    The blood-brain barrier (BBB) is formed in part by vascular endothelial cells that constitute the capillaries and microvessels of the brain. The function of this barrier is to maintain homeostasis within the brain microenvironment and buffer the brain from changes in the periphery. A dysfunction of the BBB would permit circulating molecules and pathogens typically restricted to the periphery to enter the brain and interfere with normal brain function. As increased permeability of the BBB is associated with several neuropathologies, it is important to have a reliable and sensitive method that determines BBB permeability and the degree of BBB disruption. A detailed protocol is presented for assessing the integrity of the BBB by transcardial perfusion of a 10,000 Da FITC labeled dextran molecule and its visualization to determine the degree of extravasation from brain microvessels. PMID:28398646

  3. Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y1R and Y4R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15.

    PubMed

    Liu, Mengjie; Richardson, Rachel R; Mountford, Simon J; Zhang, Lei; Tempone, Matheus H; Herzog, Herbert; Holliday, Nicholas D; Thompson, Philip E

    2016-09-21

    Traceable truncated Neuropeptide Y (NPY) analogues with Y1 receptor (Y1R) affinity and selectivity are highly desirable tools in studying receptor location, regulation, and biological functions. A range of fluorescently labeled analogues of a reported Y1R/Y4R preferring ligand BVD-15 have been prepared and evaluated using high content imaging techniques. One peptide, [Lys(2)(sCy5), Arg(4)]BVD-15, was characterized as an Y1R antagonist with a pKD of 7.2 measured by saturation analysis using fluorescent imaging. The peptide showed 8-fold lower affinity for Y4R (pKD = 6.2) and was a partial agonist at this receptor. The suitability of [Lys(2)(sCy5), Arg(4)]BVD-15 for Y1R and Y4R competition binding experiments was also demonstrated in intact cells. The nature of the label was shown to be critical with replacement of sCy5 by the more hydrophobic Cy5.5 resulting in a switch from Y1R antagonist to Y1R partial agonist.

  4. Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae).

    PubMed

    López-Abarrategui, Carlos; McBeth, Christine; Mandal, Santi M; Sun, Zhenyu J; Heffron, Gregory; Alba-Menéndez, Annia; Migliolo, Ludovico; Reyes-Acosta, Osvaldo; García-Villarino, Mónica; Nolasco, Diego O; Falcão, Rosana; Cherobim, Mariana D; Dias, Simoni C; Brandt, Wolfgang; Wessjohann, Ludger; Starnbach, Michael; Franco, Octavio L; Otero-González, Anselmo J

    2015-08-01

    Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability. © FASEB.

  5. Monocarboxylate Transporters Mediate Fluorescein Uptake in Corneal Epithelial Cells.

    PubMed

    Sun, Yi-Chen; Liou, Hau-Min; Yeh, Po-Ting; Chen, Wei-Li; Hu, Fung-Rong

    2017-07-01

    To determine the presence of monocarboxylate transporter (MCT) in human and rabbit corneal epithelium and its role in transcellular fluorescein transportation in the cornea. The presence of MCTs in human and rabbit corneal epithelium was determined by RT-PCR and immunohistochemistry. Intracellular fluorescein uptake experiment was performed using cultured human corneal epithelial cells (HCECs). The involvement of MCT in fluorescein uptake was determined by addition of MCT inhibitors to HCECs and acute dry eye model on New Zealand albino rabbits by spectrophotometry, corneal impression cytology, and external eye photographs. MCT-1 and MCT-4 were identified in both human and rabbit corneal epithelia. A longer treatment period and a lower pH value in culture medium increased fluorescein uptake in HCECs. Fluorescein uptake in HCECs was decreased following addition of MCT inhibitors in a concentration-dependent manner. Impression cytology under fluorescent microscopy showed intracellular fluorescein staining in the rabbit cornea with acute dry eye treatment that was decreased following topical treatment of MCT inhibitors. Fluorescein ingress in corneal epithelial cells is mediated by the MCT family. Further study of MCT-mediated transport on HCECs may potentially benefit differential diagnosis and contribute better understandings of ocular surface disorders.

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

    PubMed

    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.

  7. Fluorescein angiography basic science and engineering.

    PubMed

    Wolfe, D R

    1986-12-01

    Fluorescein angiography is an application of the physical phenomenon of fluorescence, which is phosphorescence in which the quantum mechanical decay curve is so rapid that it appears instantaneous, and it consequently has no afterglow. Sodium fluorescein is excited by light energy between 465 and 490 nm, and it decays into a lower state emitting light energy between 520 and 530 nm as fluorescent radiation. The free electrons available for excitation are reduced by chemical bonding between the fluorescein dye and plasma proteins to which up to 80% of the dye is bound in the bloodstream, thus reducing overall fluorescence. Optimalization of the observed and recorded fluorescence is afforded by providing exciter and barrier filters with as little overlap as possible to reduce or eliminate contrast reducing pseudofluorescence.

  8. General Synthetic Method for Si-Fluoresceins and Si-Rhodamines

    PubMed Central

    2017-01-01

    The century-old fluoresceins and rhodamines persist as flexible scaffolds for fluorescent and fluorogenic compounds. Extensive exploration of these xanthene dyes has yielded general structure–activity relationships where the development of new probes is limited only by imagination and organic chemistry. In particular, replacement of the xanthene oxygen with silicon has resulted in new red-shifted Si-fluoresceins and Si-rhodamines, whose high brightness and photostability enable advanced imaging experiments. Nevertheless, efforts to tune the chemical and spectral properties of these dyes have been hindered by difficult synthetic routes. Here, we report a general strategy for the efficient preparation of Si-fluoresceins and Si-rhodamines from readily synthesized bis(2-bromophenyl)silane intermediates. These dibromides undergo metal/bromide exchange to give bis-aryllithium or bis(aryl Grignard) intermediates, which can then add to anhydride or ester electrophiles to afford a variety of Si-xanthenes. This strategy enabled efficient (3–5 step) syntheses of known and novel Si-fluoresceins, Si-rhodamines, and related dye structures. In particular, we discovered that previously inaccessible tetrafluorination of the bottom aryl ring of the Si-rhodamines resulted in dyes with improved visible absorbance in solution, and a convenient derivatization through fluoride-thiol substitution. This modular, divergent synthetic method will expand the palette of accessible xanthenoid dyes across the visible spectrum, thereby pushing further the frontiers of biological imaging. PMID:28979939

  9. (18)F-Fluoroglucosylation of peptides, exemplified on cyclo(RGDfK).

    PubMed

    Hultsch, Christina; Schottelius, Margret; Auernheimer, Jörg; Alke, Andrea; Wester, Hans-Jürgen

    2009-09-01

    Oxime formation between an aminooxy-functionalized peptide and an (18)F-labelled aldehyde has recently been introduced as a powerful method for the rapid one-step chemoselective synthesis of radiofluorinated peptides. Here, the potential of using routinely produced and thus readily available [(18)F]fluorodeoxyglucose ([(18)F]FDG) as the aldehydic prosthetic group was investigated using an aminooxyacetyl-conjugated cyclic RGD peptide (cyclo(RGDfK(Aoa-(Boc)) as a model peptide. The use of [(18)F]FDG from routine production ([(18)F]FDGTUM) containing an excess of D: -glucose did not allow the radiosynthesis of [(18)F]FDG-RGD in activities >37 MBq in reasonable yield, rendering the direct use of clinical grade [(18)F]FDG for the routine clinical synthesis of (18)F-labelled peptides impossible. Using no-carrier-added (n.c.a.) [(18)F]FDG obtained via HPLC separation of [(18)F]FDGTUM from excess glucose, however, afforded [(18)F]FDG-RGD in yields of 56-93% (decay corrected) and activities up to 37 MBq. Suitable reaction conditions were 20 min at 120 degrees C and pH 2.5, and a peptide concentration of 5 mM. In a preliminary in vivo biodistribution study in M21 melanoma-bearing nude mice, [(18)F]FDG-RGD showed increased tumour accumulation compared to the "gold standard" [(18)F]galacto-RGD (2.18 vs 1.49 %iD/g, respectively, at 120 min after injection), but also slightly increased uptake in non-target organs, leading to comparable tumour/organ ratios for both compounds. These data demonstrate that chemoselective (18)F-labelling of aminooxy-functionalized peptides using n.c.a. [(18)F]FDG represents a radiofluorination/glycosylation strategy that allows preparation of (18)F-labelled peptides in high yield with suitable pharmacokinetics. As soon as the necessary n.c.a. preparation of [(18)F]FDG prior to reaction with the Aoa-peptide can be implemented in a fully automated [(18)F]FDG-synthesis, [(18)F]fluoroglucosylation of peptides may represent a promising alternative to

  10. O5.05FLUORESCEIN-GUIDED REMOVAL OF HIGH-GRADE GLIOMAS WITH A DEDICATED FILTER ON THE SURGICAL MICROSCOPE: PRELIMINARY RESULTS OF THE FLUOGLIO STUDY

    PubMed Central

    Acerbi, F.; Broggi, M.; Cavallo, C.; Anghileri, E.; Eoli, M.; Schiariti, M.; Corte, E. La; Pollo, B.; Boffano, C.; Ferroli, P.

    2014-01-01

    BACKGROUND: Fluorescein is a fluorescent tracer that can be used for many applications. It is able to accumulate in brain areas with blood-brain barrier disruption, and thus it can be considered an ideal dye for intraoperative visualization of high-grade gliomas (HGG). We report the preliminary results of a phase II trail (FLUOGLIO) on a new fluorescein-guided technique to remove HHG with a dedicated filter on the surgical microscope. METHODS: In September 2011 we started a prospective phase II-trial (FLUOGLIO) to evaluated safety and obtain initial indications about efficacy of fluorescein-guided surgery for HGG. Patients with suspected HGG amenable to complete resection of contrast-enhancing area were eligible to participate in this study. This report is based on the analysis of the short- and long-term results in 28 consecutive patients with HGG (age range 45-74 years), enrolled since September 2011. Fluorescein was intravenous (i.v.) injected after intubation (5-10 mg/Kg). Tumor was removed with microsurgical technique and fluorescence visualization by BLU400 or YELLOW560 filters on Pentero microscope (Carl Zeiss, Germany). The study was approved by our Ethical Committee and registered on the European Regulatory Authorities website (EudraCT No. 2011-002527-18). RESULTS: Median pre-operative tumor volume was 33.1 cm3 (2.4-87.8 cm3). We found no adverse reaction to fluorescein administration. Tumor was completely removed in 80% of the patients. Median follow-up was 10 months. 6 months Progression-free Survival (PFS) and median survival were respectively 71.4 % and 11 months. CONCLUSION: Our analysis suggested that fluorescein-guided technique with a dedicated filter on the surgical microscope is safe and allows high-rate of complete resection of contrast-enhanced tumor at the early post-operative MRI.

  11. Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of Indium-111-labeled Lactam Bridge-Cyclized α-MSH Peptides

    PubMed Central

    Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

    2011-01-01

    The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides. Methods Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. Results DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively. Conclusion The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic

  12. Spasmolytic, antimicrobial and cytotoxic activities of 5-phenylpentyl isothiocyanate, a new glucosinolate autolysis product from horseradish (Armoracia rusticana P. Gaertn., B. Mey. & Scherb., Brassicaceae).

    PubMed

    Dekić, Milan S; Radulović, Niko S; Stojanović, Nikola M; Randjelović, Pavle J; Stojanović-Radić, Zorica Z; Najman, Stevo; Stojanović, Sanja

    2017-10-01

    Detailed analyses of horseradish autolysates led to the identification of a new natural product, 5-phenylpentyl isothiocyanate (PhPeITC). The structural assignment was corroborated by synthesis, and the identity unequivocally established by spectral means. The occurrence of PhPeITC is the first direct proof of the existence of a 5-phenylpentyl glucosinolate in the aerial parts of this species as one of the possible "mustard oil" precursors. To verify its possible contribution to the horseradish functional food status, horseradish above- and underground autolysates, together with five ω-phenylalkyl isothiocyanates were tested for their spasmolytic, cytotoxic and antimicrobial activities. Specifically, the cytotoxic effect on Caco-2, HeLa (cancer) and MDCK (non-cancer) cell lines was established. Additionally, the five tested ITCs exerted significant spasmolytic activity (on rat distal colon), with PhPeITC being almost 100 times more potent than papaverine. A non-selective antimicrobial activity of all ITCs was revealed in the case of 6 bacterial and 2 fungal strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Peptides Labeled with Pyridinium Salts for Sensitive Detection and Sequencing by Electrospray Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Waliczek, Mateusz; Kijewska, Monika; Rudowska, Magdalena; Setner, Bartosz; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2016-11-01

    Mass spectrometric analysis of trace amounts of peptides may be problematic due to the insufficient ionization efficiency resulting in limited sensitivity. One of the possible ways to overcome this problem is the application of ionization enhancers. Herein we developed new ionization markers based on 2,4,6-triphenylpyridinium and 2,4,6-trimethylpyridinium salts. Using of inexpensive and commercially available pyrylium salt allows selective derivatization of primary amino groups, especially those sterically unhindered, such as ɛ-amino group of lysine. The 2,4,6-triphenylpyridinium modified peptides generate in MS/MS experiments an abundant protonated 2,4,6-triphenylpyridinium ion. This fragment is a promising reporter ion for the multiple reactions monitoring (MRM) analysis. In addition, the fixed positive charge of the pyridinium group enhances the ionization efficiency. Other advantages of the proposed ionization enhancers are the simplicity of derivatization of peptides and the possibility of convenient incorporation of isotopic labels into derivatized peptides.

  14. Comparison of /sup 125/I-labeled and /sup 14/C-Labeled peptides of the major outer membrane protein of Chlamydia Trachomatis Strain L2/434 separated by high-performance liquid chromatography

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Judd, R.C.; Caldwell, H.D.

    1985-01-01

    The objective of this study was to determine if in-gel chloramine-T radioiodination adequately labels OM proteins to allow for accurate and precise structural comparison of these molecules. Therefore, intrinsically /sup 14/C-amino acid labeled proteins and /sup 125/I-labeled proteins were cleaved with two endopeptidic reagents and the peptide fragments separated by HPLC. A comparison of retention times of the fragments, as determined by differential radiation counting, thus indicated whether /sup 125/Ilabeling identified of all the peptide peaks seen in the /sup 14/Clabeled proteins. Results demonstrated that radioiodination yields complete and accurate information about the primary structure of outer membrane proteins. Inmore » addition, it permits the use of extremely small amounts of protein allowing for method optimization and multiple separations to insure reproducibility.« less

  15. (99m)Tc-labeled SWL specific peptide for targeting EphA2 receptor.

    PubMed

    Liu, Yu; Lan, Xiaoli; Wu, Tao; Lang, Juntao; Jin, Xueyan; Sun, Xun; Wen, Qiong; An, Rui

    2014-07-01

    EphA2, one member of the Eph receptor family, is widely expressed in multiple aggressive cancers. SWL, a small peptide identified by phage display, has high binding affinity to EphA2, suggesting that it could be exploited for targeted molecular imaging. Therefore, a novel peptide-based probe, (99m)Tc-HYNIC-SWL, was developed and its potential to specifically target EphA2-positive tumors was investigated. The SWL peptide was labeled with hydrazinonicotinic acid (HYNIC), followed by (99m)Tc labeling. Immunofluorescence staining was carried out to detect the expression of EphA2 in A549 lung cancer cells and OCM-1 melanoma cells. Saturation binding experiments were performed by incubating A549 cells with increasing concentrations of radiolabeled peptide in vitro. To test the probe in vivo, nude mice bearing either A549 or OCM-1 derived tumors were established, injected with (99m)Tc-HYNIC-SWL, and subjected to SPECT imaging. Mice injected with excess unlabeled SWL were used as a specific control. Ex vivo γ-counting of dissected tissues from the mice was also performed to evaluate biodistribution. Immunofluorescence staining showed that A549 cells intensively expressed EphA2, while OCM-1 cells had little expression. (99m)Tc-HYNIC-SWL displayed high binding affinity with A549 cells (KD=2.6±0.7nM). From the SPECT images and the results of the biodistribution study, significantly higher uptake of the tracer was seen in A549 tumors (1.44±0.12 %ID/g) than in OCM-1 tumors (0.43±0.20 %ID/g) at 1h after injection. Pre-injection with excess unlabeled peptide in A549-bearing nude mice, significantly reduced tumor uptake of the radiolabeled probe (0.58±0.20 %ID/g) was seen. These data suggest that (99m)Tc-HYNIC-SWL specifically targets EphA2 in tumors. The expression of EphA2 can be noninvasively investigated using (99m)Tc-HYNIC-SWL by SPECT imaging. The in vitro and in vivo characteristics of (99m)Tc-HYNIC-SWL make it a promising probe for EphA2-positive tumor imaging

  16. In vivo fluorescence imaging of hepatocellular carcinoma xenograft using near-infrared labeled epidermal growth factor receptor (EGFR) peptide

    PubMed Central

    Li, Z.; Zhou, Q.; Zhou, J.; Duan, X.; Zhu, J.; Wang, T. D.

    2016-01-01

    Minimally-invasive surgery of hepatocellular carcinoma (HCC) can be limited by poor tumor visualization with white light. We demonstrate systemic administration of a Cy5.5-labeled peptide specific for epidermal growth factor receptor (EGFR) to target HCC in vivo in a mouse xenograft model. We attached a compact imaging module to the proximal end of a medical laparoscope to collect near-infrared fluorescence and reflectance images concurrently at 15 frames/sec. We measured a mean target-to-background ratio of 2.99 ± 0.22 from 13 surgically exposed subcutaneous human HCC tumors in vivo in 5 mice. This integrated imaging methodology is promising to guide laparoscopic resection of HCC. PMID:27699089

  17. Isoelectric focusing of proteins and peptides

    NASA Technical Reports Server (NTRS)

    Egen, N.

    1979-01-01

    Egg-white solution was chosen as the reference solution in order to assess the effects of operational parameters (voltage, flow rate, ampholine pH range and concentration, and protein concentration) of the RIEF apparatus on protein resolution. Topics of discussion include: (1) comparison of RIEF apparatus to conventional IEF techniques (column and PAG) with respect to resolution and throughput; (2) peptide and protein separation (AHF, Thymosin - Fraction 5, vasoactive peptide, L-asparaginase and ACP); and (3) detection of peptides - dansyl derivatives of amino acids and peptides, post-focusing fluorescent labeling of amino acids, peptides and proteins, and ampholine extraction from focused gels.

  18. Peptide affinity labels for thrombin and other trypsin-like proteases

    DOEpatents

    Shaw, E.N.; Kettner, C.A.

    1982-03-09

    A peptide affinity label is disclosed of the formula (I): as given in the patent wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C[sub 1]--C[sub 4] alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C[sub 1]--C[sub 6] acyl, and Q--(A)--[sub n], wherein Q = hydrogen, aroyl, or C[sub 1]--C[sub 6] acyl, n = 1--10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereofcontaining, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH[sub 2]--, --CH[sub 2]--CH[sub 2]--, --CH[sub 2]--CH[sub 2]--CH[sub 2]--, --CH[double bond]CH-- and --CH(OH)--CH[sub 2]. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent. 2 figs.

  19. Peptide affinity labels for thrombin and other trypsin-like proteases

    DOEpatents

    Shaw, Elliott N.; Kettner, Charles A.

    1982-03-09

    A peptide affinity label of the formula (I): ##STR1## wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C.sub.1 -C.sub.4 alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C.sub.1 -C.sub.6 acyl, and Q--(A)--.sub.n, wherein Q=hydrogen, aroyl, or C.sub.1 -C.sub.6 acyl, n=1-10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereof-containing, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH.sub.2 --, --CH.sub.2 --CH.sub.2 --,--CH.sub.2 --CH.sub.2 --CH.sub.2 --, --CH.dbd.CH-- and --CH(OH)--CH.sub.2. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent.

  20. Targeting PCNA Phosphorylation in Breast Cancer

    DTIC Science & Technology

    2012-04-01

    inhibitor and dyes. These basic peptides and polycationic peptoids were labeled with fluorescein and used to assess the effect of structure on cellular... substitution of a peptoid conjugate that was synthesized during the project period. Additional types of analogs were prepared as well each providing...Broceta, A., Diezmann, F., Ou-Yang, C. Y., Fara, M. A. & Bradley, M. Synthesis, penetrability and intracellular targeting of fluorescein -tagged

  1. Nerve growth factor injected into the gastric ulcer base incorporates into endothelial, neuronal, glial and epithelial cells: implications for angiogenesis, mucosal regeneration and ulcer healing.

    PubMed

    Tanigawa, T; Ahluwalia, A; Watanabe, T; Arakawa, T; Tarnawski, A S

    2015-08-01

    A previous study has demonstrated that locally administered growth factors such as epidermal growth factor, basic fibroblast growth factor and hepatocyte growth factor can accelerate healing of experimental gastric ulcers in rats. That study indicates that locally administered growth factors can exert potent biological effects resulting in enhanced gastric ulcers healing. However, the fate of injected growth factors, their retention and localization to specific cellular compartments have not been examined. In our preliminary study, we demonstrated that local injection of nerve growth factor to the base of experimental gastric ulcers dramatically accelerates ulcer healing, increases angiogenesis - new blood vessel formation, and improves the quality of vascular and epithelial regeneration. Before embarking on larger, definitive and time sequence studies, we wished to determine whether locally injected nerve growth factor is retained in gastric ulcer's tissues and taken up by specific cells during gastric ulcer healing. Gastric ulcers were induced in anesthetized rats by local application of acetic acid using standard methods; and, 60 min later fluorescein isothiocyanate-labeled nerve growth factor was injected locally to the ulcer base. Rats were euthanized 2, 5 and 10 days later. Gastric specimens were obtained and processed for histology. Unstained paraffin sections were examined under a fluorescence microscope, and the incorporation of fluorescein isothiocyanate-labeled nerve growth factor into various gastric tissue cells was determined and quantified. In addition, we performed immunostaining for S100β protein that is expressed in neural components. Five and ten days after ulcer induction labeled nerve growth factor (injected to the gastric ulcer base) was incorporated into endothelial cells of blood vessels, neuronal, glial and epithelial cells, myofibroblasts and muscle cells. This study demonstrates for the first time that during gastric ulcer healing

  2. Optimal time following fluorescein instillation to evaluate rigid gas permeable contact lens fit.

    PubMed

    Wolffsohn, James S; Tharoo, Ali; Lakhlani, Nikita

    2015-04-01

    To examine the optimum time at which fluorescein patterns of gas permeable lenses (GPs) should be evaluated. Aligned, 0.2mm steep and 0.2mm flat GPs were fitted to 17 patients (aged 20.6 ± 1.1 years, 10 male). Fluorescein was applied to their upper temporal bulbar conjunctiva with a moistened fluorescein strip. Digital slit lamp images (CSO, Italy) at 10× magnification of the fluorescein pattern viewed with blue light through a yellow filter were captured every 15s. Fluorescein intensity in central, mid peripheral and edge regions of the superior, inferior, temporal and nasal quadrants of the lens were graded subjectively using a +2 to -2 scale and using ImageJ software on the simultaneously captured images. Subjectively graded and objectively image analysed fluorescein intensity changed with time (p < 0.001), lens region (centre, mid-periphery and edge: p < 0.05) and there was interaction between lens region with lens fit (p < 0.001). For edge band width, there was a significant effect of time (F = 118.503, p < 0.001) and lens fit (F = 5.1249, p = 0.012). The expected alignment, flat and steep fitting patterns could be seen from approximately after 30 to 180 s subjectively and 15 to 105 s in captured images. Although the stability of fluorescein intensity can start to decline in as little as 45 s post fluorescein instillation, the diagnostic pattern of alignment, steep or flat fit is seen in each meridian by subjective observation from about 30s to 3 min indicating this is the most appropriate time window to evaluate GP lenses in clinical practice. Copyright © 2014 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

  3. Retcam fluorescein gonioangiography: a new modality for early detection of angle neovascularization in diabetic retinopathy.

    PubMed

    Azad, Rajvardhan; Arora, Tarun; Sihota, Ramanjit; Chandra, Parijat; Mahajan, Deepankur; Sain, Siddarth; Sharma, Yograj

    2013-10-01

    To evaluate the role of Retcam fluorescein gonioangiography in detecting neovascularization of the angle and correlate the same with gonioscopy in diabetic retinopathy. One hundred and fifty eyes of 150 patients (25 each of mild, moderate, severe, very severe nonproliferative diabetic retinopathy (NPDR) proliferative diabetic retinopathy (PDR); and PDR with high-risk characteristics) were recruited. They underwent complete ocular examination including applanation tonometry, gonioscopy, Retcam fluorescein gonioangiography, and fundus fluorescein angiography. Using Retcam fluorescein gonioangiography, of 150 eyes neovascularization of the angle was detected in 37 eyes (24.66%) compared with 22 eyes (14.66%) on gonioscopy (P = 0.04). Small newly formed vessels were evident only with Retcam fluorescein gonioangiography. In 10 of 50 patients (20%) with severe/very severe NPDR, angle neovascularization was appreciable on Retcam fluorescein angiography compared with 5 patients (10%) on gonioscopy. Similarly, 25 of 50 patients (50%) with PDR/PDR with high-risk characteristics had neovascularization of the angle on Retcam gonioangiography compared with 17 (34%) on gonioscopy. Retcam fluorescein gonioangiography is a novel technique for early detection of angle neovascularization in diabetic retinopathy and hence preventing progression to neovascular glaucoma. The objective nature of this test helps in precise decision making compared with gonioscopy for early intervention especially in cases of pre-PDR.

  4. Organic Isothiocyanates: Dietary Modulators of Doxorubicin Resistance in Breast Cancer

    DTIC Science & Technology

    2004-06-01

    al. (30) have dem- isothiocyanates: chemistry and mechanisms. Cancer Res. 54: onstrated the inhibition of renal clearance of colchicine by 1976s-1981s... Chemistry , Uni- possibility of flip-flop kinetics (absorption being slower versity at Buffalo) for his assistance in internal standard than elimination) in...Anticarcinogenic activities of organic sult fi’om interindividual variability and limited subject isothiocyanates: chemistry and mechanisms, Cancer Res. 54 (1994

  5. Fibroblast Growth Factor-Peptide Improves Barrier Function and Proliferation in Human Keratinocytes After Radiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang Kunzhong; Tian Yeping; Yin Liangjie

    2011-09-01

    Purpose: Epidermal keratinocytes, which can be severely damaged after ionizing radiation (IR), are rapid turnover cells that function as a barrier, protecting the host from pathogenic invasion and fluid loss. We tested fibroblast growth factor-peptide (FGF-P), a small peptide derived from the receptor-binding domain of FGF-2, as a potential mitigator of radiation effects via proliferation and the barrier function of keratinocytes. Methods and Materials: Keratinocytes isolated from neonatal foreskin were grown on transwells. After being exposed to 0, 5, or 10 Gy IR, the cells were treated with a vehicle or FGF-P. The permeability of IR cells was assessed bymore » using transepithelial electrical resistance (TEER) and a paracellular tracer flux of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) with Ussing chambers. The cell proliferation was measured with yellow tetrazolium salt (MTT) and tritiated thymidine ([{sup 3}H]-TdR) assays. The phosphorylation of extracellular signal-regulated kinases (ERK) was measured in an enzyme-linked immunosorbent (ELISA)-like assay, and the proteins related to tight junctions (TJ) and adherens junctions (AJ) were examined with Western blotting. We used a mouse model to assess the ability of FGF-P to promote the healing of skin {beta} burns created with a strontium applicator. Results: We found (1) FGF-P reduced the permeability of irradiated keratinocytes, as evidenced by increased TEER and decreased diffusion of FITC-BSA, both associated with the regulation of different proteins and levels of TJ and AJ; and (2) FGF-P enhanced the proliferation of irradiated keratinocytes, as evidenced by increased MTT activity and [{sup 3}H]-TdR incorporation, which was associated with activation of the ERK pathway; and (3) FGF-P promoted the healing of skin {beta} burns. Conclusions: FGF-P enhances the barrier function, including up-regulation of TJ proteins, increases proliferation of human keratinocytes, and accelerates

  6. Site-specific orientation of an α-helical peptide ovispirin-1 from isotope-labeled SFG spectroscopy.

    PubMed

    Ding, Bei; Laaser, Jennifer E; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T; Chen, Zhan

    2013-11-27

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C═O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution.

  7. Site-specific Orientation of an α-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy

    PubMed Central

    Ding, Bei; Laaser, Jennifer E.; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T.; Chen, Zhan

    2013-01-01

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single isotope labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138 degrees from the surface normal, and the transition dipole of the isotope labeled C=O group is tilted at 23 degrees from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrated that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

  8. Mechanisms of Action of Isothiocyanates in Cancer Chemoprevention: An Update

    PubMed Central

    Navarro, Sandi L.; Li, Fei; Lampe, Johanna W.

    2011-01-01

    Isothiocyanates, derived from glucosinolates, are thought to be responsible for the chemoprotective actions conferred by higher cruciferous vegetable intake. Evidence suggests that isothiocyanates exert their effects through a variety of distinct but interconnected signaling pathways important for inhibiting carcinogenesis, including those involved in detoxification, inflammation, apoptosis, and cell cycle and epigenetic regulation, among others. This article provides an update on the latest research on isothiocyanates and these mechanisms, and points out remaining gaps in our understanding of these events. Given the variety of ITC produced from glucosinolates, and the diverse pathways on which these compounds act, a systems biology approach, in vivo, may help to better characterize their integrated role in cancer prevention. In addition, the effects of dose, duration of exposure, and specificity of different ITC should be considered. PMID:21935537

  9. Dual fluorescence/contactless conductivity detection for microfluidic chip.

    PubMed

    Liu, Cui; Mo, Yun-yan; Chen, Zuan-guang; Li, Xiang; Li, Ou-lian; Zhou, Xie

    2008-07-28

    A new dual detection system for microchip is reported. Both fluorescence detector (FD) and contactless conductivity detector (CCD) were combined together and integrated on a microfluidic chip. They shared a common detection position and responded simultaneously. A blue light-emitting diode was used as excitation source and a small planar photodiode was used to collect the emitted fluorescence in fluorescence detection, which made the device more compact and portable. The coupling of the fluorescence and contactless conductivity modes at the same position of a single separation channel enhanced the detection characterization of sample and offered simultaneous detection information of both fluorescent and charged specimen. The detection conditions of the system were optimized. K(+), Na(+), fluorescein sodium, fluorescein isothiocyanate (FITC) and FITC-labeled amino acids were used to evaluate the performance of the dual detection system. The limits of detection (LOD) of FD for fluorescein Na(+), FITC, FITC-labeled arginine (Arg), glycine (Gly) and phenylalanine (Phe) were 0.02micromolL(-1), 0.05micromolL(-1), 0.16micromolL(-1), 0.15micromolL(-1), 0.12micromolL(-1) respectively, and the limits of detection (LOD) of CCD achieved 0.58micromolL(-1) and 0.39micromolL(-1) for K(+) and Na(+) respectively.

  10. Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

    NASA Astrophysics Data System (ADS)

    Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

    2009-02-01

    Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

  11. Development of a (99m)Tc-labeled lactam bridge-cyclized alpha-MSH derivative peptide as a possible single photon imaging agent for melanoma tumors.

    PubMed

    Shamshirian, Danial; Erfani, Mostafa; Beiki, Davood; Fallahi, Babak; Shafiei, Mohammad

    2015-10-01

    Melanocortin-1 (MC1) receptor is an attractive melanoma-specific target which has been used for melanoma imaging and therapy. In this work, a new lactam bridge α-MSH analog was labeled with (99m)Tc via HYNIC and EDDA/tricine as coligands including gamma aminobutyric acid (GABA) as a three carbon chain spacer between HYNIC and the N-terminus of the cyclic peptide. Also, stability in human serum, receptor bound internalization, in vivo tumor uptake, and tissue biodistribution were thoroughly investigated. HYNIC-GABA-Nle-CycMSHhept was synthesized using a standard Fmoc strategy. Labeling was performed at 95 °C and analysis involved instant thin layer chromatography and high performance liquid chromatography methods. The receptor bound internalization rate was studied in MC1 receptor expressing B16/F10 cells. Biodistribution of radiopeptide was studied in nude mice bearing B16/F10 tumor. Labeling yield of >98 % (n = 3) was obtained corresponding to a specific activity of 81 MBq/nmol. Peptide conjugate showed efficient stability in the presence of human serum. The radioligand showed specific internalization into B16/F10 cells (12.45 ± 1.1 % at 4 h). In biodistribution studies, a receptor-specific uptake was observed in MC1 receptor-positive organs so that after 2 h the uptake in mouse tumor was 5.10 ± 0.08 % ID/g, while low accumulation in the kidney uptake was observed (4.58 ± 0.68 % ID/g at 2 h after injection). The obtained results show that the presented new designed labeled peptide conjugate may be a suitable candidate for diagnosis of malignant tumors.

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

    PubMed

    Bache, Nicolai; Rand, Kasper D; Roepstorff, Peter; Ploug, Michael; Jørgensen, Thomas J D

    2008-12-01

    We have previously shown that peptide amide hydrogens undergo extensive intramolecular migration (i.e., complete hydrogen scrambling) upon collisional activation of protonated peptides (Jørgensen et al. J. Am. Chem. Soc. 2005, 127, 2785-2793). The occurrence of hydrogen scrambling enforces severe limitations on the application of gas-phase fragmentation as a convenient method to obtain information about the site-specific deuterium uptake for proteins and peptides in solution. To investigate whether deprotonated peptides exhibit a lower level of scrambling relative to their protonated counterparts, we 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/(2)H) have migrated extensively in the anionic peptide, thereby erasing the original regioselective deuteration pattern obtained in solution.

  13. Characterization, in vitro cytotoxicity assessment, and in vivo visualization of multimodal, RITC-labeled, silica-coated magnetic nanoparticles for labeling human cord blood-derived mesenchymal stem cells.

    PubMed

    Park, Ki-Soo; Tae, Jinsung; Choi, Bongkum; Kim, Young-Seok; Moon, Cheol; Kim, Sa-Hyun; Lee, Han-Sin; Kim, Jinhyun; Kim, Junsung; Park, Jaeberm; Lee, Jung-Hee; Lee, Jong Eun; Joh, Jae-Won; Kim, Sungjoo

    2010-04-01

    Live imaging is a powerful technique that can be used to characterize the fate and location of stem cells in animal models. Here we investigated the characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, MNPs@SiO2(RITC). We also conducted various in vivo-uptake tests with nanoparticle-labeled human MSCs. MNPs@SiO2(RITC) showed photostability against ultraviolet light exposure and were nontoxic to human MSCs, based on the MTT, apoptosis, and cell cycle arrest assays. In addition, MNPs@SiO2(RITC) did not affect the surface phenotype or morphology of human MSCs. We also demonstrated that MNPs@SiO2(RITC) have stable retention properties in MSCs in vitro. Furthermore, using optical and magnetic resonance imaging, we successfully detected a visible signal from labeled human MSCs that were transplanted into NOD.CB17-Prkdc(SCID) (NOD-SCID) mice. These results demonstrate that MNPs@SiO2(RITC) are biocompatible and useful tools for human MSC labeling and bioimaging. The characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, RITC were investigated in this study. RITC showed photostability against ultraviolet light exposure and was nontoxic to human MSCs. Using both optical and magnetic resonance imaging, successful detection of signal from labeled human MSCs transplanted into mice is demonstrated. Copyright 2010 Elsevier Inc. All rights reserved.

  14. 27 CFR 5.32 - Mandatory label information.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Distilled Spirits § 5.32 Mandatory label information. There shall be stated: (a) On the brand label: (1) Brand name. (2) Class and type, in accordance with § 5.35. (3) Alcoholic content, in accordance with § 5... prescribed in § 5.47, net contents in accordance with § 5.38(b) or § 5.38a(b)(2). (b) On the brand label or...

  15. Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae)

    PubMed Central

    López-Abarrategui, Carlos; McBeth, Christine; Mandal, Santi M.; Sun, Zhenyu J.; Heffron, Gregory; Alba-Menéndez, Annia; Migliolo, Ludovico; Reyes-Acosta, Osvaldo; García-Villarino, Mónica; Nolasco, Diego O.; Falcão, Rosana; Cherobim, Mariana D.; Dias, Simoni C.; Brandt, Wolfgang; Wessjohann, Ludger; Starnbach, Michael; Franco, Octavio L.; Otero-González, Anselmo J.

    2015-01-01

    Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability.—López-Abarrategui, C., McBeth, C., Mandal, S. M., Sun, Z. J., Heffron, G., Alba-Menéndez, A., Migliolo, L., Reyes-Acosta, O., García-Villarino, M., Nolasco, D. O., Falcão, R., Cherobim, M. D., Dias, S. C., Brandt, W., Wessjohann, L., Starnbach, M., Franco, O. L., Otero-González, A. J. Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae). PMID:25921828

  16. Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease.

    PubMed

    Di Pardo, Alba; Castaldo, Salvatore; Capocci, Luca; Amico, Enrico; Vittorio, Maglione

    2017-11-08

    Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies. Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy. Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

  17. Competitive immunoassay of phenobarbital by microchip electrophoresis with laser induced fluorescence detection.

    PubMed

    Huang, Yong; Zhao, Shulin; Shi, Ming; Liu, Jinwen; Liang, Hong

    2011-05-23

    A microchip electrophoresis method with laser induced fluorescence detection was developed for the immunoassay of phenobarbital. The detection was based on the competitive immunoreaction between analyte phenobarbital and fluorescein isothiocyanate (FITC) labeled phenobarbital with a limited amount of antibody. The assay was developed by varying the borate concentration, buffer pH, separation voltage, and incubation time. A running buffer system containing 35 mM borate and 15 mM sodium dodecyl sulfate (pH 9.5), and 2800 V separation voltage provided analysis conditions for a high-resolution, sensitive, and repeatable assay of phenobarbital. Free FITC-labeled phenobarbital and immunocomplex were separated within 30s. The calibration curve for phenobarbital had a detection limit of 3.4 nM and a range of 8.6-860.0 nM. The assay could be used to determine the phenobarbital plasma concentration in clinical plasma sample. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Quantification of FITC-labelled probiotic Lactobacillus salivarius DSPV 001P during gastrointestinal transit in broilers.

    PubMed

    Blajman, J E; Astesana, D M; Zimmermann, J A; Rossler, E; Scharpen, A Romero; Berisvil, A P; Zbrun, M V; Soto, L P; Rosmini, M R; Frizzo, L S

    2017-02-07

    The knowledge related to the fate of probiotics in the complex environment of the intestinal microbiota in broilers is just beginning to be elucidated; however, it is not yet well understood. A good method to investigate the mechanisms by which probiotics mediate their effects is to mark probiotic bacteria and trace them. The aim of this research was to develop a new method to estimate in vivo fluorescein isothiocyanate (FITC)-labelled Lactobacillus salivarius DSPV 001P counts during passage through the gastrointestinal tract (GIT) of broilers. Forty-five, 1 d old Cobb broilers were used in this trial. Programmed necropsies were performed 30 min, 6 h, and 12 h after the administration of the probiotic bacterium, and samples of liver, crop, duodenum, caecum, and bursa of fabricius were collected. To determine the spatial and temporal transit of L. salivarius DSPV 001P in broilers, the number of bacteria as well as its respective fluorescent signal produced by FITC were measured. In order to observe the relationship between the variables, a logistic regression analysis was applied. The amount of fluorescence could be used as an indicator of fluorescent probiotic bacteria in the crop and duodenum 30 min after probiotic bacterium supplementation. In addition, the fluorescent signal could be used to estimate bacterial counts in caecum 6 and 12 h after L. salivarius DSPV 001P administration. To the best of our knowledge, this research is the first in vivo trial to employ the bacterial FITC-labelling technique in order to enumerate probiotic bacteria during gastrointestinal transit in broilers.

  19. [Endoscopic endonasal detection of cerebrospinal fluid leakage with topical fluorescein].

    PubMed

    Sato, Taku; Kishida, Yugo; Watanabe, Tadashi; Tani, Akiko; Tada, Yasuhiro; Tamura, Takamitsu; Ichikawa, Masahiro; Sakuma, Jun; Omori, Koichi; Saito, Kiyoshi

    2013-08-01

    We evaluated the effectiveness of intraoperative topical application of fluorescein to detect the leakage point of cerebrospinal fluid(CSF)rhinorrhea. Three patients with CSF rhinorrhea were treated with an endoscopic endonasal technique. Ten percent fluorescein was topically used for intraoperative localization of the leak site. A change of the fluorescein color from brown to green due to dilation of CSF were recognized as evidence of CSF rhinorrhea. We repeated the procedure to detect any small defects. All CSF rhinorrheas were successfully repaired by this endoscopic endonasal approach. Topical application of fluorescein is simple and sensitive for identifying intraoperative CSF rhinorrhea.

  20. Effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized alpha-MSH peptides.

    PubMed

    Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

    2011-04-01

    The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of (111)In-labeled lactam bridge-cyclized DOTA-[X]-CycMSH(hex) {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH(2); X = GGNle, GENle, or NleGE; GG = -Gly-Gly- and GE = -Gly-Glu-} peptides. Three novel peptides (DOTA-GGNle-CycMSH(hex), DOTA-GENle-CycMSH(hex), and DOTA-NleGE-CycMSH(hex)) were designed and synthesized. The melanocortin-1 (MC1) receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of (111)In-DOTA-GGNle-CycMSH(hex) and (111)In-DOTA-GENle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. DOTA-GGNle-CycMSH(hex) and DOTA-GENle-CycMSH(hex) displayed 2.1 and 11.5 nM MC1 receptor-binding affinities, whereas DOTA-NleGE-CycMSH(hex) showed 873.4 nM MC1 receptor-binding affinity. The introduction of the -GG- linker maintained high melanoma uptake while decreasing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex). The tumor uptake of (111)In-DOTA-GGNle-CycMSH(hex) was 19.05 ± 5.04 and 18.6 ± 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. (111)In-DOTA-GGNle-CycMSH(hex) exhibited 28%, 32%, and 42% less kidney uptake than (111)In-DOTA-Nle-CycMSH(hex) we reported previously, and 61%, 65%, and 68% less liver uptake than (111)In-DOTA-Nle-CycMSH(hex) at 2, 4, and 24 h after injection, respectively. The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the (111)In-labeled lactam bridge-cyclized α-melanocyte-stimulating hormone peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex), highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide

  1. Validity of fluorexon disodium versus sodium fluorescein for use in Goldmann tonometry.

    PubMed

    Ng, Loretta T; Tong, Judy W; De Land, Paul N

    2006-07-01

    To evaluate the safety, validity, and comfort of 0.35% fluorexon disodium and 0.4% benoxinate (Flura-Safe) compared with the gold standard of 0.25% sodium fluorescein and 0.4% benoxinate for Goldmann applanation tonometry (GAT). This was a double-masked, randomized, crossover clinical trial. Subjects received either the standard or study formulation for GAT on visit 1 and the other formulation 1 week later. At each visit, tonometer mire quality, adequacy of fluorescence, ease of intraocular pressure (IOP) measurements, the IOP value, and anesthetizing efficacy of the formulation were assessed. Subjects graded general comfort, soreness and irritation, and burning and stinging of each formulation at 1 and 5 minutes after drop instillation. Sixty-seven subjects completed the study. The mean IOP was 13.9 +/- 2.7 with fluorexon and 13.9 +/- 2.8 mm Hg with fluorescein OD and 14.0 +/- 2.8 with fluorexon and 13.9 +/- 2.5 mm Hg with fluorescein OS. The measurements with the 2 formulations were highly correlated for OD and OS, and the differences between the 2 measurements were not clinically significant. There was also no significant difference between the 2 drops in mire clarity, adequacy of fluorescence, or corneal anesthesia. However, fluorexon was statistically more comfortable (P = 0.039) and caused less stinging and burning (P = 0.014) at 1 minute versus the fluorescein formulation. Not only was the new fluorexon product accurate and effective in GAT, it was also statistically more comfortable and had a less stinging and burning effect at 1 minute after drop instillation than the traditional fluorescein formulation. Because fluorexon is less likely to stain soft contact lenses, this may be the dye-anesthetic formulation of choice for practices that routinely perform GAT.

  2. Transcriptomic alterations in human prostate cancer cell LNCaP tumor xenograft modulated by dietary phenethyl isothiocyanate

    USDA-ARS?s Scientific Manuscript database

    Temporal growth of tumor xenografts in mice on a control diet was compared to mice supplemented daily with 3 µmol/g of the cancer preventive compound phenethyl isothiocyanate. Phenethyl isothiocyanate decreased the rate of tumor growth. The effects of phenethyl isothiocyanate on tumor growth were ex...

  3. Urease from Helicobacter pylori is inactivated by sulforaphane and other isothiocyanates.

    PubMed

    Fahey, Jed W; Stephenson, Katherine K; Wade, Kristina L; Talalay, Paul

    2013-05-24

    Infections by Helicobacter pylori are very common, causing gastroduodenal inflammation including peptic ulcers, and increasing the risk of gastric neoplasia. The isothiocyanate (ITC) sulforaphane [SF; 1-isothiocyanato-4-(methylsulfinyl)butane] derived from edible crucifers such as broccoli is potently bactericidal against Helicobacter, including antibiotic-resistant strains, suggesting a possible dietary therapy. Gastric H. pylori infections express high urease activity which generates ammonia, neutralizes gastric acidity, and promotes inflammation. The finding that SF inhibits (inactivates) urease (jack bean and Helicobacter) raised the issue of whether these properties might be functionally related. The rates of inactivation of urease activity depend on enzyme and SF concentrations and show first order kinetics. Treatment with SF results in time-dependent increases in the ultraviolet absorption of partially purified Helicobacter urease in the 260-320 nm region. This provides direct spectroscopic evidence for the formation of dithiocarbamates between the ITC group of SF and cysteine thiols of urease. The potencies of inactivation of Helicobacter urease by isothiocyanates structurally related to SF were surprisingly variable. Natural isothiocyanates closely related to SF, previously shown to be bactericidal (berteroin, hirsutin, phenethyl isothiocyanate, alyssin, and erucin), did not inactivate urease activity. Furthermore, SF is bactericidal against both urease positive and negative H. pylori strains. In contrast, some isothiocyanates such as benzoyl-ITC, are very potent urease inactivators, but are not bactericidal. The bactericidal effects of SF and other ITC against Helicobacter are therefore not obligatorily linked to urease inactivation, but may reduce the inflammatory component of Helicobacter infections. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Urease from Helicobacter pylori is inactivated by sulforaphane and other isothiocyanates

    PubMed Central

    Fahey, Jed W.; Stephenson, Katherine K.; Wade, Kristina L.; Talalay, Paul

    2013-01-01

    Infections by Helicobacter pylori are very common, causing gastroduodenal inflammation including peptic ulcers, and increasing the risk of gastric neoplasia. The isothiocyanate (ITC) sulforaphane [SF; 1-isothiocyanato-4-(methylsulfinyl)butane] derived from edible crucifers such as broccoli is potently bactericidal against Helicobacter, including antibiotic-resistant strains, suggesting a possible dietary therapy. Gastric H. pylori infections express high urease activity which generates ammonia, neutralizes gastric acidity, and promotes inflammation. The finding that SF inhibits (inactivates) urease (jack bean and Helicobacter) raised the issue of whether these properties might be functionally related. The rates of inactivation of urease activity depend on enzyme and SF concentrations and show first order kinetics. Treatment with SF results in time-dependent increases in the ultraviolet absorption of partially purified Helicobacter urease in the 280–340 nm region. This provides direct spectroscopic evidence for the formation of dithiocarbamates between the ITC group of SF and cysteine thiols of urease. The potencies of inactivation of Helicobacter urease by isothiocyanates structurally related to SF were surprisingly variable. Natural isothiocyanates closely related to SF, previously shown to be bactericidal (berteroin, hirsutin, phenethyl isothiocyanate, alyssin, and erucin), did not inactivate urease activity. Furthermore, SF is bactericidal against both urease positive and negative H. pylori strains. In contrast, some isothiocyanates such as benzoyl-ITC, are very potent urease inactivators, but are not bactericidal. The bactericidal effects of SF and other ITC against Helicobacter are therefore not obligatorily linked to urease inactivation, but may reduce the inflammatory component of Helicobacter infections. PMID:23583386

  5. Antibody-based bacterial toxin detection

    NASA Astrophysics Data System (ADS)

    Menking, Darrell E.; Heitz, Jonathon M.; Anis, Nabil A.; Thompson, Roy G.

    1994-03-01

    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a one step assay, antibodies against Cholera toxin or Staphylococcus Enterotoxin B were noncovalently immobilized on quartz fibers and probed with fluorescein-isothiocyanate (FITC)-labeled toxins. In the two-step assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified antitoxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or antitoxin IgG in a dose-dependent manner and the detection of the toxins was in the nanomolar range.

  6. Influence of ionization states of antigen on anti-fluorescein antibodies

    NASA Astrophysics Data System (ADS)

    Fukunishi, Hiroaki

    2012-10-01

    Ratios of anion and di-anion states of fluorescein (FLU(-1) and FLU(-2)) are 21.2% and 78.8%, respectively, in the neutral pH. We investigated the influence of ionization states of antigen on anti-fluorescein antibodies. For this purpose, steered molecular dynamics (SMD) simulations were performed. Potential of mean forces (PMF) based on Jarzynski equality showed that wild-type (4-4-20) more strongly binds to FLU(-1) than FLU(-2), whereas its femtomolar-affinity mutant (4M5.3) more strongly binds to FLU(-2) than FLU(-1). It was speculated that the environment or the process of in vivo antibody production had been different from those of the protein engineering.

  7. Alleviation by garlic of antitumor drug-induced damage to the intestine.

    PubMed

    Horie, T; Awazu, S; Itakura, Y; Fuwa, T

    2001-03-01

    Antitumour drugs such as methotrexate (MTX) and 5-fluorouracil (5-FU) induce intestinal damage. This is a serious side effect of cancer chemotherapy. The present studies examined whether or not aged garlic extract (AGE) protects against damage from these antitumor drugs. Both drugs were administered orally for 4 or 5 d to rats fed a standard laboratory diet with and without 2% AGE. The small intestinal absorption of the poorly absorbable compound, fluorescein isothiocyanate--labeled dextran (FD-4; average molecular weight, 4400) was used to evaluate the damage to the intestine using the in vitro everted intestine technique and the in situ intestinal loop technique. FD-4 absorption increased in the antitumour drug-treated rats fed the diet without garlic. Interestingly, FD-4 absorption was depressed in rats fed the diet containing AGE. These results suggest that AGE may protect the small intestine of rats from antitumour drug-induced damage.

  8. 27 CFR 5.32 - Mandatory label information.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Mandatory label information. 5.32 Section 5.32 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY ALCOHOL LABELING AND ADVERTISING OF DISTILLED SPIRITS Labeling Requirements for...

  9. Evaluating Kinase ATP Uptake and Tyrosine Phosphorylation using Multiplexed Quantification of Chemically Labeled and Post-Translationally Modified Peptides

    PubMed Central

    Fang, Bin; Hoffman, Melissa A.; Mirza, Abu-Sayeef; Mishall, Katie M.; Li, Jiannong; Peterman, Scott M.; Smalley, Keiran S. M.; Shain, Kenneth H.; Weinberger, Paul M.; Wu, Jie; Rix, Uwe; Haura, Eric B.; Koomen, John M.

    2015-01-01

    Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease. PMID:25782629

  10. Enhanced Cellular Uptake of Short Polyarginine Peptides through Fatty Acylation and Cyclization

    PubMed Central

    2015-01-01

    Many of the reported arginine-rich cell-penetrating peptides (CPPs) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties. Herein, we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines, namely, R5 and R6. We further explored the effect of acylation with long chain fatty acids (i.e., octanoic acid, dodecanoic acid, and hexadecanoic acid) and cyclization on the cell penetrating properties of the peptides. The fluorescence-labeled acylated cyclic peptide dodecanoyl-[R5] and linear peptide dodecanoyl-(R5) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein, respectively. The mechanism of the peptide internalization into cells was found to be energy-dependent endocytosis. Dodecanoyl-[R5] and dodecanoyl-[R6] enhanced the intracellular uptake of a fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F′-GpYEEI) in human ovarian cancer cells (SK-OV-3) by 3.4-fold and 5.5-fold, respectively, as shown by flow cytometry. The cellular uptake of F′-GpYEEI in the presence of hexadecanoyl-[R5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[R5] and dodecanoyl-[R5], respectively. Dodecanoyl-[R5] enhanced the cellular uptake of the phosphopeptide by 1.4–2.5-fold higher than the corresponding linear peptide dodecanoyl-(R5) and those of representative CPPs, such as hepta-arginine (CR7) and TAT peptide. These results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine-containing peptides can improve their cell-penetrating property, possibly through efficient interaction of rigid positively charged R and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids. PMID:24978295

  11. Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

    PubMed

    Keiderling, Timothy A

    2017-12-01

    Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra. © 2017 Wiley Periodicals, Inc.

  12. Biochemical characterization and anti-inflammatory properties of an isothiocyanate-enriched moringa (Moringa oleifera) seed extract.

    PubMed

    Jaja-Chimedza, Asha; Graf, Brittany L; Simmler, Charlotte; Kim, Youjin; Kuhn, Peter; Pauli, Guido F; Raskin, Ilya

    2017-01-01

    Moringa oleifera Lam. is a tropical plant, used for centuries as food and traditional medicine. The aim of this study was to develop, validate and biochemically characterize an isothiocyanate-enriched moringa seed extract (MSE), and to compare the anti-inflammatory effects of MSE-containing moringa isothiocyanate-1 (MIC-1) with a curcuminoid-enriched turmeric extract (CTE), and a material further enriched in its primary phytochemical, curcumin (curcumin-enriched material; CEM). MSE was prepared by incubating ground moringa seeds with water to allow myrosinase-catalyzed enzymatic formation of bioactive MIC-1, the predominant isothiocyanate in moringa seeds. Optimization of the extraction process yielded an extract of 38.9% MIC-1. Phytochemical analysis of MSE revealed the presence of acetylated isothiocyanates, phenolic glycosides unique to moringa, flavonoids, fats and fatty acids, proteins and carbohydrates. MSE showed a reduction in the carrageenan-induced rat paw edema (33% at 500 mg/kg MIC-1) comparable to aspirin (27% at 300 mg/kg), whereas CTE did not have any significant effect. In vitro, MIC-1 at 1 μM significantly reduced the production of nitric oxide (NO) and at 5 μM, the gene expression of LPS-inducible nitric oxide synthase (iNOS) and interleukins 1β and 6 (IL-1β and IL-6), whereas CEM did not show any significant activity at all concentrations tested. MIC-1 (10μM) was also more effective at upregulating the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) target genes NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase pi 1 (GSTP1), and heme oxygenase 1 (HO1) than the CEM. Thus, in contrast to CTE and CEM, MSE and its major isothiocyanate MIC-1 displayed strong anti-inflammatory and antioxidant properties in vivo and in vitro, making them promising botanical leads for the mitigation of inflammatory-mediated chronic disorders.

  13. Biochemical characterization and anti-inflammatory properties of an isothiocyanate-enriched moringa (Moringa oleifera) seed extract

    PubMed Central

    Simmler, Charlotte; Kim, Youjin; Kuhn, Peter; Pauli, Guido F.; Raskin, Ilya

    2017-01-01

    Moringa oleifera Lam. is a tropical plant, used for centuries as food and traditional medicine. The aim of this study was to develop, validate and biochemically characterize an isothiocyanate-enriched moringa seed extract (MSE), and to compare the anti-inflammatory effects of MSE-containing moringa isothiocyanate-1 (MIC-1) with a curcuminoid-enriched turmeric extract (CTE), and a material further enriched in its primary phytochemical, curcumin (curcumin-enriched material; CEM). MSE was prepared by incubating ground moringa seeds with water to allow myrosinase-catalyzed enzymatic formation of bioactive MIC-1, the predominant isothiocyanate in moringa seeds. Optimization of the extraction process yielded an extract of 38.9% MIC-1. Phytochemical analysis of MSE revealed the presence of acetylated isothiocyanates, phenolic glycosides unique to moringa, flavonoids, fats and fatty acids, proteins and carbohydrates. MSE showed a reduction in the carrageenan-induced rat paw edema (33% at 500 mg/kg MIC-1) comparable to aspirin (27% at 300 mg/kg), whereas CTE did not have any significant effect. In vitro, MIC-1 at 1 μM significantly reduced the production of nitric oxide (NO) and at 5 μM, the gene expression of LPS-inducible nitric oxide synthase (iNOS) and interleukins 1β and 6 (IL-1β and IL-6), whereas CEM did not show any significant activity at all concentrations tested. MIC-1 (10μM) was also more effective at upregulating the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) target genes NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase pi 1 (GSTP1), and heme oxygenase 1 (HO1) than the CEM. Thus, in contrast to CTE and CEM, MSE and its major isothiocyanate MIC-1 displayed strong anti-inflammatory and antioxidant properties in vivo and in vitro, making them promising botanical leads for the mitigation of inflammatory-mediated chronic disorders. PMID:28792522

  14. Acetone-Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling.

    PubMed

    Assem, Naila; Ferreira, David J; Wolan, Dennis W; Dawson, Philip E

    2015-07-20

    Macrocyclization is a broadly applied approach for overcoming the intrinsically disordered nature of linear peptides. Herein, it is shown that dichloroacetone (DCA) enhances helical secondary structures when introduced between peptide nucleophiles, such as thiols, to yield an acetone-linked bridge (ACE). Aside from stabilizing helical structures, the ketone moiety embedded in the linker can be modified with diverse molecular tags by oxime ligation. Insights into the structure of the tether were obtained through co-crystallization of a constrained S-peptide in complex with RNAse S. The scope of the acetone-linked peptides was further explored through the generation of N-terminus to side chain macrocycles and a new approach for generating fused macrocycles (bicycles). Together, these studies suggest that acetone linking is generally applicable to peptide macrocycles with a specific utility in the synthesis of stabilized helices that incorporate functional tags. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Proteases in doping control analysis.

    PubMed

    Thevis, M; Maurer, J; Kohler, M; Geyer, H; Schänzer, W

    2007-07-01

    Urine manipulation in sports drug testing has become a serious problem for doping control laboratories, and recent scandals in elite endurance sports have revealed the problem of urine manipulation presumably using proteases, which will impede the detection of drugs such as erythropoietin (EPO) or other peptide hormones. Using commonly accepted analytical strategies, a protocol was developed enabling the determination of elevated protease activities in doping control specimens followed by the visualization of protein degradation and identification of proteases such as chymotrypsin, trypsin and papain. Therefore, protease detection kits based on fluorescein isothiocyanate-labeled casein were employed, and protease concentrations greater than 15 microg/mL of urine entailed subsequent 1-dimensional gel electrophoretic visualization of urinary proteins. The presence of 20 microg of proteases per mL of urine caused a complete degradation of proteins usually observed in urinary matrices ("trace of burning"), while respective proteases were still detected in spiked urine samples after 10 days of storage at + 4 and - 20 degrees C. Identification of target proteases at respective molecular weights was accomplished using bottom-up sequencing approaches based on in-gel digestion of separated enzymes followed by capillary liquid chromatography--Orbitrap tandem mass spectrometry.

  16. Molecular engineering of fluorescein dyes as complementary absorbers in dye co-sensitized solar cells

    DOE PAGES

    Pepe, Giulio; Cole, Jacqueline M.; Waddell, Paul G.; ...

    2016-09-22

    Fluorescein dye derivatives exhibit extended optical absorption up to 500 nm, rendering these compounds suitable as co-absorbers in dye-sensitized solar cells (DSCs). A molecular engineering approach is presented, which embraces this intrinsic optical attribute of fluoresceins, while modifying the dye chemistry to enhance their light harvesting efficiency, in order to effectively tailor them for DSC applications. This approach first realizes relationships between the molecular structure and the optoelectronic properties for a series of five a priori known (parent) fluorescein dyes: 5-carboxyfluorescein (1), a mixture of m-carboxyfluorescein where m = 5 or 6 (2), 5-carboxyfluorescein diacetate (3), 6-carboxyfluorescein diacetate (4), amore » mixture of n-carboxy-2',7'-dichlorofluorescein diacetate where n = 5 or 6 (5). The first step in this approach combines, where available, experimental and computational methods so that electronic structure calculations can also be validated for representative fluorescein dyes. Such calculations can then be used reliably to predict the structure and properties of fluorescein dyes for cases where experimental data are lacking. Structure-function relationships established from this initial step inform the selection of parent dye 1 that is taken forward to the second step in molecular engineering: in silico chemical derivation to re-functionalize 1 for DSC applications. For this purpose, computational calculations are used to extend the charge conjugation in 1 between its donor and acceptor moieties. These structural modifications result in a bathochromic shift of the lowest excitation by ~1.3-1.9 eV (100-170 nm), making the dye optically absorb in the visible region. Further calculations on dye molecules adsorbed onto the surface of a TiO 2 cluster are used to investigate the dye sensitization behavior via dye adsorption energies and anchoring modes. The results of this theoretical investigation lead to two molecularly engineered

  17. Molecular engineering of fluorescein dyes as complementary absorbers in dye co-sensitized solar cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pepe, Giulio; Cole, Jacqueline M.; Waddell, Paul G.

    Fluorescein dye derivatives exhibit extended optical absorption up to 500 nm, rendering these compounds suitable as co-absorbers in dye-sensitized solar cells (DSCs). A molecular engineering approach is presented, which embraces this intrinsic optical attribute of fluoresceins, while modifying the dye chemistry to enhance their light harvesting efficiency, in order to effectively tailor them for DSC applications. This approach first realizes relationships between the molecular structure and the optoelectronic properties for a series of five a priori known (parent) fluorescein dyes: 5-carboxyfluorescein (1), a mixture of m-carboxyfluorescein where m = 5 or 6 (2), 5-carboxyfluorescein diacetate (3), 6-carboxyfluorescein diacetate (4), amore » mixture of n-carboxy-2',7'-dichlorofluorescein diacetate where n = 5 or 6 (5). The first step in this approach combines, where available, experimental and computational methods so that electronic structure calculations can also be validated for representative fluorescein dyes. Such calculations can then be used reliably to predict the structure and properties of fluorescein dyes for cases where experimental data are lacking. Structure-function relationships established from this initial step inform the selection of parent dye 1 that is taken forward to the second step in molecular engineering: in silico chemical derivation to re-functionalize 1 for DSC applications. For this purpose, computational calculations are used to extend the charge conjugation in 1 between its donor and acceptor moieties. These structural modifications result in a bathochromic shift of the lowest excitation by ~1.3-1.9 eV (100-170 nm), making the dye optically absorb in the visible region. Further calculations on dye molecules adsorbed onto the surface of a TiO 2 cluster are used to investigate the dye sensitization behavior via dye adsorption energies and anchoring modes. The results of this theoretical investigation lead to two molecularly engineered

  18. Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability.

    PubMed

    Birch, Ditlev; Christensen, Malene Vinther; Staerk, Dan; Franzyk, Henrik; Nielsen, Hanne Mørck

    2017-12-01

    Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC 50 values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Matrix- and plasma-derived peptides promote tissue-specific injury responses and wound healing in diabetic swine.

    PubMed

    Sheets, Anthony R; Massey, Conner J; Cronk, Stephen M; Iafrati, Mark D; Herman, Ira M

    2016-07-02

    Non-healing wounds are a major global health concern and account for the majority of non-traumatic limb amputations worldwide. However, compared to standard care practices, few advanced therapeutics effectively resolve these injuries stemming from cardiovascular disease, aging, and diabetes-related vasculopathies. While matrix turnover is disrupted in these injuries, debriding enzymes may promote healing by releasing matrix fragments that induce cell migration, proliferation, and morphogenesis, and plasma products may also stimulate these processes. Thus, we created matrix- and plasma-derived peptides, Comb1 and UN3, which induce cellular injury responses in vitro, and accelerate healing in rodent models of non-healing wounds. However, the effects of these peptides in non-healing wounds in diabetes are not known. Here, we interrogated whether these peptides stimulate healing in a diabetic porcine model highly reminiscent of human healing impairments in type 1 and type 2-diabetes. After 3-6 weeks of streptozotocin-induced diabetes, full-thickness wounds were surgically created on the backs of adult female Yorkshire swine under general anesthesia. Comb1 and UN3 peptides or sterile saline (negative control) were administered to wounds daily for 3-7 days. Following sacrifice, wound tissues were harvested, and quantitative histological and immunohistochemical analyses were performed for wound closure, angiogenesis and granulation tissue deposition, along with quantitative molecular analyses of factors critical for angiogenesis, epithelialization, and dermal matrix remodeling. Comb1 and UN3 significantly increase re-epithelialization and angiogenesis in diabetic porcine wounds, compared to saline-treated controls. Additionally, fluorescein-conjugated Comb1 labels keratinocytes, fibroblasts, and vascular endothelial cells in porcine wounds, and Far western blotting reveals these cell populations express multiple fluorescein-Comb1-interacting proteins in vitro. Further

  20. Fluorescein sodium-guided surgery of a brain abscess: A case report

    PubMed Central

    Höhne, Julius; Brawanski, Alexander; Schebesch, Karl-Michael

    2016-01-01

    Background: Up to now, the feasibility and benefit of using fluorescein sodium under a dedicated surgical microscope filter (YE560, YELLOW 560 nm filter, Carl Zeiss Meditec, Germany) has never been clinically evaluated in infectious disorders of the brain. Case Description: Here, we report the case of a male patient with a brain abscess in the right parietal lobe that was removed under fluorescence-guidance (intravenous administration of fluorescein sodium 10%, 5 mg/kg bodyweight). The abscess capsule showed intensive yellow fluorescent staining, while − under white light − the cortex appeared normal. Conclusion: This technique may improve the identification and surgical removal of brain abscesses. PMID:28031990

  1. Isothiocyanates from Wasabia japonica activate transient receptor potential ankyrin 1 channel.

    PubMed

    Uchida, Kunitoshi; Miura, Yosuke; Nagai, Masashi; Tominaga, Makoto

    2012-11-01

    6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) and 6-(methylthio)hexyl isothiocyanate (6-MTITC) have low pungency and are responsible for the fresh flavor of wasabi (Wasabia japonica [Miq] Matsumura). In this study, we found that these two isothiocyanates activate transient receptor potential ankyrin 1 (TRPA1), and 6-MSITC activates transient receptor potential vanilloid 1 (TRPV1), but not other transient receptor potential channels expressed in sensory neurons. Both 6-MSITC and 6-MTITCinduced intracellular Ca(2+) increases in human embryonic kidney-derived 293 cells expressing mouse TRPA1 (mTRPA1) as measured by Ca(2+) imaging. In whole-cell patch-clamp recordings, 6-MSITC and 6-MTITC dose-dependently activated both mTRPA1 (EC(50) = 147±26 µM for 6-MSITC and 30±3 µM for 6-MTITC) and human TRPA1 (hTRPA1; EC(50) = 39±4 µM for 6-MSITC and 34±3 µM for 6-MTITC). In addition, TRPA1 N-terminal cysteines, which are reported to be important for channel activation by electrophilic ligands, were involved in 6-MSITC- and 6-MTITC-evoked TRPA1 activation. These isothiocyanates also activated endogenous TRPA1 expressed in mouse dorsal root ganglion neurons and intraplantar injection of 10-30 mM 6-MSITC-evoked pain-related behaviors in mice. These results indicate the following: 1) 6-MSITC and 6-MTITC activate both mTRPA1 and hTRPA1; 2) 6-MSITC activates mTRPV1; and 3) the pharmacological functions of these isothiocyanates could be derived from TRPA1 activation.

  2. A (99m) Tc-tricine-HYNIC-labeled peptide targeting the neurotensin receptor for single-photon imaging in malignant tumors.

    PubMed

    Erfani, Mostafa; Zarrabi Ahrabi, Nakisa; Shafiei, Mohammad; Shirmardi, Seyed Pezhman

    2014-03-01

    In this study, a new neurotensin (NT) analog was labeled with (99m) Tc via HYNIC chelator and tricine as coligand and investigated further. An NT (7-13) analog was prepared, and labeling with (99m) Tc was performed. The internalization rate and biodistribution of radiopeptide were studied in HT-29 cells and nude mice bearing tumor, respectively. Radiolabeling with (99m) Tc was performed at high specific activities (54 MBq/nmol) with an acceptable labeling yield (>95%). In vitro cell line studies showed a specific internalization uptake up to 13.23 ± 0.45% during 4 h which was blocked in the presence of excess cold peptide to 0.83 ± 0.15%. In biodistribution studies, uptake was observed in NT receptor-positive organs so that after 1 h the uptakes in mouse intestine and tumor were 1.23 ± 0.16% ID/g and 1.12 ± 0.11% ID/g, respectively. In animals co-injected with excess cold peptide, reduction uptake in tumor and intestines were 73% (1.10% vs. 0.29% ID/g at 4 h) and 61% (1.22% vs. 0.47% ID/g at 4 h) respectively. Predominant renal excretion pathway with a highest accumulation of activity in bladder was observed for this radiopeptide. This radiolabeled peptide could be a candidate for detection of NT positive tumors. Copyright © 2014 John Wiley & Sons, Ltd.

  3. Active packaging of cheese with allyl isothiocyanate, an alternative to modified atmosphere packaging.

    PubMed

    Winther, Mette; Nielsen, Per Vaeggemose

    2006-10-01

    The natural antimicrobial compound allyl isothiocyanate (AITC), found in mustard oil, is effective against cheese-related fungi both on laboratory media and cheese. Penicillium commune, Penicillium roqueforti, and Aspergillus flavus were more sensitive to AITC when it was added just after the spores had completed 100% germination and branching had started on Czapek yeast extract agar than were spores in the dormant phase. The use of 1 AITC label (Wasaouro interior labels, LD30D, 20 by 20 mm) in combination with atmospheric air in the packaging extended the shelf life of Danish Danbo cheese from 4 1/2 to 13 weeks. Two AITC labels extended the shelf life from 4 1/2 to 28 weeks. Both 1 and 2 labels in combination with modified atmosphere packaging extended the shelf life of the cheese from 18 to 28 weeks. This study showed that AITC was absorbed in the cheese, but it was not possible to detect any volatile breakdown products from AITC in the cheese. Cheese stored for up to 12 weeks with an AITC label had an unacceptable mustard flavor. The mustard flavor decreased to an acceptable level between weeks 12 and 28. Cheese stored in atmospheric air had a fresher taste without a CO2 off-flavor than did cheese stored in modified atmosphere packaging. AITC may be a good alternative to modified atmosphere packaging for cheese. The extended shelf life of cheese in the package is very desirable: the cheese can be transported longer distances, and the packaging can be used for the final maturing of the cheese. Furthermore, AITC can address problems such as pinholes and leaking seals in cheese packaging.

  4. Antimicrobial isothiocyanates from the seeds of Moringa oleifera Lam.

    PubMed

    Padla, Eleanor P; Solis, Ludivina T; Levida, Ruel M; Shen, Chien-Chang; Ragasa, Consolacion Y

    2012-01-01

    4-(alpha-L-Rhamnosyloxy)benzyl isothiocyanate (1) and 4-(4'-O-acetyl-alpha-L-rhamnosyloxy)-benzyl isothiocyanate (2) isolated from Moringa oleifera seeds were screened for their antibacterial activities against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, and Pseudomonas aeruginosa, and for their antifungal activities against Candida albicans, Trichophyton rubrum, and Epidermophyton floccosum using the disk diffusion method. Isothiocyanates 1 and 2 were found active at the lowest inhibitory concentration of 1 mg/ml against all Gram-positive bacteria tested (S. aureus, S. epidermidis, B. subtilis) and against the dermatophytic fungi E. floccosum and T. rubrum. Statistically significant differences were found between the mean inhibition zones (IZ) of 1 and 2 and the standard drugs, ofloxacin and clotrimazole. The minimum inhibitory concentration (MIC) values confirmed the good antimicrobial activity of 1 and 2 against S. aureus, good to moderate activity against S. epidermidis, moderate activity against B. subtilis, and weak activity against E. floccosum and T. rubrum. The in vitro bactericidal effect of 1 and 2 against the Gram-positive bacterial strains tested is suggested by MBC:MIC ratios of 2:1.

  5. Matrix-analyte-interaction in MALDI-MS: Pellet and nano-electrospray preparations

    NASA Astrophysics Data System (ADS)

    Horneffer, Verena; Glückmann, Matthias; Krüger, Ralf; Karas, Michael; Strupat, Kerstin; Hillenkamp, Franz

    2006-03-01

    The incorporation of analytes into matrix crystals and even more so its mechanistic aspects as a prerequisite for a successful MALDI-MS has been discussed controversially in the literature. Solventless sample preparation techniques can shed new light on this question. In order to investigate some crucial aspects of these preparation techniques, lyophylized peptides and proteins were ground or milled with the powder of two different matrices, 2,5-DHB as incorporating matrix and 2,6-DHB for which protein incorporation was definitely excluded in a prior study, and pressed into pellets. The dependence of the quality of the UV-MALDI-spectra on the mass (up to 12,360 Da) and the milling time in a ball mill is reported. For mellitin different initial axial ion velocities were found, when desorbed from 2,5-DHB-pellets as prepared and after wetting and re-drying. Velocities of 150 and 580 m s-1 for dry and wetted pellets are taken as representative for hard desorption from a surface and soft desorption of matrix-incorporated analytes, respectively. Proteins labeled with either fluorescein isothiocyanate (FITC) or Texas Red (TR) were nano-electrosprayed onto a bed of ferulic acid in a [`]dry' or [`]wet' mode. All [`]dry' deposits exhibit strong fluorescence but do not yield MALDI-ion signals. All [`]wet' deposits yield MALDI-signals of the proteins; the fluorescence of FITC is quenched in [`]wet' deposits because of the low matrix pH.

  6. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-06

    Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.

  7. Application of fluorescently labeled tracer technique for detection of natural active macromolecules in Chinese medicine.

    PubMed

    Zeng, Qiao-Hui; Zhang, Xue-Wu; Xu, Kai-Peng; Jiang, Jian-Guo

    2014-02-01

    Active substances in traditional Chinese Medicine (TCM) contain not only a variety of small molecules, but also many other macromolecules (TCMMs), such as proteins, peptides and polysaccharides. Active TCMM can achieve good therapeutic effects by regulating the body's overall function with lower side effects. This review summarized the literatures published in recent years on the application of fluorescently labeled tracer technique for detection of natural active macromolecules in TCM. Classified by fluorescent markers, applications of fluorescein, rhodamine, and quantum dots (QDs) in TCMM active tracer are reviewed, and the methods and principles of TCMM fluorescent marker are illustrated. Studies on active TCMMs and their action mechanism are quite difficult due to a multitarget, multicomponent, and multipath system of TCM. However, the development of fluorescently labeled active tracer technique (FLATT) provides this research with new tools. Traditional fluorescent markers have many deficiencies, such as easily quenched, short luminous cycle, and intrinsic toxicity. Relatively, FLATT has many obvious advantages, and its application in TCMM is still at the early stage. In order to improve the overall level of fluorescence labeling in TCMM active tracer, the improvement on FLATT's detection sensitivity and biological affinity is urgent and critical to allow study of these interesting molecules.

  8. Myrosinase-dependent and –independent formation and control of isothiocyanate products of glucosinolate hydrolysis

    USDA-ARS?s Scientific Manuscript database

    Brassicales contain a myrosinase enzyme that hydrolyzes glucosinolates to form toxic isothiocyanates, as a defense against bacteria, fungi, insects and herbivores including man. Low levels of isothiocyanates trigger a host defense system in mammals that protects them against chronic diseases. Becaus...

  9. Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

    PubMed Central

    Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy. PMID:11773123

  10. iAMP-2L: a two-level multi-label classifier for identifying antimicrobial peptides and their functional types.

    PubMed

    Xiao, Xuan; Wang, Pu; Lin, Wei-Zhong; Jia, Jian-Hua; Chou, Kuo-Chen

    2013-05-15

    Antimicrobial peptides (AMPs), also called host defense peptides, are an evolutionarily conserved component of the innate immune response and are found among all classes of life. According to their special functions, AMPs are generally classified into ten categories: Antibacterial Peptides, Anticancer/tumor Peptides, Antifungal Peptides, Anti-HIV Peptides, Antiviral Peptides, Antiparasital Peptides, Anti-protist Peptides, AMPs with Chemotactic Activity, Insecticidal Peptides, and Spermicidal Peptides. Given a query peptide, how can we identify whether it is an AMP or non-AMP? If it is, can we identify which functional type or types it belong to? Particularly, how can we deal with the multi-type problem since an AMP may belong to two or more functional types? To address these problems, which are obviously very important to both basic research and drug development, a multi-label classifier was developed based on the pseudo amino acid composition (PseAAC) and fuzzy K-nearest neighbor (FKNN) algorithm, where the components of PseAAC were featured by incorporating five physicochemical properties. The novel classifier is called iAMP-2L, where "2L" means that it is a 2-level predictor. The 1st-level is to answer the 1st question above, while the 2nd-level is to answer the 2nd and 3rd questions that are beyond the reach of any existing methods in this area. For the conveniences of users, a user-friendly web-server for iAMP-2L was established at http://www.jci-bioinfo.cn/iAMP-2L. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Fluress, fluorescein and benoxinate: recovery from bacterial contamination.

    PubMed

    Yolton, D P; German, C J

    1980-05-01

    The ability to recover from bacterial contaminations with Staphylococcus auresus and Pseudomonas aeruginosa was determined for three optometric DPAs: Fluress, fluorescein and benoxinate. Results show that Fluress recovers from contamination more rapidly than benoxinate or fluorescein. This ability to recover from contamination and the relative ease of use of Fluress may make it the DPA choice for a number of optometric procedures including applanation tonometry.

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

    PubMed Central

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

    2012-01-01

    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. PMID:22916074

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

    PubMed

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

    2012-01-01

    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/(64)Cu dual-labeled cyclic RGD peptide. The integrin α(v)β(3) 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. The dual-labeled probe (64)Cu-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). 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. Synthesis and comparative in vivo evaluation of 99m Tc(CO)3 -labeled PEGylated and non-PEGylated cRGDfK peptide monomers.

    PubMed

    Vats, Kusum; Satpati, Drishty; Sharma, Rohit; Sarma, Haladhar D; Banerjee, Sharmila

    2017-03-01

    This work aimed at studying the effect of insertion of medium PEG (PEG 7 ) on the pharmacokinetic behavior of cRGDfK peptide in comparison with the non-PEGylated analogue. The cRGDfK peptide has thus been derivatized at ε-amino group of lysine by conjugation with N 3 -PEG 7 -COOH/N 3 -CH 2 -COOH to prepare a PEGylated and a non-PEGylated analogue of cRGDfK. A tridentate chelator was then incorporated by click chemistry conjugation of the two peptide azides for radiolabeling with [ 99m Tc(CO) 3 (H 2 O) 3 ] + precursor. Comparative in vivo evaluation of the two 99m Tc(CO) 3 -labeled radiotracers, 99m Tc(CO) 3 -Pra-Tz-CH 2 -cRGDfK 5 and 99m Tc(CO) 3 -Pra-Tz-PEG 7 -cRGDfK 6, was carried out in C57BL/6 mice bearing α v β 3 -positive melanoma tumors to determine their potential toward targeting integrin α v β 3 receptors. The radiotracers exhibited excellent stability in saline as well as in serum. Maximum tumor uptake for the two radiotracers was observed at 30 min p.i. (5: 3.0 ± 0.7% ID/g; 6: 4.1 ± 0.5% ID/g). The two neutral 99m Tc(CO) 3 radiotracers prepared exhibited receptor-mediated uptake in melanoma tumor. The increase in the tumor uptake on introduction of PEG 7 unit was accompanied by slower clearance from other organs which resulted in decreased target-to-background ratios. The in vivo kinetics of 99m Tc(CO) 3 -labeled radiotracer, 99m Tc(CO) 3 -Pra-Tz-CH 2 -cRGDfK 5 with only methylene unit as the spacer, was found to be more favorable due to higher tumor/blood, tumor/liver, tumor/kidney, and tumor/lung ratios. © 2016 John Wiley & Sons A/S.

  15. Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

    PubMed Central

    Stagge, Franziska; Mitronova, Gyuzel Y.; Belov, Vladimir N.; Wurm, Christian A.; Jakobs, Stefan

    2013-01-01

    Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of 1H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae. PMID:24205303

  16. Tc-99m Radiolabeled Peptide p5 + 14 is an Effective Probe for SPECT Imaging of Systemic Amyloidosis.

    PubMed

    Kennel, Stephen J; Stuckey, Alan; McWilliams-Koeppen, Helen P; Richey, Tina; Wall, Jonathan S

    2016-08-01

    Systemic peripheral amyloidosis is a rare disease in which misfolded proteins deposit in various organs. We have previously developed I-124 labeled peptide p5 + 14 as a tracer for positron emission tomography imaging of amyloid in patients. In this report, we now document the labeling efficiency, bioactivity, and stability of Tc-99m labeled p5 + 14 for single-photon emission computed tomography (SPECT) imaging of amyloidosis, validated in a mouse model of systemic amyloidosis. Radiochemical yield, purity, and biological activity of [(99m)Tc]p5 + 14 were documented by instant thin-layer chromatography (ITLC), SDS-PAGE and a quantitative amyloid fibril pulldown assay. The efficacy and stability were documented in serum amyloid protein A (AA) amyloid-bearing or wild-type (WT) control mice imaged with SPECT/X-ray computed tomography (CT) at two time points. The uptake and retention of [(99m)Tc]p5 + 14 in hepatosplenic amyloid was evaluated using region of interest (ROI) and tissue counting measurements. Tc-99m p5 + 14 was produced with a radiochemical yield of 75 % with greater than 90 % purity and biological activity comparable to that of radioiodinated peptide. AA amyloid was visualized by SPECT/CT imaging with specific uptake seen in amyloid-laden organs at levels ∼5 folds higher than in healthy mice. ROI analyses of decay-corrected SPECT/CT images showed <20 % loss of radiolabel from the 1 to 4 h imaging time points. Biodistribution data confirmed the specificity of the probe accumulation by amyloid-laden organs as compared to non-diseased tissues. [(99m)Tc]p5 + 14 is a specific and stable radiotracer for systemic amyloid in mice and may provide a convenient and inexpensive alternative to imaging of peripheral amyloidosis in patients.

  17. Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex.

    PubMed

    Ihling, Christian; Schmidt, Andreas; Kalkhof, Stefan; Schulz, Daniela M; Stingl, Christoph; Mechtler, Karl; Haack, Michael; Beck-Sickinger, Annette G; Cooper, Dermot M F; Sinz, Andrea

    2006-08-01

    For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural

  18. Is fluorescein-guided technique able to help in resection of high-grade gliomas?

    PubMed

    Acerbi, Francesco; Broggi, Morgan; Eoli, Marica; Anghileri, Elena; Cavallo, Claudio; Boffano, Carlo; Cordella, Roberto; Cuppini, Lucia; Pollo, Bianca; Schiariti, Marco; Visintini, Sergio; Orsi, Chiara; La Corte, Emanuele; Broggi, Giovanni; Ferroli, Paolo

    2014-02-01

    Fluorescein, a dye that is widely used as a fluorescent tracer, accumulates in cerebral areas where the blood-brain barrier is damaged. This quality makes it an ideal dye for the intraoperative visualization of high-grade gliomas (HGGs). The authors report their experience with a new fluorescein-guided technique for the resection of HGGs using a dedicated filter on the surgical microscope. The authors initiated a prospective Phase II trial (FLUOGLIO) in September 2011 with the objective of evaluating the safety of fluorescein-guided surgery for HGGs and obtaining preliminary evidence regarding its efficacy for this purpose. To be eligible for participation in the study, a patient had to have suspected HGG amenable to complete resection of the contrast-enhancing area. The present report is based on the analysis of the short- and long-term results in 20 consecutive patients with HGGs (age range 45-74 years), enrolled in the study since September 2011. In all cases fluorescein (5-10 mg/kg) was injected intravenously after intubation. Tumor resection was performed with microsurgical technique and fluorescence visualization by means of BLUE 400 or YELLOW 560 filters on a Pentero microscope. The median preoperative tumor volume was 30.3 cm(3) (range 2.4-87.8 cm(3)). There were no adverse reactions related to fluorescein administration. Complete removal of contrast-enhanced tumor was achieved in 80% of the patients. The median duration of follow-up was 10 months. The 6-months progression-free survival rate was 71.4% and the median survival was 11 months. Analysis of these 20 cases suggested that fluorescein-guided technique with a dedicated filter on the surgical microscope is safe and allows a high rate of complete resection of contrast-enhanced tumor as determined on early postoperative MRI. Clinical trial registration no.: 2011-002527-18 (EudraCT).

  19. A fluorescently labeled undecapeptide derived from a protein in royal jelly of the honeybee-royalisin-for specific detection of oxidized low-density lipoprotein.

    PubMed

    Sato, Akira; Unuma, Hiroto; Yamazaki, Yoji; Ebina, Keiichi

    2018-06-01

    The probes for detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to facilitate the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that a heptapeptide (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled through the ε-amino group of N-terminal Lys to fluorescein isothiocyanate (FITC), (FITC)KP6, can be useful as a fluorescent probe for specific detection of ox-LDL. In the present study, to develop a novel fluorescent peptide for specific detection of ox-LDL, we investigated the interaction (with ox-LDL) of an undecapeptide corresponding to positions 41 to 51 of a potent antimicrobial protein (royalisin, which consists of 51 residues; from royal jelly of honeybees), conjugated at the N-terminus to FITC in the presence of 6-amino-n-caproic acid (AC) linker, (FITC-AC)-royalisin P11, which contains both sequences, Phe-Lys-Asp and Asp-Lys-Tyr, similar to Tyr-Lys-Asp in (FITC)KP6. The (FITC-AC)-royalisin P11 bound with high specificity to ox-LDL in a dose-dependent manner, through the binding to major lipid components in ox-LDL (lysophosphatidylcholine and oxidized phosphatidylcholine). In contrast, a (FITC-AC)-shuffled royalisin P11 peptide, in which sequences Phe-Lys-Asp and Asp-Lys-Tyr were modified to Lys-Phe-Asp and Asp-Tyr-Lys, respectively, hardly bound to LDL and ox-LDL. These findings strongly suggest that (FITC-AC)-royalisin P11 may be an effective fluorescent probe for specific detection of ox-LDL and that royalisin from the royal jelly of honeybees may play a role in the treatment of atherosclerosis through the specific binding of the region at positions 41 to 51 to ox-LDL. Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.

  20. Fluorescein angiography of the bladder: technique and relevance to bladder cancer and interstitial cystitis patients.

    PubMed

    Zimmern, P E; Laub, D; Leach, G E

    1995-07-01

    Fluorescein angiography has been used in the study of bleeding vessels, neovascularity, tumors and ischemic tissues in a variety of disorders. This pilot study was designed to evaluate the feasibility, safety and relevance of this interesting technology for the evaluation of bladder wall vessels in patients with interstitial cystitis and bladder cancer. Five patients with National Institutes of Health defined interstitial cystitis symptoms and 10 with bladder cancer were studied during cytoscopy while they were under general anesthesia. A yellow-green barrier filter (520 nm.) was placed over the cystoscope eyepiece and a blue exciter filter (465 nm.) was attached to the light source. Patients received a 5 ml. bolus of 10% fluorescein intravenously. After hydrodistension, glomerulations in interstitial cystitis patients were more prominent with fluorescein angiography and occurred in the venule phase. Areas of papillary transitional cell tumor and carcinoma in situ developed a brilliant yellow-green fluorescence. Adjacent normal urothelium was nonfluorescent and provided a contrasting dark background facilitating the detection of all lesions. No allergic reaction or other adverse effect related to the fluorescein injection was observed. These unique observations in a limited number of patients suggest that fluorescein angiography of the bladder is a safe and simple procedure. This preliminary report underscores the relevance of fluorescein angiography in the detection of bladder tumor and offers a new approach to the evaluation of bladder wall vessels in interstitial cystitis patients.

  1. Development of PEGylated carboxylic acid-modified polyamidoamine dendrimers as bone-targeting carriers for the treatment of bone diseases.

    PubMed

    Yamashita, Shugo; Katsumi, Hidemasa; Hibino, Nozomi; Isobe, Yugo; Yagi, Yumiko; Kusamori, Kosuke; Sakane, Toshiyasu; Yamamoto, Akira

    2017-09-28

    In this study, we aimed to develop a polyethylene glycol (PEG)-conjugated third generation polyamidoamine (PAMAM) dendrimer with multiple carboxylic acids as a bone-targeting carrier for the treatment of bone diseases. We conjugated PAMAM backbones to various carboxylic acids [aspartic acid (Asp), glutamic acid (Glu), succinic acid (Suc), or aconitic acid (Aco)] to obtain four different types of carboxylic acid-modified PAMAMs. PEG was covalently bound to carboxylic acid-modified PAMAMs to obtain PEGylated carboxylic acid-modified PAMAMs. In a tissue distribution study, the amount of 111 In-labeled unmodified PAMAM taken up by the bone after intravenous injection in mice was 11.3%. In contrast, the dose of 111 In-labeled PEG(5)-Asp-PAMAM, PEG(5)-Glu-PAMAM, PEG(5)-Suc-PAMAM, or PEG(5)-Aco-PAMAM that accumulated in the bone after injection was approximately 46.0, 15.6, 22.6, and 24.5%, respectively. The bone clearance rates of 111 In-labeled PEGylated carboxylic acid-modified PAMAMs were proportional to their affinities to hydroxyapatite and Ca 2+ . An intra-bone distribution study showed that fluorescein isothiocyanate-labeled PEG(5)-Asp-PAMAM predominantly accumulated on eroded and quiescent surfaces, a pattern associated with the pathogenesis of bone diseases, such as rheumatoid arthritis and osteoporosis. Our findings indicate that PEG(5)-Asp-PAMAM is a promising drug carrier for efficient drug targeting to the bones. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. The utilization of fluorescein in brain tumor surgery: a systematic review.

    PubMed

    Cavallo, Claudio; De Laurentis, Camilla; Vetrano, Ignazio G; Falco, Jacopo; Broggi, Morgan; Schiariti, Marco; Ferroli, Paolo; Acerbi, Francesco

    2018-05-22

    Sodium Fluorescein (SF) is a green, water-soluble dye with the capacity to accumulate in cerebral areas as a result of damaged blood-brain barrier (BBB); this property allows SF to concentrate specifically at the tumor site of various types of brain neoplasms, making the tumor tissue more clearly visible. A literature search (1947-2018) was conducted with the keywords "fluorescein neurosurgery", "YELLOW neurosurgery", "fluorescein brain tumor", "YELLOW brain tumor". We included clinical studies, clinical trials, observational studies, only conducted on humans and concerning surgery; in addition, we have included 3 articles derived from the analysis of the references of other papers. Ultimately, 57 articles were included for further analysis. Fluorescein as a fluorescent tracer in neuro-oncology is gaining a wider acceptance in the neurosurgical literature: until February 1st, 2018, at least 1099 neuro-oncological patients have been operated through fluorescein-assistance, mostly only after 2012. The most important application remains the aim to improve tumor visualization and extent of resection for high-grade gliomas (HGG), but the nonspecific mechanism of action is the theoretical base for its use also for tumors different from HGG. Nevertheless, no homogenous protocol of fluorescein utilization in neurosurgical oncology can be found in literature. Fluorescein-guided surgery is a safe and effective technique to improve visualization and resection of different CNS tumors and conditions, based on BBB alteration, with a growing evidence-based background.

  3. Fluorescein-Guided Resection of Intramedullary Spinal Cord Tumors: Results from a Preliminary, Multicentric, Retrospective Study.

    PubMed

    Acerbi, Francesco; Cavallo, Claudio; Schebesch, Karl-Michael; Akçakaya, Mehmet Osman; de Laurentis, Camilla; Hamamcioglu, Mustafa Kemal; Broggi, Morgan; Brawanski, Alexander; Falco, Jacopo; Cordella, Roberto; Ferroli, Paolo; Kiris, Talat; Höhne, Julius

    2017-12-01

    Intramedullary spinal cord tumors (IMSCTs) are rare, heterogenous lesions that are usually enhanced on preoperative magnetic resonance imaging (MRI) because of a damaged blood-brain barrier. Sodium fluorescein is a dye that accumulates in areas of the central nervous system with a damaged BBB. Given the pattern of MRI contrast enhancement of the majority of IMSCTs, the use of this fluorescent tracer could improve tumor visualization and quality of resection. In this article, we present the first experience with the application of fluorescein-guided technique for surgical removal of IMSCTs. Eleven patients (6 men, 5 women; mean age, 50.1 years), harboring 5 ependymomas, 3 hemangioblastomas, 1 astrocytoma, 1 pilocytic astrocytoma, and 1 glioneuronal tumor forming rosettes were included. Sodium fluorescein (5 mg/kg) was injected immediately after patient intubation. Tumors were removed with microsurgical technique and standard neurophysiological monitoring, under YELLOW 560 filter (Pentero 900) visualization. Surgical reports were reviewed regarding usefulness and grade of fluorescein staining. Postoperative MRI was performed within 72 hours after surgery, and postoperative clinical outcome was registered. No adverse events were registered. Fluorescent staining was reported in 9 of 11 cases (82%), all of them enhancing on preoperative MRI (100% of ependymomas, 100% of pilocytic astrocytomas, 100% of hemangioblastomas). No fluorescence was reported in 1 astrocytoma and 1 glioneuronal tumor-forming rosette. Intraoperative fluorescence was considered helpful for tumor resection in 9 of 11 cases (82%). Gross total resection was obtained in 8 of 11 cases (72.7%). Our results suggest that fluorescein-guided surgery is a safe and effective technique that can be used during the surgical resection of IMSCTs presenting with contrast-enhancement on preoperative MRI. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Simplified Synthesis of Isotopically Labeled 5,5-Dimethyl-pyrroline N-Oxide

    PubMed Central

    Leinisch, Fabian; Jiang, JinJie; Deterding, Leesa J.; Mason, Ronald P.

    2011-01-01

    5,5-Dimethylpyrroline N-oxide (15N) and 5,5-di(trideuteromethyl)pyrroline N-oxide were synthesized from the respective isotopically labeled 2-nitropropane analogs obtained from the reaction of sodium nitrate with 2-halopropanes. This facile, straightforward process allows synthesizing isotopically labeled DMPO analogs in a 4-step reaction without special equipment. PMID:21986521

  5. Case Report of Bullous Pemphigoid following Fundus Fluorescein Angiography

    PubMed Central

    Demirci, Goktug; Demirci, Gulsen Tukenmez; Gulkilik, Gokhan

    2010-01-01

    Purpose To report a first case of bullous pemphigoid (BP) following intravenous fluorescein for fundus angiography. Clinical Features: A 70-year-old male patient was admitted to the intensive care unit with BP and sepsis. He reported a history of fundus fluorescein angiography with a pre-diagnosis of senile macular degeneration 2 months prior to presentation. At that time, fluorescein extravasated at the antecubital region. Following the procedure, pruritus and erythema began at the wrists bilaterally, and quickly spread to the entire body. The patient also reported a history of allergy to human albumin solution (Plamasteril®; Abbott) 15 years before, during bypass surgery. On dermatologic examination, erythematous patches were present on the scalp, chest and anogenital region. Vesicles and bullous lesions were present on upper and lower extremities. On day 2 of hospitalization, tense bullae appeared on the upper and lower extremities. The patient was treated with oral methylprednisolone 48 mg (Prednol®; Mustafa Nevzat), topical clobetasol dipropionate 0.05% cream (Dermovate®; Glaxo SmithKline), and topical 4% urea lotion (Excipial Lipo®; Orva) for presumptive bullous pemphigoid. Skin punch biopsy provided tissue for histopathology, direct immunofluorescence examination, and salt extraction, which were all consistent with BP. After 1 month, the patient was transferred to the intensive care unit with sepsis secondary to urinary tract infection; he died 2 weeks later from sepsis and cardiac failure. Conclusions To our knowledge, this is the first reported case of BP following fundus fluorescein angiography in a patient with known human albumin solution allergy. Consideration should be made to avoid fluorescein angiography, change administration route, or premedicate with antihistamines in patients with known human albumin solution allergy. The association between fundus fluorescein angiography and BP should be further investigated. PMID:20737052

  6. Detection of DNA–protein crosslinks (DPCs) by novel direct fluorescence labeling methods: distinct stabilities of aldehyde and radiation-induced DPCs

    PubMed Central

    Shoulkamy, Mahmoud I.; Nakano, Toshiaki; Ohshima, Makiko; Hirayama, Ryoichi; Uzawa, Akiko; Furusawa, Yoshiya; Ide, Hiroshi

    2012-01-01

    Proteins are covalently trapped on DNA to form DNA–protein crosslinks (DPCs) when cells are exposed to DNA-damaging agents. DPCs interfere with many aspects of DNA transactions. The current DPC detection methods indirectly measure crosslinked proteins (CLPs) through DNA tethered to proteins. However, a major drawback of such methods is the non-linear relationship between the amounts of DNA and CLPs, which makes quantitative data interpretation difficult. Here we developed novel methods of DPC detection based on direct CLP measurement, whereby CLPs in DNA isolated from cells are labeled with fluorescein isothiocyanate (FITC) and quantified by fluorometry or western blotting using anti-FITC antibodies. Both formats successfully monitored the induction and elimination of DPCs in cultured cells exposed to aldehydes and mouse tumors exposed to ionizing radiation (carbon-ion beams). The fluorometric and western blotting formats require 30 and 0.3 μg of DNA, respectively. Analyses of the isolated genomic DPCs revealed that both aldehydes and ionizing radiation produce two types of DPC with distinct stabilities. The stable components of aldehyde-induced DPCs have half-lives of up to days. Interestingly, that of radiation-induced DPCs has an infinite half-life, suggesting that the stable DPC component exerts a profound effect on DNA transactions over many cell cycles. PMID:22730301

  7. N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging.

    PubMed

    Gourni, Eleni; Mansi, Rosalba; Jamous, Mazen; Waser, Beatrice; Smerling, Christiane; Burian, Antje; Buchegger, Franz; Reubi, Jean Claude; Maecke, Helmut R

    2014-10-01

    Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated

  8. Goat anti-rabbit IgG conjugated fluorescent dye-doped silica nanoparticles for human breast carcinoma cell recognition.

    PubMed

    Chen, Min-Yan; Chen, Ze-Zhong; Wu, Ling-Ling; Tang, Hong-Wu; Pang, Dai-Wen

    2013-11-12

    We report an indirect method for cancer cell recognition using photostable fluorescent silica nanoprobes as biological labels. The dye-doped fluorescent silica nanoparticles were synthesized using the water-in-oil (W/O) reverse microemulsion method. The silica matrix was produced by the controlled hydrolysis of tetraethylorthosilicate (TEOS) in water nanodroplets with the initiation of ammonia (NH3·H2O). Fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate conjugated with dextran (RBITC-Dextran) was doped in silica nanoparticles (NPs) with a size of 60 ± 5 nm as a fluorescent signal element by covalent bonding and steric hindrance, respectively. The secondary antibody, goat anti-rabbit IgG, was conjugated on the surface of the PEG-terminated modified FITC-doped or RBITC-Dextran-doped silica nanoparticles (PFSiNPs or PBSiNPs) by covalent binding to the PEG linkers using the cyanogen bromide method. The concentrations of goat anti-rabbit IgG covering the nanoprobes were quantified via the Bradford method. In the proof-of-concept experiment, an epithelial cell adhesion molecule (EpCAM) on the human breast cancer SK-Br-3 cell surface was used as the tumor marker, and the nanoparticle functionalized with rabbit anti-EpCAM antibody was employed as the nanoprobe for cancer cell recognition. Compared with fluorescent dye labeled IgG (FITC-IgG and RBITC-IgG), the designed nanoprobes display dramatically increased stability of fluorescence as well as photostability under continuous irradiation.

  9. Optimized synthesis of phosphorothioate oligodeoxyribonucleotides substituted with a 5′-protected thiol function and a 3′-amino group

    PubMed Central

    Aubert, Yves; Bourgerie, Sylvain; Meunier, Laurent; Mayer, Roger; Roche, Annie-Claude; Monsigny, Michel; Thuong, Nguyen T.; Asseline, Ulysse

    2000-01-01

    A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphorothioate oligonucleotides substituted with a protected thiol function at their 5′-ends and an amino group at their 3′-ends in good yield (up to 72 OD units/µmol for a 19mer phosphorothioate). Syntheses of 3′-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphoramidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2′-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This procedure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3′-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5′-terminal S-acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3′-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligonucleotide; the 5′-dithiopyridyl group was then quantitatively reduced to give a free thiol group which was then substituted by reaction with an Nα-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide–oligonucleotide conjugate. PMID:10637335

  10. Comparative evaluation of p5+14 with SAP and peptide p5 by dual-energy SPECT imaging of mice with AA amyloidosis.

    PubMed

    Martin, Emily B; Williams, Angela; Richey, Tina; Stuckey, Alan; Heidel, R Eric; Kennel, Stephen J; Wall, Jonathan S

    2016-03-03

    Amyloidosis is a protein-misfolding disorder characterized by the extracellular deposition of amyloid, a complex matrix composed of protein fibrils, hyper-sulphated glycosaminoglycans and serum amyloid P component (SAP). Accumulation of amyloid in visceral organs results in the destruction of tissue architecture leading to organ dysfunction and failure. Early differential diagnosis and disease monitoring are critical for improving patient outcomes; thus, whole body amyloid imaging would be beneficial in this regard. Non-invasive molecular imaging of systemic amyloid is performed in Europe by using iodine-123-labelled SAP; however, this tracer is not available in the US. Therefore, we evaluated synthetic, poly-basic peptides, designated p5 and p5+14, as alternative radiotracers for detecting systemic amyloidosis. Herein, we perform a comparative effectiveness evaluation of radiolabelled peptide p5+14 with p5 and SAP, in amyloid-laden mice, using dual-energy SPECT imaging and tissue biodistribution measurements. All three radiotracers selectively bound amyloid in vivo; however, p5+14 was significantly more effective as compared to p5 in certain organs. Moreover, SAP bound principally to hepatosplenic amyloid, whereas p5+14 was broadly distributed in numerous amyloid-laden anatomic sites, including the spleen, liver, pancreas, intestines and heart. These data support clinical validation of p5+14 as an amyloid radiotracer for patients in the US.

  11. A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis

    NASA Technical Reports Server (NTRS)

    Mozdziak, P. E.; Fassel, T. A.; Schultz, E.; Greaser, M. L.; Cassens, R. G.

    1996-01-01

    A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

  12. Interaction of β(3) /β(2) -peptides, consisting of Val-Ala-Leu segments, with POPC giant unilamellar vesicles (GUVs) and white blood cancer cells (U937)--a new type of cell-penetrating peptides, and a surprising chain-length dependence of their vesicle- and cell-lysing activity.

    PubMed

    Kolesinska, Beata; Eyer, Klaus; Robinson, Tom; Dittrich, Petra S; Beck, Albert K; Seebach, Dieter; Walde, Peter

    2015-05-01

    Many years ago, β(2) /β(3) -peptides, consisting of alternatively arranged β(2) - and β(3) h-amino-acid residues, have been found to undergo folding to a unique type of helix, the 10/12-helix, and to exhibit non-polar, lipophilic properties (Helv. Chim. Acta 1997, 80, 2033). We have now synthesized such 'mixed' hexa-, nona-, dodeca-, and octadecapeptides, consisting of Val-Ala-Leu triads, with N-terminal fluorescein (FAM) labels, i.e., 1-4, and studied their interactions with POPC (=1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow-cytometry assay, a membrane-toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All β(3) /β(2) -peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric β(3) /β(2) -, β(3) -, and β(2) -nonamers, 2, 5, and 6, respectively, the derivatives 5 and 6 consisting exclusively of β(3) - or β(2) -amino-acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the β(3) /β(2) -nonamer and/or the β(3) /β(2) -dodecamer derivative, 2 and/or 3, respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host-defense antimicrobial peptides. Possible sources of the chain-length-dependent destructive potential of the β(3) /β(2) -nona- and β(3) /β(2) -dodecapeptide derivatives, and a possible relationship with the phosphate-to-phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of 'mixed' β(3) /β(2) -peptides with

  13. Evaluation of 111In-Labeled Cyclic RGD Peptides: Effects of Peptide and Linker Multiplicity on Their Tumor Uptake, Excretion Kinetics and Metabolic Stability

    PubMed Central

    Shi, Jiyun; Zhou, Yang; Chakraborty, Sudipta; Kim, Young-Seung; Jia, Bing; Wang, Fan; Liu, Shuang

    2011-01-01

    Purpose: The purpose of this study was to demonstrate the valence of cyclic RGD peptides, P-RGD (PEG4-c(RGDfK): PEG4 = 15-amino-4,710,13-tetraoxapentadecanoic acid), P-RGD2 (PEG4-E[c(RGDfK)]2, 2P-RGD4 (E{PEG4-E[c(RGDfK)]2}2, 2P4G-RGD4 (E{PEG4-E[G3-c(RGDfK)]2}2: G3 = Gly-Gly-Gly) and 6P-RGD4 (E{PEG4-E[PEG4-c(RGDfK)]2}2) in binding to integrin αvβ3, and to assess the impact of peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of their corresponding 111In radiotracers. Methods: Five new RGD peptide conjugates (DOTA-P-RGD (DOTA =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTA-P-RGD2, DOTA-2P-RGD4, DOTA-2P4G-RGD4, DOTA-6P-RGD4), and their 111In complexes were prepared. The integrin αvβ3 binding affinity of cyclic RGD conjugates were determined by a competitive displacement assay against 125I-c(RGDyK) bound to U87MG human glioma cells. Biodistribution, planar imaging and metabolism studies were performed in athymic nude mice bearing U87MG human glioma xenografts. Results: The integrin αvβ3 binding affinity of RGD conjugates follows the order of: DOTA-6P-RGD4 (IC50 = 0.3 ± 0.1 nM) ~ DOTA-2P4G-RGD4 (IC50 = 0.2 ± 0.1 nM) ~ DOTA-2P-RGD4 (IC50 = 0.5 ± 0.1 nM) > DOTA-3P-RGD2 (DOTA-PEG4-E[PEG4-c(RGDfK)]2: IC50 = 1.5 ± 0.2 nM) > DOTA-P-RGD2 (IC50 = 5.0 ± 1.0 nM) >> DOTA-P-RGD (IC50 = 44.3 ± 3.5 nM) ~ c(RGDfK) (IC50 = 49.9 ± 5.5 nM) >> DOTA-6P-RGK4 (IC50 = 437 ± 35 nM). The fact that DOTA-6P-RGK4 had much lower integrin αvβ3 binding affinity than DOTA-6P-RGD4 suggests that the binding of DOTA-6P-RGD4 to integrin αvβ3 is RGD-specific. This conclusion is consistent with the lower tumor uptake for 111In(DOTA-6P-RGK4) than that for 111In(DOTA-6P-RGD4). It was also found that the G3 and PEG4 linkers between RGD motifs have a significant impact on the integrin αvβ3-targeting capability, biodistribution characteristics, excretion kinetics and metabolic stability of 111In-labeled cyclic RGD

  14. [Nursing care in fluorescein angiography].

    PubMed

    Santos-Blanco, Feliciano

    2008-01-01

    Fluoresceinic angiography of the ocular fundus is a diagnostic technique to study retinal and choroidal circulation. This technique consists of parenteral administration of 500 mg of sodium fluorescein 10% and photographing the fluorescence in the eye vessels. Although this substance is fairly safe, it may also produce mild, moderate or severe local and/or general adverse reactions. The nursing process is routinely used in hospital units but not always in outpatient clinics, even through the use of invasive procedures with intravenous medication administration is common. Therefore, nurses, as those reponsible for intravenous administration, should use the nursing process to guarantee the quality of care required by the patient. To do this, we describe an individualized care plan based on evaluation by Marjorie Gordon's functional health patterns, NANDA's nursing diagnoses Taxonomy II, Nursing Outcomes Classification (NOC), Nursing Interventions Classifications (NIC) and potential complications of the procedure.

  15. Enhancement in ex vivo phagocytic capacity of peritoneal leukocytes in mice by oral delivery of various lactic-acid-producing bacteria.

    PubMed

    Lee, Yeonhee; Lee, Taik-Soo

    2005-01-01

    Lactic-acid-producing bacteria (LABs) are known to have immunomodulating activity. In the current study, various LABs were tested for their immunity-enhancing activity, especially the phagocytic activity of leukocytes. Viable but not heat-killed cells of Weissella kimchii strain PL9001, Lactobacillus fermentum strain PL9005, and L. plantarum strain PL9011 significantly increased the ex vivo phagocytic capacity of mouse peritoneal leukocytes to ingest fluorescein isothiocyanate (FITC)-labeled Escherichia coli in a strain-dependent manner. Results of this and previous studies suggest these LABs as candidates for new probiotics. This is the first report of the enhancement of peritoneal leukocyte activity of these species.

  16. Carbachol alleviates rat cytokine release and organ dysfunction induced by lipopolysaccharide.

    PubMed

    Zhou, Guoyong; Hu, Sen; Lv, Yi; Song, Qi; Zou, Xiaofang; Sheng, Zhiyong

    2011-07-01

    To observe the influence of carbachol on inflammatory cytokine release and its protective role on organ function in rat endotoxemia model, and, furthermore, to investigate its receptor mechanism in rat peritoneal macrophages in vitro. In the animal experiments, Wistar rats were subjected to lipopolysaccharide (LPS) injection (5 mg/kg body weight) to establish an endotoxemia animal model, and carbachol/nicotine was given 15 minutes after LPS injection. Serum contents of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were determined with enzyme-linked immunosorbent assay 4 hours after LPS injection. Plasma alanine aminotransferase, creatine kinase-MB, and diamine oxidase contents were detected 24 hours after LPS injection. In cell experiments, rat peritoneal macrophages were collected and initially pretreated with atropine (muscarinic cholinergic receptor antagonist) or α-Bungarotoxin (an antagonist that specifically binds α7 subunit of nicotinic cholinergic receptor), then with carbachol or nicotine, and finally stimulated with LPS. Contents of TNF-α, IL-6, and IL-10 in supernatant were assayed by enzyme-linked immunosorbent assay. Furthermore, macrophages were exposed to nicotine and carbachol of high concentration and then stained with fluorescein isothiocyanate-labeled α-bungarotoxin and observed with fluorescent confocal microscopy. Carbachol inhibited expression of TNF-α and IL-6 after LPS injection and had no significant effect on IL-10 in rat endotoxemia model. It also inhibited the increase of plasma alanine aminotransferase and creatine kinase-MB contents whereas restored the inhibited plasma diamine oxidase activity. Cell experiments also showed that increases of TNF-α and IL-6 after LPS stimulation could be significantly inhibited by carbachol or nicotine, whereas IL-10 was not apparently altered. Atropine did not downregulate the inhibitive effects of both carbachol and nicotine, whereas α-bungarotoxin significantly downregulated

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

    PubMed

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

    2017-07-01

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

  18. Fluorescein dye improves microscopic evaluation and counting of demodex in blepharitis with cylindrical dandruff.

    PubMed

    Kheirkhah, Ahmad; Blanco, Gabriela; Casas, Victoria; Tseng, Scheffer C G

    2007-07-01

    To show whether fluorescein dye helps detect and count Demodex embedded in cylindrical dandruff (CD) of epilated eyelashes from patients with blepharitis. Two eyelashes with CD were removed from each lid of 10 consecutive patients with blepharitis and subjected to microscopic examination with and without fluorescein solution to detect and count Demodex mites. Of 80 eyelashes examined, 36 (45%) lashes retained their CD after removal. Before addition of the fluorescein solution, the mean total Demodex count per patient was 14.9 +/- 10 and the mean Demodex count per lash was 3.1 +/- 2.5 and 0.8 +/- 0.7 in epilated eyelashes with and without retained CD, respectively (P < 0.0001). After addition of the fluorescein solution, opaque and compact CD instantly expanded to reveal embedded mites in a yellowish and semitransparent background. As a result, the mean total Demodex count per patient was significantly increased to 20.2 +/- 13.8 (P = 0.003), and the mean count per lash was significantly increased to 4.4 +/- 2.8 and 1 +/- 0.8 in eyelashes with and without retained CD (P < 0.0001 and P = 0.007), respectively. This new method yielded more mites in 8 of 10 patients and allowed mites to be detected in 3 lashes with retained CD and 1 lash without retained CD that had an initial count of zero. Addition of fluorescein solution after mounting further increases the proficiency of detecting and counting mites embedded in CD of epilated eyelashes.

  19. Fluorescein sodium-guided resection of cerebral metastases-an update.

    PubMed

    Höhne, Julius; Hohenberger, Christoph; Proescholdt, Martin; Riemenschneider, Markus J; Wendl, Christina; Brawanski, Alexander; Schebesch, Karl-Michael

    2017-02-01

    Cerebral metastasis (CM) is the most common malignancy affecting the brain. In patients eligible for surgery, complete tumor removal is the most important predictor of overall survival and neurological outcome. The emergence of surgical microscopes fitted with a fluorescein-specific filter have facilitated fluorescein-guided microsurgery and identification of tumor tissue. In 2012, we started evaluating fluorescein (FL) with the dedicated microscope filter in cerebral metastases (CM). After describing the treatment results of our first 30 patients, we now retrospectively report on 95 patients. Ninety-five patients with CM of different primary cancers were included (47 women, 48 men, mean age, 60 years, range, 25-85 years); 5 mg/kg bodyweight of FL was intravenously injected at induction of anesthesia. A YELLOW 560-nm filter (Pentero 900, ZEISS Meditec, Germany) was used for microsurgical tumor resection and resection control. The extent of resection (EOR) was assessed by means of early postoperative contrast-enhanced MRI and the grade of fluorescent staining as described in the surgical reports. Furthermore, we evaluated information on neurological outcome and surgical complications as well as any adverse events. Ninety patients (95%) showed bright fluorescent staining that markedly enhanced tumor visibility. Five patients (5%); three with adenocarcinoma of the lung, one with melanoma of the skin, and one with renal cell carcinoma) showed insufficient FL staining. Thirteen patients (14%) showed residual tumor tissue on the postoperative MRI. Additionally, the MRI of three patients did not confirm complete resection beyond doubt. Thus, gross-total resection had been achieved in 83% (n = 79) of patients. No adverse events were registered during the postoperative course. FL and the YELLOW 560-nm filter are safe and feasible tools for increasing the EOR in patients with CM. Further prospective evaluation of the FL-guided technique in CM-surgery is in planning.

  20. Quantitative and Qualitative Analysis of Bacteria in Er(III) Solution by Thin-Film Magnetopheresis

    PubMed Central

    Zborowski, Maciej; Tada, Yoko; Malchesky, Paul S.; Hall, Geraldine S.

    1993-01-01

    Magnetic deposition, quantitation, and identification of bacteria reacting with the paramagnetic trivalent lanthanide ion, Er3+, was evaluated. The magnetic deposition method was dubbed thin-film magnetopheresis. The optimization of the magnetic deposition protocol was accomplished with Escherichia coli as a model organism in 150 mM NaCl and 5 mM ErCl3 solution. Three gram-positive bacteria, Staphylococcus epidermidis, Staphylococcus saprophyticus, and Enterococcus faecalis, and four gram-negative bacteria, E. coli, Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, were subsequently investigated. Quantitative analysis consisted of the microscopic cell count and a scattered-light scanning of the magnetically deposited material aided by the computer data acquisition system. Qualitative analysis consisted of Gram stain differentiation and fluorescein isothiocyanate staining in combination with selected antisera against specific types of bacteria on the solid substrate. The magnetic deposition protocol allowed quantitative detection of E. coli down to the concentration of 105 CFU ml-1, significant in clinical diagnosis applications such as urinary tract infections. Er3+ did not interfere with the typical appearance of the Gram-stained bacteria nor with the antigen recognition by the antibody in the immunohistological evaluations. Indirect antiserum-fluorescein isothiocyanate labelling correctly revealed the presence of E. faecalis and P. aeruginosa in the magnetically deposited material obtained from the mixture of these two bacterial species. On average, the reaction of gram-positive organisms was significantly stronger to the magnetic field in the presence of Er3+ than the reaction of gram-negative organisms. The thin-film magnetophoresis offers promise as a rapid method for quantitative and qualitative analysis of bacteria in solutions such as urine or environmental water. Images PMID:16348916

  1. One-step synthesis of fluorescein modified nano-carbon for Pd(II) detection via fluorescence quenching.

    PubMed

    Panchompoo, Janjira; Aldous, Leigh; Baker, Matthew; Wallace, Mark I; Compton, Richard G

    2012-05-07

    Carbon black (CB) nanoparticles modified with fluorescein, a highly fluorescent molecule, were prepared using a facile and efficient methodology. Simply stirring CB in aqueous solution containing fluorescein resulted in the strong physisorption of fluorescein onto the CB surface. The resulting Fluorescein/CB was then characterised by means of X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV), fluorescence microscopy and fluorescence spectroscopy. The optimum experimental conditions for fluorescence of Fluorescein/CB viz. fluorescence excitation and emission wavelengths, O(2) removal and the amount of Fluorescein/CB used, were investigated. The Fluorescein/CB was used as a fluorescent probe for the sensitive detection of Pd(II) in water, based on fluorescence quenching. The results demonstrated that the fluorescence intensity of Fluorescein/CB decreased with increasing Pd(II) concentration, and the fluorescence quenching process could be described by the Stern-Volmer equation. The limit of detection (LOD) for the fluorescence quenching of Fluorescein/CB by Pd(II) in aqueous solution was found to be 1.07 μM (based on 3σ). Last, approaches were studied for the removal of Fe(III) which interferes with the fluorescence quenching of Fluorescein/CB. Complexation of Fe(III) with salicylic acid was used to enhance and control the selectivity of Fluorescein/CB sensor towards Pd(II) in the presence of Fe(III).

  2. Histopathological confirmation of similar intramucosal distribution of fluorescein in both intravenous administration and local mucosal application for probe-based confocal laser endomicroscopy of the normal stomach.

    PubMed

    Nonaka, Kouichi; Ohata, Ken; Ban, Shinichi; Ichihara, Shin; Takasugi, Rumi; Minato, Yohei; Tashima, Tomoaki; Matsuyama, Yasushi; Takita, Maiko; Matsuhashi, Nobuyuki; Neumann, Helmut

    2015-12-16

    Probe-based confocal laser endomicroscopy (pCLE) is capable of acquiring in vivo magnified cross-section images of the gastric mucosa. Intravenous injection of fluorescein sodium is used for confocal imaging. However, it is still under debate if local administration of the dye to the mucosa is also effective for confocal imaging as it is not yet clear if topical application also reveals the intramucosal distribution of fluorescein. The objective of this study was to evaluate the intramucosal distribution of fluorescein sodium after topical application and to compare the distribution to the conventional intravenous injection used for confocal imaging. pCLE of the stomach uninfected with Helicobacter pylori was performed in a healthy male employing intravenous administration and local mucosal application of fluorescein. The mucosa of the lower gastric body was biopsied 1 min and 5 min after intravenous administration or local mucosal application of fluorescein, and the distribution of fluorescein in the biopsy samples was examined histologically. Green fluorescence was already observed in the cytoplasm of fundic glandular cells in the biopsied deep mucosa 1 min after local mucosal application of fluorescein. It was also observed in the foveolar lumen and inter-foveolar lamina propria, although it was noted at only a few sites. In the tissue biopsied 5 min after the local mucosal application of fluorescein, green fluorescence was more frequently noted in the cytoplasm of fundic glandular cells than in that 1 min after the local mucosal application of fluorescein, although obvious green fluorescence was not identified in the foveolar lumen or inter-foveolar lamina propria. The distribution of intravenously administered fluorescein in the cytoplasm of fundic glandular cells was also clearly observed similarly to that after local mucosal application of fluorescein. Green fluorescence in more cells was observed in many cells 5 min after intravenous administration compared

  3. Acute Toxicity of Sodium Fluorescein to Ashy Pebblesnails Fluminicola fuscus

    USGS Publications Warehouse

    Stockton, Kelly A.; Moffitt, Christine M.; Blew, David L.; Farmer, C. Neil

    2011-01-01

    Water resource agencies and groundwater scientists use fluorescein dyes to trace ground water flows that supply surface waters that may contain threatened or endangered mollusk species. Since little is known of the toxicity of sodium fluorescein to mollusks, we tested the toxicity of sodium fluorescein to the ashy pebblesnail Fluminicola fuscus. The pebblesnail was selected as a surrogate test species for the threatened Bliss Rapid snail Taylorcocha serpenticola that is endemic to the Snake River and its tributaries in the Hagerman Valley, Idaho. In laboratory tests, we expose replicated groups of snails to a series of concentrations of fluorescein in a static 24 h exposure at 15 degrees C. Following the exposure, we removed snails, rinsed them, and allowed a 48 h recovery in clean water before recording mortality. We estimated 377 mg/L as the median lethal dose. Mortality to snails occurred at concentrations well above those expected in test wells during the monitoring efforts.

  4. Sungsanpin, a lasso peptide from a deep-sea streptomycete.

    PubMed

    Um, Soohyun; Kim, Young-Joo; Kwon, Hyuknam; Wen, He; Kim, Seong-Hwan; Kwon, Hak Cheol; Park, Sunghyouk; Shin, Jongheon; Oh, Dong-Chan

    2013-05-24

    Sungsanpin (1), a new 15-amino-acid peptide, was discovered from a Streptomyces species isolated from deep-sea sediment collected off Jeju Island, Korea. The planar structure of 1 was determined by 1D and 2D NMR spectroscopy, mass spectrometry, and UV spectroscopy. The absolute configurations of the stereocenters in this compound were assigned by derivatizations of the hydrolysate of 1 with Marfey's reagents and 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate, followed by LC-MS analysis. Careful analysis of the ROESY NMR spectrum and three-dimensional structure calculations revealed that sungsanpin possesses the features of a lasso peptide: eight amino acids (-Gly(1)-Phe-Gly-Ser-Lys-Pro-Ile-Asp(8)-) that form a cyclic peptide and seven amino acids (-Ser(9)-Phe-Gly-Leu-Ser-Trp-Leu(15)) that form a tail that loops through the ring. Sungsanpin is thus the first example of a lasso peptide isolated from a marine-derived microorganism. Sungsanpin displayed inhibitory activity in a cell invasion assay with the human lung cancer cell line A549.

  5. Fluorescein as a Visible-Light-Induced Oxidase Mimic for Signal-Amplified Colorimetric Assay of Carboxylesterase by an Enzymatic Cascade Reaction.

    PubMed

    Liu, Li; Sun, Chaoqun; Yang, Juan; Shi, Ying; Long, Yijuan; Zheng, Huzhi

    2018-04-20

    We have found that fluorescein possesses high visible-light-induced oxidase mimetic activity and could transform colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB) without unstable and destructive H 2 O 2 under visible-light illumination. Instead, fluorescein uses oxygen as a mild and green electron acceptor, and its activity can be easily controlled by the switching "on/off" of visible light. In addition, the visible-light-induced catalytic mechanism was elucidated in detail and, as the main reactive species h + and O 2 .- accounted for TMB oxidation. Based on the fact that fluorescein diacetate (FDA) possessed no activity and generated active fluorescein in situ in the presence of carboxylesterase (CaE), a signal-amplified sensing platform through a cascade reaction for CaE detection was constructed. Our proposed sensing system displayed excellent analytical performance for the detection of CaE in a wide linear range from 0.040 to 20 U L -1 with a low detection limit of 0.013 U L -1 . This work not only changes the conventional concept that fluorescein is generally considered to be photocatalytically inert, but also provides a novel sensing strategy by tailoring the enzyme mimetic activity of fluorescein derivatives with analyte. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. A compactly integrated laser-induced fluorescence detector for microchip electrophoresis.

    PubMed

    Li, Hai-Fang; Lin, Jin-Ming; Su, Rong-Guo; Uchiyama, Katsumi; Hobo, Toshiyuki

    2004-06-01

    A simple and easy-to-use integrated laser-induced fluorescence detector for microchip electrophoresis was constructed and evaluated. The fluid channels and optical fiber channels in the glass microchip were fabricated using standard photolithographic techniques and wet chemical etching. A 473 nm diode-pumped laser was used as the excitation source, and the collimation and collection optics and mirrors were discarded by using a multimode optical fiber to couple the excitation light straight into the microchannel and placing the microchip directly on the top of the photomultiplier tube. A combination of filter systems was incorporated into a poly(dimethylsiloxane) layer, which was reversibly sealed to the bottom of the microchip to eliminate the scattering excitation light reaching to the photomultiplier tube. Fluorescein/calcein samples were taken as model analytes to evaluate the performance with respect to design factors. The detection limits were 0.05 microM for fluorescein and 0.18 microM for calcein, respectively. The suitability of this simple detector for fluorescence detection was demonstrated by baseline separation of fluorescein isothiocyanate (FITC)-labeled arginine, phenylalanine, and glycine and FITC within 30 s at separation length of 3.8 cm and electrical field strength of 600 V/cm.

  7. Label-free quantitation of peptide release from neurons in a microfluidic device with mass spectrometry imaging

    PubMed Central

    Zhong, Ming; Lee, Chang Young; Croushore, Callie A.; Sweedler, Jonathan V.

    2013-01-01

    Microfluidic technology allows the manipulation of mass-limited samples and when used with cultured cells, enables control of the extracellular microenvironment, making it well suited for studying neurons and their response to environmental perturbations. While matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) provides for off-line coupling to microfluidic devices for characterizing small-volume extracellular releasates, performing quantitative studies with MALDI is challenging. Here we describe a label-free absolute quantitation approach for microfluidic devices. We optimize device fabrication to prevent analyte losses before measurement and then incorporate a substrate that collects the analytes as they flow through a collection channel. Following collection, the channel is interrogated using MS imaging. Rather than quantifying the sample present via MS peak height, the length of the channel containing appreciable analyte signal is used as a measure of analyte amount. A linear relationship between peptide amount and band length is suggested by modeling the adsorption process and this relationship is validated using two neuropeptides, acidic peptide (AP) and α-bag cell peptide [1-9] (αBCP). The variance of length measurement, defined as the ratio of standard error to mean value, is as low as 3% between devices. The limit of detection (LOD) of our system is 600 fmol for AP and 400 fmol for αBCP. Using appropriate calibrations, we determined that an individual Aplysia bag cell neuron secretes 0.15 ± 0.03 pmol of AP and 0.13 ± 0.06 pmol of αBCP after being stimulated with elevated KCl. This quantitation approach is robust, does not require labeling, and is well suited for miniaturized off-line characterization from microfluidic devices. PMID:22508372

  8. Quantitative phosphoproteomics using acetone-based peptide labeling: Method evaluation and application to a cardiac ischemia/reperfusion model

    PubMed Central

    Wijeratne, Aruna B.; Manning, Janet R.; Schultz, Jo El J.; Greis, Kenneth D.

    2013-01-01

    Mass spectrometry (MS) techniques to globally profile protein phosphorylation in cellular systems that are relevant to physiological or pathological changes have been of significant interest in biological research. In this report, an MS-based strategy utilizing an inexpensive acetone-based peptide labeling technique known as reductive alkylation by acetone (RABA) for quantitative phosphoproteomics was explored to evaluate its capacity. Since the chemistry for RABA-labeling for phosphorylation profiling had not been previously reported, it was first validated using a standard phosphoprotein and identical phosphoproteomes from cardiac tissue extracts. A workflow was then utilized to compare cardiac tissue phosphoproteomes from mouse hearts not expressing FGF2 vs. hearts expressing low molecular weight fibroblast growth factor-2 (LMW FGF2) to relate low molecular weight fibroblast growth factor-2 (LMW FGF2) mediated cardioprotective phenomena induced by ischemia/reperfusion (I/R) injury of hearts, with downstream phosphorylation changes in LMW FGF2 signaling cascades. Statistically significant phosphorylation changes were identified at 14 different sites on 10 distinct proteins including some with mechanisms already established for LMW FGF2-mediated cardioprotective signaling (e.g. connexin-43), some with new details linking LMW FGF2 to the cardioprotective mechanisms (e.g. cardiac myosin binding protein C or cMyBPC), and also several new downstream effectors not previously recognized for cardio-protective signaling by LMW FGF2. Additionally, one of the phosphopeptides, cMyBPC/pSer-282, identified was further verified with site-specific quantification using an SRM (selected reaction monitoring)-based approach that also relies on isotope labeling of a synthetic phosphopeptide with deuterated acetone as an internal standard. Overall, this study confirms that the inexpensive acetone-based peptide labeling can be used in both exploratory and targeted quantification

  9. Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, wasabi.

    PubMed

    Morimitsu, Y; Hayashi, K; Nakagawa, Y; Horio, F; Uchida, K; Osawa, T

    2000-01-01

    6-Methylsulfinylhexyl isothiocyanate (MS-ITC) was isolated from wasabi (Wasabia japonica, Japanese domestic horseradish) as a potential inhibitor of human platelet aggregation in vitro through our extensive screening of vegetables and fruits. In the course of another screening for the induction of glutathione S-transferase (GST) activity in RL34 cells. MS-ITC was inadvertently isolated from wasabi as a potential inducer of GST. MS-ITC administered to rats or mice also showed both activities in vivo. As a result from elucidation of the platelet aggregation inhibition and the GST induction mechanisms of MS-ITC, the isothiocyanate moiety of MS-ITC plays an important role for antiplatelet and anticancer activities because of its highly reactivity with sulfhydryl (-SH) groups in biomolecules (GSH, cysteine residue in a certain protein, etc.).

  10. Antiplatelet and anticancer isothiocyanates in Japanese domestic horseradish, Wasabi.

    PubMed

    Morimitsu, Y; Hayashi, K; Nakagawa, Y; Fujii, H; Horio, F; Uchida, K; Osawa, T

    2000-07-31

    6-Methylsulfinylhexyl isothiocyanate (MS-ITC) was isolated from wasabi (Wasabia japonica, Japanese domestic horseradish) as a potential inhibitor of human platelet aggregation in vitro through our extensive screening of vegetables and fruits. In the course of an another screening for the induction of glutathione S-transferase (GST) activity in RL34 cells, MS-ITC was inadvertently isolated from wasabi as a potential inducer of GST. MS-ITC administered to rats or mice also showed both activities in vivo. As a result from elucidation of the platelet aggregation inhibition and the GST induction mechanisms of MS-ITC, the isothiocyanate moiety of MS-ITC plays an important role for antiplatelet and anticancer activities because of its high reactivity with sulfhydryl (RSH) groups in biomolecules (GSH, cysteine residue in a certain protein, etc.).

  11. 16 CFR 300.5 - Required label and method of affixing.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Required label and method of affixing. 300.5 Section 300.5 Commercial Practices FEDERAL TRADE COMMISSION REGULATIONS UNDER SPECIFIC ACTS OF CONGRESS RULES AND REGULATIONS UNDER THE WOOL PRODUCTS LABELING ACT OF 1939 Labeling § 300.5 Required label and...

  12. [Quenched fluorescein: a reference dye for instrument response function of TCSPC].

    PubMed

    Pan, Hai-feng; Ding, Jing-xin; Liang, Rong-rong; Tao, Zhan-dong; Liu, Meng-wei; Zhang, San-jun; Xu, Jian-hua

    2014-08-01

    Measuring the instrument response function (IRF) and fitting by reconvolution algorithms are routines to improve time resolution in fluorescence lifetime measurements. Iodide ions were successfully used to quench the fluorescence of fluorescein in this study. By systematically adding saturated NaI water solution in basic fluorescein solution, the lifetimes of fluorescein were reduced from 4 ns to 24 ps. The quenched lifetime of fluorescein obtained from the analysis of Time-Correlated Single Photon Counting (TCSPC) measurement agrees well with that from femtosecond frequency up-conversion measurement. In time resolved excitation spectra measurements, the IRF should be measured at various detection wavelengths providing scattring materials are used. This study could not only reduce the complexity of IRF measurement, but also avoid the existing color effect in system. This study should have wide applications in time resolved fluorescence spectroscopy and fluorescence lifetime imaging.

  13. Fluorescein transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejunum.

    PubMed

    Berginc, Katja; Zakelj, Simon; Levstik, Lea; Ursic, Darko; Kristl, Albin

    2007-05-01

    Membrane transport characteristics of a paracellular permeability marker fluorescein were evaluated using artificial membrane, Caco-2 cell monolayers and rat jejunum, all mounted in side-by-side diffusion cells. Modified Ringer buffers with varied pH values were applied as incubation salines on both sides of artificial membrane, cell culture monolayers or rat jejunum. Passive transport according to pH partition theory was determined using all three permeability models. In addition to that, active transport of fluorescein in the M-S (mucosal-to-serosal) direction through rat jejunum was observed. The highest M-S P(app) values regarding the active transport through the rat jejunum were observed in incubation saline with pH 6.5. Fluorescein transport through the rat jejunum was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and alpha-CHC (alpha-cyano-4-hydroxycinnamic acid). Thus, we assume that two pH-dependent influx transporters could be involved in the fluorescein membrane transport through the intestinal (jejunal) epithelium. One is very likely an MCT (monocarboxylic acid cotransporter) isoform, inhibited by specific MCT inhibitor alpha-CHC, while the involvement of the second one with overlapping substrate/inhibitor specificities (most probably a member of the organic anion-transporting polypeptide family, inhibited at least partially by DIDS) could not be excluded.

  14. Red-free light for measurement of intraocular pressure using Goldmann applanation tonometer without fluorescein.

    PubMed

    Ghoneim, Ehab M

    2014-01-01

    To evaluate the use of red-free light for the measurement of intraocular pressure (IOP) using a Goldmann applanation tonometer without fluorescein. This cross-sectional study was carried out on 500 eyes in 250 patients attending the Ophthalmology Outpatient Clinic at Suez Canal University Hospital. The IOP was measured using a Goldmann applanation tonometer mounted on a Haag-Streit slit-lamp. The measurements were performed first using red-free light without fluorescein. Then the measurements were repeated with cobalt blue light and topical fluorescein on the same eyes. The mean IOP was 15.23 ± 3.3 (SD) mm Hg using the red-free light without fluorescein, whereas it was 15.78 ± 3.7 (SD) mm Hg when measured using cobalt blue light after the application of fluorescein to the conjunctival sac. This difference was not statistically significant. Measurement of IOP with a Goldmann applanationtonometer with red-free light and without the use of fluorescein is simple, saves time, and gives an accurate IOP measurement relative to the traditional measurement technique with cobalt blue light and topical fluorescein.

  15. 40 CFR 205.55-5 - Labeling-exterior. [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Labeling-exterior. [Reserved] 205.55-5 Section 205.55-5 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS TRANSPORTATION EQUIPMENT NOISE EMISSION CONTROLS Medium and Heavy Trucks § 205.55-5 Labeling...

  16. Fabrication of triple-labeled polyelectrolyte microcapsules for localized ratiometric pH sensing.

    PubMed

    Song, Xiaoxue; Li, Huanbin; Tong, Weijun; Gao, Changyou

    2014-02-15

    Encapsulation of pH sensitive fluorophores as reporting molecules provides a powerful approach to visualize the transportation of multilayer capsules. In this study, two pH sensitive dyes (fluorescein and oregon green) and one pH insensitive dye (rhodamine B) were simultaneously labeled on the microcapsules to fabricate ratiometric pH sensors. The fluorescence of the triple-labeled microcapsule sensors was robust and nearly independent of other intracellular species. With a dynamic pH measurement range of 3.3-6.5, the microcapsules can report their localized pH at a real time. Cell culture experiments showed that the microcapsules could be internalized by RAW 246.7 cells naturally and finally accumulated in acidic organelles with a pH value of 5.08 ± 0.59 (mean ± s.d.; n=162). Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Temporal Characteristics of Sodium Fluorescein in the Tear Meniscus.

    PubMed

    Markoulli, Maria; Isa, Nur Amalina M D; Papas, Eric B

    2017-02-01

    To observe the emission intensity profile of sodium fluorescein in the human tear film as a function of time and concentration. Twenty-two participants with no dry eye signs or symptoms were randomly allocated to receive 1 μL of either a 2 or 10% concentration of fluorescein to one eye. Images of the inferior tear meniscus were captured at regular intervals over 30 minutes and the process repeated for the other eye with the alternate concentration. Fluorescence intensity was quantified on the basis of the grayscale pixel values in the tear meniscus images. The fluorescein-decay profile over time and between concentrations was determined. Peak fluorescence intensity was reached in 3.9 ± 3.0 and 8.7 ± 4.4 minutes after instillation for the 2 and 10% concentrations, respectively. The 10% concentration of fluorescein maintained its peak fluorescence intensity longer than the 2% concentration (about 9 and 2 minutes, respectively). The peak fluorescence intensity was not significantly different between the higher and lower concentrations (44 ± 37 vs. 38 ± 32 units, P = .22). For both concentrations, the observed intensity did not return to baseline levels by the end of the 30-minute observation time. The fluorescence intensity of fluorescein in a clinical setting varies with time such that both the onset and duration of maximum brightness are concentration dependent. At low concentration (2%), maximum brightness occurs almost immediately after instillation and lasts about 2 minutes. With a higher concentration (10%), the effective working window is delayed for about 7 to 8 minutes. Irrespective of initial concentration, observable fluorescence remains in the tear film beyond 30 minutes post-instillation.

  18. Analysis of chemical equilibrium of silicon-substituted fluorescein and its application to develop a scaffold for red fluorescent probes.

    PubMed

    Hirabayashi, Kazuhisa; Hanaoka, Kenjiro; Takayanagi, Toshio; Toki, Yuko; Egawa, Takahiro; Kamiya, Mako; Komatsu, Toru; Ueno, Tasuku; Terai, Takuya; Yoshida, Kengo; Uchiyama, Masanobu; Nagano, Tetsuo; Urano, Yasuteru

    2015-09-01

    Fluorescein is a representative green fluorophore that has been widely used as a scaffold of practically useful green fluorescent probes. Here, we report synthesis and characterization of a silicon-substituted fluorescein, i.e., 2-COOH TokyoMagenta (2-COOH TM), which is a fluorescein analogue in which the O atom at the 10' position of the xanthene moiety of fluorescein is replaced with a Si atom. This fluorescein analogue forms a spirolactone ring via intramolecular nucleophilic attack of the carboxylic group in a pH-dependent manner. Consequently, 2-COOH TM exhibits characteristic large pH-dependent absorption and fluorescence spectral changes: (1) 2-COOH TM is colorless at acidic pH, whereas fluorescein retains observable absorption and fluorescence even at acidic pH, and the absorption maximum is also shifted; (2) the absorption spectral change occurs above pH 7.0 for 2-COOH TM and below pH 7.0 for fluorescein; (3) 2-COOH TM shows a much sharper pH response than fluorescein because of its pKa inversion, i.e., pKa1 > pKa2. These features are also different from those of a compound without the carboxylic group, 2-Me TokyoMagenta (2-Me TM). Analysis of the chemical equilibrium between pH 3.0 and 11.0 disclosed that 2-COOH TM favors the colorless and nonfluorescent lactone form, compared with fluorescein. Substitution of Cl atoms at the 4' and 5' positions of the xanthene moiety of 2-COOH TM to obtain 2-COOH DCTM shifted the equilibrium so that the new derivative exists predominantly in the strongly fluorescent open form at physiological pH (pH 7.4). To demonstrate the practical utility of 2-COOH DCTM as a novel scaffold for red fluorescent probes, we employed it to develop a probe for β-galactosidase.

  19. A sensitive electrochemiluminescent biosensor based on AuNP-functionalized ITO for a label-free immunoassay of C-peptide.

    PubMed

    Liu, Xiang; Fang, Chen; Yan, Jilin; Li, Huiling; Tu, Yifeng

    2018-05-23

    The C-peptide is a co-product of pancreatic β-cells during insulin secretion; its content in body fluid is closely related to diabetes. This paper reports an immune-sensing strategy for a simple and effective assay of C-peptide based on label-free electrochemiluminescent (ECL) signaling, with high sensitivity and specificity. The basal electrode was constructed of an indium tin oxide (ITO) glass as a conductive substrate, which was decorated by Au nanoparticles (AuNPs) with hydrolysed (3-aminopropyl)trimethoxysilane as the linker. The characteristics of the fabricated electrode were investigated by electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. After immobilizing the C-peptide antibody, which takes great advantage of AuNPs' binding capacity, this immunosensor can quantify C-peptide using luminol as the ECL probe. By measuring ECL inhibition, calibration can be established to report the C-peptide concentration between 0.05 ng mL -1 and 100 ng mL -1 with a detection limit of 0.0142 ng mL -1 . As a proof of concept, the proposed strategy is a promising and versatile platform for the clinical diagnosis, classification, and research of diabetes. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

    PubMed

    Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

    2002-02-01

    We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

  1. Inhibition of Bladder Cancer by Broccoli Isothiocyanates Sulforaphane and Erucin: Characterization, Metabolism and Interconversion

    PubMed Central

    Abbaoui, Besma; Riedl, Kenneth M; Ralston, Robin A; Thomas-Ahner, Jennifer M; Schwartz, Steven J; Clinton, Steven K; Mortazavi, Amir

    2013-01-01

    Epidemiologic evidence suggests diets rich in cruciferous vegetables, particularly broccoli, are associated with lower bladder cancer risk. Our objectives are to investigate these observations and determine the role of isothiocyanates in primary or secondary bladder cancer prevention. We initially investigate the mechanisms whereby broccoli and broccoli sprout extracts and pure isothiocyanates inhibit normal, non-invasive (RT4) and invasive (J82, UMUC3) human urothelial cell viability. Sulforaphane (IC50= 5.66±1.2μM) and erucin (IC50= 8.79±1.3μM) are found to be the most potent inhibitors and normal cells are least sensitive. This observation is associated with downregulation of survivin, EGFR and HER2/neu, G2/M cell cycle accumulation and apoptosis. In a murine UMUC3 xenograft model, we fed semipurified diets containing 4% broccoli sprouts, or 2% broccoli sprout isothiocyanate extract; or gavaged pure sulforaphane or erucin (each at 295 μmol/kg, similar to dietary exposure); and report tumor weight reduction of 42% (p=0.02), 42% (p=0.04), 33% (p=0.04) and 58% (p<0.0001), respectively. Sulforaphane and erucin metabolites are present in mouse plasma (micromolar range) and tumor tissue, with N-acetyl cysteine conjugates as the most abundant. Interconversion of sulforaphane and erucin metabolites was observed. This work supports development of fully characterized, novel food products for phase I/II human studies targeting bladder cancer prevention. PMID:23038615

  2. A novel facile method of labeling octreotide with (18)F-fluorine.

    PubMed

    Laverman, Peter; McBride, William J; Sharkey, Robert M; Eek, Annemarie; Joosten, Lieke; Oyen, Wim J G; Goldenberg, David M; Boerman, Otto C

    2010-03-01

    Several methods have been developed to label peptides with (18)F. However, in general these are laborious and require a multistep synthesis. We present a facile method based on the chelation of (18)F-aluminum fluoride (Al(18)F) by 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The method is characterized by the labeling of NOTA-octreotide (NOTA-d-Phe-cyclo[Cys-Phe-d-Trp-Lys-Thr-Cys]-Throl (MH(+) 1305) [IMP466]) with (18)F. Octreotide was conjugated with the NOTA chelate and labeled with (18)F in a 2-step, 1-pot method. The labeling procedure was optimized with regard to the labeling buffer, peptide, and aluminum concentration. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of (18)F-IMP466 was studied in AR42J tumor-bearing mice and compared with that of (68)Ga-labeled IMP466. In addition, small-animal PET/CT images were acquired. IMP466 was labeled with Al(18)F in a single step with 50% yield. The labeled product was purified by high-performance liquid chromatography to remove unbound Al(18)F and unlabeled peptide. The radiolabeling, including purification, was performed in 45 min. The specific activity was 45,000 GBq/mmol, and the peptide was stable in serum for 4 h at 37 degrees C. Labeling was performed at pH 4.1 in sodium citrate, sodium acetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 2-(N-morpholino)ethanesulfonic acid buffer and was optimal in sodium acetate buffer. The apparent 50% inhibitory concentration of the (19)F-labeled IMP466 determined on AR42J cells was 3.6 nM. Biodistribution studies at 2 h after injection showed a high tumor uptake of (18)F-IMP466 (28.3 +/- 5.2 percentage injected dose per gram [%ID/g]; tumor-to-blood ratio, 300 +/- 90), which could be blocked by an excess of unlabeled peptide (8.6 +/- 0.7 %ID/g), indicating that the accumulation in the tumor was receptor-mediated. Biodistribution of (68)Ga-IMP466 was similar to that of (18)F-IMP466. (18)F

  3. Functional expression of the 11 human Organic Anion Transporting Polypeptides in insect cells reveals that sodium fluorescein is a general OATP substrate.

    PubMed

    Patik, Izabel; Kovacsics, Daniella; Német, Orsolya; Gera, Melinda; Várady, György; Stieger, Bruno; Hagenbuch, Bruno; Szakács, Gergely; Özvegy-Laczka, Csilla

    2015-12-15

    Organic Anion Transporting Polypeptides (OATPs), encoded by genes of the Solute Carrier Organic Anion (SLCO) family, are transmembrane proteins involved in the uptake of various compounds of endogenous or exogenous origin. In addition to their physiological roles, OATPs influence the pharmacokinetics and drug-drug interactions of several clinically relevant compounds. To examine the function and molecular interactions of human OATPs, including several poorly characterized family members, we expressed all 11 human OATPs at high levels in the baculovirus-Sf9 cell system. We measured the temperature- and inhibitor-sensitive cellular accumulation of sodium fluorescein and fluorescein-methotrexate, two fluorescent substrates of the OATPs, OATP1B1 and 1B3. OATP1B1 and 1B3 were functional in Sf9 cells, showing rapid uptake (t1/2(fluorescein-methotrexate) 2.64 and 4.16 min, and t1/2(fluorescein) 6.71 and 5.58 min for OATP1B1 and 1B3, respectively) and high-affinity transport (Km(fluorescein-methotrexate) 0.23 and 0.53 μM, and Km(fluorescein) 25.73 and 38.55 μM for OATP1B1 and 1B3, respectively) of both substrates. We found that sodium fluorescein is a general substrate of all human OATPs: 1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, while fluorescein-methotrexate is only transported by 1B1, 1B3, 1A2 and 2B1. Acidic extracellular pH greatly facilitated fluorescein uptake by all OATPs, and new molecular interactions were detected (between OATP2B1 and Imatinib, OATP3A1, 5A1 and 6A1 and estradiol 17-β-d-glucuronide, and OATP1C1 and 4C1 and prostaglandin E2). These studies demonstrate, for the first time, that the insect cell system is suitable for the functional analysis of the entire human OATP family, and for drug-OATP interaction screening. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Labeling Thiols on Proteins, Living Cells, and Tissues with Enhanced Emission Induced by FRET

    PubMed Central

    Yuan, Yue; Wang, Xijun; Mei, Bin; Zhang, Dongxin; Tang, Anming; An, Linna; He, Xiaoxiao; Jiang, Jun; Liang, Gaolin

    2013-01-01

    Using N-(2-Aminoethyl)maleimide-cysteine(StBu) (Mal-Cys) as a medium, protein thiols were converted into N-terminal cysteines. After a biocompatible condensation reaction between the N-terminal cysteine and fluorescent probe 2-cyanobenzothiazole-Gly-Gly-Gly-fluorescein isothiocyanate (CBT-GGG-FITC), a new fluorogenic structure Luciferin-GGG-FITC was obtained. The latter exhibits near one order of magnitude (7 folds) enhanced fluorescence emission compared to the precursor moiety due to fluorescence resonance energy transfer (FRET) effect between the newly formed luciferin structure and the FITC motif. Theoretical investigations revealed the underlying mechanism that satisfactorily explained the experimental results. With this method, enhanced fluorescence imaging of thiols on proteins, outer membranes of living cells, translocation of membrane proteins, and endothelial cell layers of small arteries was successfully achieved. PMID:24343586

  5. Labeling Thiols on Proteins, Living Cells, and Tissues with Enhanced Emission Induced by FRET

    NASA Astrophysics Data System (ADS)

    Yuan, Yue; Wang, Xijun; Mei, Bin; Zhang, Dongxin; Tang, Anming; An, Linna; He, Xiaoxiao; Jiang, Jun; Liang, Gaolin

    2013-12-01

    Using N-(2-Aminoethyl)maleimide-cysteine(StBu) (Mal-Cys) as a medium, protein thiols were converted into N-terminal cysteines. After a biocompatible condensation reaction between the N-terminal cysteine and fluorescent probe 2-cyanobenzothiazole-Gly-Gly-Gly-fluorescein isothiocyanate (CBT-GGG-FITC), a new fluorogenic structure Luciferin-GGG-FITC was obtained. The latter exhibits near one order of magnitude (7 folds) enhanced fluorescence emission compared to the precursor moiety due to fluorescence resonance energy transfer (FRET) effect between the newly formed luciferin structure and the FITC motif. Theoretical investigations revealed the underlying mechanism that satisfactorily explained the experimental results. With this method, enhanced fluorescence imaging of thiols on proteins, outer membranes of living cells, translocation of membrane proteins, and endothelial cell layers of small arteries was successfully achieved.

  6. Application of higher energy collisional dissociation (HCD) to the fragmentation of new DOTA-based labels and N-termini DOTA-labeled peptides.

    PubMed

    El-Khatib, A H; He, Y; Esteban-Fernández, D; Linscheid, M W

    2017-08-01

    1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) derivatives are applied in quantitative proteomics owing to their ability to react with different functional groups, to harbor lanthanoides and hence their compatibility with molecular and elemental mass spectrometry. The new DOTA derivatives, namely Ln-MeCAT-Click and Ln-DOTA-Dimedone, allow efficient thiol labeling and targeting sulfenation as an important post-translational modification, respectively. Quantitative applications require the investigation of fragmentation behavior of these reagents. Therefore, the fragmentation behavior of Ln-MeCAT-Click and Ln-DOTA-Dimedone was studied using collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and higher-energy collision dissociation (HCD) using different energy levels, and the efficiency of reporter ion production was estimated. The efficiency of characteristic fragment formation was in the order IRMPD > HCD (normal energy level) > CID. On the other hand, the application of HCD at high energy levels (HCD@HE; NCE > 250%) resulted in a significant increase in reporter ion production (33-54%). This new strategy was successfully applied to generate label-specific reporter ions for DOTA amino labeling at the N-termini and in a quantitative fashion for the estimation of amino:thiol ratio in peptides. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  7. Optimizing isothiocyanate formation during enzymatic glucosinolate breakdown by adjusting pH value, temperature and dilution in Brassica vegetables and Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Hanschen, Franziska S.; Klopsch, Rebecca; Oliviero, Teresa; Schreiner, Monika; Verkerk, Ruud; Dekker, Matthijs

    2017-01-01

    Consumption of glucosinolate-rich Brassicales vegetables is associated with a decreased risk of cancer with enzymatic hydrolysis of glucosinolates playing a key role. However, formation of health-promoting isothiocyanates is inhibited by the epithiospecifier protein in favour of nitriles and epithionitriles. Domestic processing conditions, such as changes in pH value, temperature or dilution, might also affect isothiocyanate formation. Therefore, the influences of these three factors were evaluated in accessions of Brassica rapa, Brassica oleracea, and Arabidopsis thaliana. Mathematical modelling was performed to determine optimal isothiocyanate formation conditions and to obtain knowledge on the kinetics of the reactions. At 22 °C and endogenous plant pH, nearly all investigated plants formed nitriles and epithionitriles instead of health-promoting isothiocyanates. Response surface models, however, clearly demonstrated that upon change in pH to domestic acidic (pH 4) or basic pH values (pH 8), isothiocyanate formation considerably increases. While temperature also affects this process, the pH value has the greatest impact. Further, a kinetic model showed that isothiocyanate formation strongly increases due to dilution. Finally, the results show that isothiocyanate intake can be strongly increased by optimizing the conditions of preparation of Brassicales vegetables.

  8. Peptide library synthesis on spectrally encoded beads for multiplexed protein/peptide bioassays

    NASA Astrophysics Data System (ADS)

    Nguyen, Huy Q.; Brower, Kara; Harink, Björn; Baxter, Brian; Thorn, Kurt S.; Fordyce, Polly M.

    2017-02-01

    Protein-peptide interactions are essential for cellular responses. Despite their importance, these interactions remain largely uncharacterized due to experimental challenges associated with their measurement. Current techniques (e.g. surface plasmon resonance, fluorescence polarization, and isothermal calorimetry) either require large amounts of purified material or direct fluorescent labeling, making high-throughput measurements laborious and expensive. In this report, we present a new technology for measuring antibody-peptide interactions in vitro that leverages spectrally encoded beads for biological multiplexing. Specific peptide sequences are synthesized directly on encoded beads with a 1:1 relationship between peptide sequence and embedded code, thereby making it possible to track many peptide sequences throughout the course of an experiment within a single small volume. We demonstrate the potential of these bead-bound peptide libraries by: (1) creating a set of 46 peptides composed of 3 commonly used epitope tags (myc, FLAG, and HA) and single amino-acid scanning mutants; (2) incubating with a mixture of fluorescently-labeled antimyc, anti-FLAG, and anti-HA antibodies; and (3) imaging these bead-bound libraries to simultaneously identify the embedded spectral code (and thus the sequence of the associated peptide) and quantify the amount of each antibody bound. To our knowledge, these data demonstrate the first customized peptide library synthesized directly on spectrally encoded beads. While the implementation of the technology provided here is a high-affinity antibody/protein interaction with a small code space, we believe this platform can be broadly applicable to any range of peptide screening applications, with the capability to multiplex into libraries of hundreds to thousands of peptides in a single assay.

  9. 46 CFR 160.037-5 - Labeling and marking.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Hand Orange Smoke Distress Signals § 160.037-5 Labeling and marking. (a) Labeling. Each hand orange smoke distress signal shall bear a label securely affixed thereto...: (Company brand or style designation) Hand Orange Smoke Distress Signal For daytime use—50 seconds burning...

  10. 46 CFR 160.037-5 - Labeling and marking.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Hand Orange Smoke Distress Signals § 160.037-5 Labeling and marking. (a) Labeling. Each hand orange smoke distress signal shall bear a label securely affixed thereto...: (Company brand or style designation) Hand Orange Smoke Distress Signal For daytime use—50 seconds burning...

  11. 46 CFR 160.037-5 - Labeling and marking.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Hand Orange Smoke Distress Signals § 160.037-5 Labeling and marking. (a) Labeling. Each hand orange smoke distress signal shall bear a label securely affixed thereto...: (Company brand or style designation) Hand Orange Smoke Distress Signal For daytime use—50 seconds burning...

  12. 46 CFR 160.037-5 - Labeling and marking.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Hand Orange Smoke Distress Signals § 160.037-5 Labeling and marking. (a) Labeling. Each hand orange smoke distress signal shall bear a label securely affixed thereto...: (Company brand or style designation) Hand Orange Smoke Distress Signal For daytime use—50 seconds burning...

  13. Investigation of the weak binding of a tetrahistidine-tagged peptide to quantum dots by using capillary electrophoresis with fluorescence detection.

    PubMed

    Qin, Haifang; Jiang, Xiyuan; Fan, Jie; Wang, Jianpeng; Liu, Li; Qiu, Lin; Wang, Jianhao; Jiang, Pengju

    2017-01-01

    Capillary electrophoresis with fluorescence detection was utilized to probe the self-assembly between cyanine group dye labeled tetrahistidine containing peptide and CdSe/ZnS quantum dots, inside the capillary. Quantum dots and cyanine group dye labeled tetrahistidine containing peptide were injected into the capillary one after the other and allowed to self-assemble. Their self-assembly resulted into a measurable Förster resonance energy transfer signal between quantum dots and cyanine group dye labeled tetrahistidine containing peptide. The Förster resonance energy transfer signal increased upon increasing the cyanine group dye labeled tetrahistidine containing peptide/quantum dot molar ratio and reached a plateau at the 32/1 molar ratio. Additionally, the Förster resonance energy transfer signal was also affected by the increment of the interval time of injection and the sampling time. Online ligand exchange experiments were used to assess, the potential of a monovalent ligand of imidazole and a hexavalent ligand peptide, to displace surface bound cyanine group dye labeled peptide ligands from the quantum dots surface. Under optimal conditions, a linear relationship between the integrated peak areas and hexavalent ligand peptide was obtained at a hexavalent ligand concentration range of 0-0.5 mM. Therefore, the present assay has the potential to be applied in the online ligands detection. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Rapid, High Affinity Binding by a Fluorescein Templated Copolymer Combining Covalent, Hydrophobic, and Acid–Base Noncovalent Crosslinks

    PubMed Central

    Timberman, Anthony; Yang, Rongfang; Papantones, Alex; Seitz, W. Rudolf

    2018-01-01

    A new type of biomimetic templated copolymer has been prepared by reverse addition fragmentation chain transfer polymerization (RAFT) in dioxane. The initial formulation includes the template fluorescein, N-isopropylacrylamide (NIPAM, 84 mol %), methacrylic acid (MAA, 5-mol %), 4-vinylpyridine (4-VP, 9 mmol %), and N,N′-methylenebis(acrylamide) (MBA, 2 mol %). PolyNIPAM is a thermosensitive polymer that comes out of aqueous solution above its lower critical solution temperature forming hydrophobic ‘crosslinks’. MAA and 4-VP interact in dioxane forming acid–base crosslinks. The excess 4-VP serves as a recognition monomer organizing around the template fluorescein to form a binding site that is held in place by the noncovalent and covalent crosslinks. The MBA is a covalent crosslinker. The RAFT agent in the resulting copolylmer was reduced to a thiol and attached to gold nanoparticles. The gold nanoparticle bound copolymer binds fluorescein completely in less than two seconds with an affinity constant greater than 108 M−1. A reference copolymer prepared with the same monomers by the same procedure binds fluorescein much more weakly. PMID:29693601

  15. Improved Facial Nerve Identification During Parotidectomy With Fluorescently Labeled Peptide

    PubMed Central

    Hussain, Timon; Nguyen, Linda T.; Whitney, Michael; Hasselmann, Jonathan; Nguyen, Quyen T.

    2016-01-01

    Objectives/Hypothesis Additional intraoperative guidance could reduce the risk of iatrogenic injury during parotid gland cancer surgery. We evaluated the intraoperative use of fluorescently labeled nerve binding peptide NP41 to aid facial nerve identification and preservation during parotidectomy in an orthotopic model of murine parotid gland cancer. We also quantified the accuracy of intraoperative nerve detection for surface and buried nerves in the head and neck with NP41 versus white light (WL) alone. Study Design Twenty-eight mice underwent parotid gland cancer surgeries with additional fluorescence (FL) guidance versus WL reflectance (WLR) alone. Eight mice were used for additional nerve-imaging experiments. Methods Twenty-eight parotid tumor-bearing mice underwent parotidectomy. Eight mice underwent imaging of both sides of the face after skin removal. Postoperative assessment of facial nerve function measured by automated whisker tracking were compared between FL guidance (n = 13) versus WL alone (n = 15). In eight mice, nerve to surrounding tissue contrast was measured under FL versus WLR for all nerve branches detectable in the field of view. Results Postoperative facial nerve function after parotid gland cancer surgery tended to be better with additional FL guidance. Fluorescent labeling significantly improved nerve to surrounding tissue contrast for both large and smaller buried nerve branches compared to WLR visualization and improved detection sensitivity and specificity. Conclusions NP41 FL imaging significantly aids the intraoperative identification of nerve braches otherwise nearly invisible to the naked eye. Its application in a murine model of parotid gland cancer surgery tended to improve functional preservation of the facial nerve. PMID:27171862

  16. The effect of the hydrophilic/hydrophobic ratio of polymeric micelles on their endocytosis pathways into cells.

    PubMed

    Zhang, Zhao; Qu, Qianqian; Li, Jinrong; Zhou, Shaobing

    2013-06-01

    Fluorescein isothiocyanate (FITC), a fluorescent probe, is coupled to amphiphilic monomethoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) copolymers. FITC-labeled mPEG-PCL copolymers self-assemble into micelles through the solvent evaporation method. The cellular internalization is examined using fluorescence microscopy on incubation of NIH-3T3 fibroblasts with micelles or free FITC solution. The effect of the hydrophilic/hydrophobic ratio on the endocytosis mechanisms is evaluated by fluorescence microscopy on culturing of human hepatoblastoma cells and human umbilical vein endothelial cells, individually, mixed with the micelles holding the same parameters including micelle size, shape, and surface charges. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Use of indocyanine green and sodium fluorescein for anterior segment angiography in ophthalmologically normal cats.

    PubMed

    Pirie, Chris G; Alario, Anthony

    2015-10-01

    To assess and compare results of anterior segment angiography of ophthalmologically normal cats following IV injection with indocyanine green and sodium fluorescein dyes. 10 client-owned cats. Anterior segment angiography was performed in anesthetized cats following administration of 0.25% indocyanine green (1.0 mg/kg, IV) or 10% sodium fluorescein (20 mg/kg, IV) solution. All cats received both treatments. Imaging (1 eye/cat) was performed with a full-spectrum digital single-lens reflex camera equipped with an adaptor (1 image/s for 30 seconds) immediately following IV dye injection and 1, 2, 3, 4, and 5 minutes after injection. Onset and duration of arterial, capillary, and venous phases of iris vasculature were identified and compared statistically between treatments. Degree of iridal pigmentation, leakage of dye from iris vasculature, and image quality were subjectively assessed. No differences were found in onset or duration of vascular phases between treatments. Visibility of the iris vasculature was not impaired by poor or moderate iridal pigmentation with either method. Indocyanine green provided subjectively better vascular detail and image contrast than sodium fluorescein. No vascular dye leakage was observed following indocyanine green administration. Leakage of dye from blood vessels in the stroma (in 10 cats) and presence of dye in the anterior chamber (in 5 cats) were detected after sodium fluorescein administration. Images obtained with either fluorescent dye were considered to be of diagnostic quality. Lack of leakage following indocyanine green administration suggested this treatment may have better diagnostic utility for anterior segment angiography. The photographic equipment used provided a cost-effective alternative to existing imaging systems.

  18. Ultrasonic irradiation enhanced the ability of Fluorescein-DA-Fe(III) on sonodynamic and sonocatalytic damages of DNA molecules.

    PubMed

    Wu, Qiong; Chen, Xia; Jia, Lizhen; Wang, Yi; Sun, Ying; Huang, Xingjun; Shen, Yuxiang; Wang, Jun

    2017-11-01

    The interaction of DNA with Bis [N,N-bis (carboxymethyl) aminomethyl] fluorescein-Ferrous(III) (Fluorescein-DA-Fe(III)) with dual functional (sonodynamic and sonocatalytic) activity was studied by UV-vis spectroscopy, fluorescence spectroscopy, FT-IR spectroscopy, circular dichroism (CD) spectroscopy and viscosity measurements. And then, the damage of DNA caused by Fluorescein-DA-Fe(III) under ultrasonic irradiation (US) was researched by agarose gel electrophoresis and cytotoxicity assay. Meanwhile, some influenced factors such as ultrasonic irradiation time and Fluorescein-DA-Fe(III) concentration on the damage degree of DNA molecules were also examined. As a control, for Bis [N,N-bis (carboxymethyl) aminomethyl] fluorescein (Fluorescein-DA), the same experiments were carried out. The results showed that both Fluorescein-DA-Fe(III) and Fluorescein-DA can interact with DNA molecules. Under ultrasonic irradiation, Fluorescein-DA shows sonodynamic activity, which can damage DNA molecules. While, in the presence of Fe(III) ion, the Fluorescein-DA-Fe(III) displays not only sonodynamic activity but also sonocatalytic activity under ultrasonic irradiation, which injures DNA more serious than Fluorescein-DA. The researches confirmed the dual function (sonodynamic activity and sonocatalytic activity) of Fluorescein-DA-Fe(III) and expanded the usage of Fluorescein-DA-Fe(III) as a sonosensitizer in sonodynamic therapy (SDT). Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates.

    PubMed

    Teixeira, Luis Gustavo D; Malavolta, Luciana; Bersanetti, Patrícia A; Schreier, Shirley; Carmona, Adriana K; Nakaie, Clovis R

    2015-01-01

    Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the

  20. Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

    PubMed Central

    Teixeira, Luis Gustavo D.; Malavolta, Luciana; Bersanetti, Patrícia A.; Schreier, Shirley; Carmona, Adriana K.; Nakaie, Clovis R.

    2015-01-01

    Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the

  1. Labeled Antimicrobial Peptides for Detection of Microorganisms

    DTIC Science & Technology

    2008-12-01

    1. INTRODUCTION Antimicrobial peptides (AMPs) are part of the innate defense system found in all organisms to protect them from microbial infection...2005) with antimicrobial activity against predominantly gram-negative bacteria. SMAP29 is from the cathelicidin family of peptides found in sheep ...in buffer, milk and apple juice. Cells were grown and prepared in PBST as described above. 20 III anti-£. coli 0157 paramagnetic Dyna-beads (Dynal

  2. 9 CFR 317.5 - Generically approved labeling.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Section 317.5 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE..., without such labeling being submitted for approval to the Food Safety and Inspection Service in Washington... particular. (2) The Food Safety and Inspection Service shall select samples of generically approved labeling...

  3. Targeting of follicle stimulating hormone peptide-conjugated dendrimers to ovarian cancer cells

    NASA Astrophysics Data System (ADS)

    Modi, Dimple A.; Sunoqrot, Suhair; Bugno, Jason; Lantvit, Daniel D.; Hong, Seungpyo; Burdette, Joanna E.

    2014-02-01

    Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles.Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side

  4. Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jacobs, R.M.; Boyce, J.T.; Kociba, G.J.

    This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation timemore » studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats.« less

  5. Magneto-actuated immunoassay for the detection of Mycobacterium fortuitum in hemodialysis water.

    PubMed

    Brugnera, Michelle Fernanda; Bundalian, Reynaldo; Laube, Tamara; Julián, Esther; Luquin, Marina; Zanoni, Maria Valnice Boldrin; Pividori, Maria Isabel

    2016-06-01

    This paper addresses a sensitive method for the detection of mycobacteria in hemodialysis water samples based on a magneto-actuated immunoassay with optical readout. In this approach, micro (2.8μm) sized magnetic particles were modified with an antibody against the lipoarabinomannan (LAM) located in the mycobacterial cell wall. The system relies on the immunocapturing of the mycobacteria with the tailored antiLAM magnetic particles to pre-concentrate the bacteria from the hemodialysis samples throughout an immunological reaction. The performance of the immunomagnetic separation on the magnetic carrier was evaluated using confocal microscopy to study the binding pattern, as well as a magneto-actuated immunoassay with optical readout for the rapid detection of the bacteria in spiked hemodialysis samples. In this approach, the antiLAM polyclonal antibody was labeled with fluorescein isothiocyanate. The optical readout was achieved by the incubation with a secondary anti-fluorescein antibody labeled with peroxidase as optical reporter. The magneto-actuated immunoassay was able to detect mycobacteria contamination in hemodialysis water at a limit of detection of 13CFUmL(-1) in a total assay time of 3h without any previous culturing pre-enrichment step. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Isothiocyanates, Nitriles, and Epithionitriles from Glucosinolates Are Affected by Genotype and Developmental Stage in Brassica oleracea Varieties.

    PubMed

    Hanschen, Franziska S; Schreiner, Monika

    2017-01-01

    Vegetables of the Brassica oleracea group, such as broccoli, cauliflower, and cabbage, play an important role for glucosinolate consumption in the human diet. Upon maceration of the vegetable tissue, glucosinolates are degraded enzymatically to form volatile isothiocyanates, nitriles, and epithionitriles. However, only the uptake of isothiocyanates is linked to the cancer-preventive effects. Thus, it is of great interest to evaluate especially the isothiocyanate formation. Here, we studied the formation of glucosinolates and their respective hydrolysis products in sprouts and fully developed vegetable heads of different genotypes of the five B. oleracea varieties: broccoli, cauliflower as well as white, red, and savoy cabbages. Further, the effect of ontogeny (developmental stages) during the head development on the formation of glucosinolates and their respective hydrolysis products was evaluated at three different developmental stages (mini, fully developed, and over-mature head). Broccoli and red cabbage were mainly rich in 4-(methylsulfinyl)butyl glucosinolate (glucoraphanin), whereas cauliflower, savoy cabbage and white cabbage contained mainly 2-propenyl (sinigrin) and 3-(methylsulfinyl)propyl glucosinolate (glucoiberin). Upon hydrolysis, epithionitriles or nitriles were often observed to be the main hydrolysis products, with 1-cyano-2,3-epithiopropane being most abundant with up to 5.7 μmol/g fresh weight in white cabbage sprouts. Notably, sprouts often contained more than 10 times more glucosinolates or their hydrolysis products compared to fully developed vegetables. Moreover, during head development, both glucosinolate concentrations as well as hydrolysis product concentrations changed and mini heads contained the highest isothiocyanate concentrations. Thus, from a cancer-preventive point of view, consumption of mini heads of the B. oleracea varieties is recommended.

  7. Isothiocyanates, Nitriles, and Epithionitriles from Glucosinolates Are Affected by Genotype and Developmental Stage in Brassica oleracea Varieties

    PubMed Central

    Hanschen, Franziska S.; Schreiner, Monika

    2017-01-01

    Vegetables of the Brassica oleracea group, such as broccoli, cauliflower, and cabbage, play an important role for glucosinolate consumption in the human diet. Upon maceration of the vegetable tissue, glucosinolates are degraded enzymatically to form volatile isothiocyanates, nitriles, and epithionitriles. However, only the uptake of isothiocyanates is linked to the cancer-preventive effects. Thus, it is of great interest to evaluate especially the isothiocyanate formation. Here, we studied the formation of glucosinolates and their respective hydrolysis products in sprouts and fully developed vegetable heads of different genotypes of the five B. oleracea varieties: broccoli, cauliflower as well as white, red, and savoy cabbages. Further, the effect of ontogeny (developmental stages) during the head development on the formation of glucosinolates and their respective hydrolysis products was evaluated at three different developmental stages (mini, fully developed, and over-mature head). Broccoli and red cabbage were mainly rich in 4-(methylsulfinyl)butyl glucosinolate (glucoraphanin), whereas cauliflower, savoy cabbage and white cabbage contained mainly 2-propenyl (sinigrin) and 3-(methylsulfinyl)propyl glucosinolate (glucoiberin). Upon hydrolysis, epithionitriles or nitriles were often observed to be the main hydrolysis products, with 1-cyano-2,3-epithiopropane being most abundant with up to 5.7 μmol/g fresh weight in white cabbage sprouts. Notably, sprouts often contained more than 10 times more glucosinolates or their hydrolysis products compared to fully developed vegetables. Moreover, during head development, both glucosinolate concentrations as well as hydrolysis product concentrations changed and mini heads contained the highest isothiocyanate concentrations. Thus, from a cancer-preventive point of view, consumption of mini heads of the B. oleracea varieties is recommended. PMID:28690627

  8. PET Imaging of VEGFR-2 Expression in Lung Cancer with 64Cu-Labeled Ramucirumab.

    PubMed

    Luo, Haiming; England, Christopher G; Graves, Stephen A; Sun, Haiyan; Liu, Glenn; Nickles, Robert J; Cai, Weibo

    2016-02-01

    Lung cancer accounts for 17% of cancer-related deaths worldwide, and most patients present with locally advanced or metastatic disease. Novel PET imaging agents for assessing vascular endothelial growth factor receptor-2 (VEGFR-2) expression can be used for detecting VEGFR-2-positive malignancies and subsequent monitoring of therapeutic response to VEGFR-2-targeted therapies. Here, we report the synthesis and characterization of an antibody-based imaging agent for PET imaging of VEGFR-2 expression in vivo. Ramucirumab (named RamAb), a fully humanized IgG1 monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and labeled with (64)Cu. Flow cytometry analysis and microscopy studies were performed to compare the VEGFR-2 binding affinity of RamAb and NOTA-RamAb. PET imaging and biodistribution studies were performed in nude mice bearing HCC4006 and A549 xenograft tumors. Ex vivo histopathology was performed to elucidate the expression patterns of VEGFR-2 in different tissues and organs to validate in vivo results. Flow cytometry examination revealed the specific binding capacity of fluorescein isothiocyanate-RamAb to VEGFR-2, and no difference in VEGFR-2 binding affinity was seen between RamAb and NOTA-RamAb. After being labeled with (64)Cu, PET imaging revealed specific and prominent uptake of (64)Cu-NOTA-RamAb in VEGFR-2-positive HCC4006 tumors (9.4 ± 0.5 percentage injected dose per gram at 48 h after injection; n = 4) and significantly lower uptake in VEGFR-2-negative A549 tumors (4.3 ± 0.2 percentage injected dose per gram at 48 h after injection; n = 3). Blocking experiments revealed significantly lower uptake in HCC4006 tumors, along with histology analysis, further confirming the VEGFR-2 specificity of (64)Cu-NOTA-RamAb. This study provides initial evidence that (64)Cu-NOTA-RamAb can function as a PET imaging agent for visualizing VEGFR-2 expression in vivo, which may also find

  9. Phenethyl Isothiocyanate: A comprehensive review of anti-cancer mechanisms

    PubMed Central

    Gupta, Parul; Wright, Stephen E.; Kim, Sung-Hoon; Srivastava, Sanjay K.

    2014-01-01

    The epidemiological evidence suggests a strong inverse relationship between dietary intake of cruciferous vegetables and the incidence of cancer. Among other constituents of cruciferous vegetables, isothiocyanates (ITC) are the main bioactive chemicals present. Phenethyl isothiocyanate (PEITC) is present as gluconasturtiin in many cruciferous vegetables with remarkable anti-cancer effects. PEITC is known to not only prevent the initiation phase of carcinogenesis process but also to inhibit the progression of tumorigenesis. PEITC targets multiple proteins to suppress various cancer-promoting mechanisms such as cell proliferation, progression and metastasis. Pre-clinical evidence suggests that combination of PEITC with conventional anti-cancer agents is also highly effective in improving overall efficacy. Based on accumulating evidence, PEITC appears to be a promising agent for cancer therapy and is already under clinical trials for leukemia and lung cancer. This is the first review which provides a comprehensive analysis of known targets and mechanisms along with a critical evaluation of PEITC as a future anti-cancer agent. PMID:25152445

  10. Lumican Peptides: Rational Design Targeting ALK5/TGFBRI

    NASA Astrophysics Data System (ADS)

    Gesteira, Tarsis Ferreira; Coulson-Thomas, Vivien J.; Yuan, Yong; Zhang, Jianhua; Nader, Helena B.; Kao, Winston W.-Y.

    2017-02-01

    Lumican, a small leucine rich proteoglycan (SLRP), is a component of extracellular matrix which also functions as a matrikine regulating multiple cell activities. In the cornea, lumican maintains corneal transparency by regulating collagen fibrillogenesis, promoting corneal epithelial wound healing, regulating gene expression and maintaining corneal homeostasis. We have recently shown that a peptide designed from the 13 C-terminal amino acids of lumican (LumC13) binds to ALK5/TGFBR1 (type1 receptor of TGFβ) to promote wound healing. Herein we evaluate the mechanism by which this synthetic C-terminal amphiphilic peptide (LumC13), binds to ALK5. These studies clearly reveal that LumC13-ALK5 form a stable complex. In order to determine the minimal amino acids required for the formation of a stable lumican/ALK5 complex derivatives of LumC13 were designed and their binding to ALK5 investigated in silico. These LumC13 derivatives were tested both in vitro and in vivo to evaluate their ability to promote corneal epithelial cell migration and corneal wound healing, respectively. These validations add to the therapeutic value of LumC13 (Lumikine) and aid its clinical relevance of promoting the healing of corneal epithelium debridement. Moreover, our data validates the efficacy of our computational approach to design active peptides based on interactions of receptor and chemokine/ligand.

  11. A new automated NaCl based robust method for routine production of gallium-68 labeled peptides

    PubMed Central

    Schultz, Michael K.; Mueller, Dirk; Baum, Richard P.; Watkins, G. Leonard; Breeman, Wouter A. P.

    2017-01-01

    A new NaCl based method for preparation of gallium-68 labeled radiopharmaceuticals has been adapted for use with an automated gallium-68 generator system. The method was evaluated based on 56 preparations of [68Ga]DOTATOC and compared to a similar acetone-based approach. Advantages of the new NaCl approach include reduced preparation time (< 15 min) and removal of organic solvents. The method produces high peptide-bound % (> 97%), and specific activity (> 40 MBq nmole−1 [68Ga]DOTATOC) and is well-suited for clinical production of radiopharmaceuticals. PMID:23026223

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

    PubMed

    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.

  13. Investigation on interaction and sonodynamic damage of fluorescein derivants to bovine serum albumin (BSA) under ultrasonic irradiation

    NASA Astrophysics Data System (ADS)

    Zou, Mingming; Zhang, Lei; Wang, Jun; Wang, Qi; Gao, Jingqun; Fan, Ping

    2013-06-01

    The fluorescein derivants (Fluorescein: (2-(6-Hydroxy-3-oxo-(3H)-xanthen-9-yl) benzoic acid), Fluorescein-DA: (Bis [N,N-bis (carboxymethyl) aminomethyl] fluorescein) and Fluorescein-DAsbnd Fe(III): (Bis [N,N-bis (carboxymethyl) aminomethyl] fluoresceinsbnd Ferrous(III)) with a tricyclic plane structure were used to study the interaction and sonodynamic damage to bovine serum albumin (BSA) under ultrasonic irradiation through fluorospectrometry and UV-vis spectrophotometry. Besides, because of the existence of Fe(III) ion in Fluorescein-DAsbnd Fe(III), under ultrasonic irradiation the sonocatalytic activity in the damage of BSA molecules was also found. Three-dimensional fluorescence spectra and three-dimensional fluorescence contour profile spectra were mentioned to determine the fluorescence quenching and the conformation change of BSA in the absence and presence of these fluorescein derivants. As judged from the experimental results, the fluorescence quenching of BSA in aqueous solution caused by these fluorescein derivants were all attributed to static quenching process. The damage degree and mode were related to some factors such as ultrasonic irradiation time, fluorescein derivant concentration and ionic strength. Finally, several quenchers were used to determine the amount and kind of generated reactive oxygen species (ROS) during sonodynamic and sonocatalytic reaction processes. It suggests that these fluorescein derivants induce protein damage via various ROS, at least, including singlet oxygen (1O2) and hydroxyl radicals (rad OH). Perhaps, this paper may offer some important subjects for broadening the application of these fluorescein derivants in sonodynamic therapy (SDT) and sonocatalytic therapy (SCT) technologies for tumor treatment.

  14. Collision-induced dissociation of diazirine-labeled peptide ions. Evidence for Brønsted-acid assisted elimination of nitrogen.

    PubMed

    Marek, Aleš; Tureček, František

    2014-05-01

    Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile diazirine ring. Collisional activation of gas-phase peptide cations resulted in facile N₂ elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N₂ elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N₂ from the diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed diazirine ring cleavage and N₂ loss is proposed as a thermometer dissociation for peptide ion dissociations.

  15. Targeted delivery of polyoxometalate nanocomposites.

    PubMed

    Geisberger, Georg; Paulus, Susann; Gyenge, Emina Besic; Maake, Caroline; Patzke, Greta R

    2011-10-04

    Polyoxometalate/carboxymethyl chitosan nanocomposites with an average diameter of 130 nm are synthesized and labeled with fluorescein isothiocyanate (FITC) for a combined drug-carrier and cellular-monitoring approach. [Eu(β(2) -SiW(11) O(39) )(2) ](13-) /CMC nanospheres as a representative example do not display cytotoxicity for POM concentrations up to 2 mg mL(-1) . Cellular uptake of fluoresecently labelled {EuSiW(11) O(39) }/FITC-CMC nanoparticles is monitored with confocal laser scanning microscopy. Nanoparticle uptake occurs after incubation times of around 1 h and no cyctotoxic effects are observed upon prolonged treatment. The preferential location of the POM/CMC nanocomposites in the perinuclear region is furthermore verified with transmission electron microscopy investigations on unlabeled nanoparticles. Therefore, this approach is a promising dual strategy for the safe cellular transfer and monitoring of bioactive POMs. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Low-dose intrathecal fluorescein for diagnosis of cerebrospinal fluid rhinorrhea using the scanning fiber endoscope in the human nasal cavities

    NASA Astrophysics Data System (ADS)

    Hou, Vivian W.; Davis, Calvin G.; Davis, Greg E.; Seibel, Eric J.

    2016-03-01

    Intrathecal fluorescein (ITF) enhances detection of cerebrospinal fluid rhinorrhea (CSFR). Clinically administered doses fall in the range of 0.1ml to 0.5ml of 5% to 10% fluorescein (1.3×10-3M to 1.3×10-2M). Though uncommon, significant morbidities associated with high doses of fluorescein have been reported. High concentrations are necessary for white light visual assessment; in contrast, fluorescent imaging enhances signal contrast and requires lower ITF concentrations for visualization. The ultrathin and flexible, multimodal scanning fiber endoscope (SFE) can visualize nanomolar concentrations of fluorescein as pseudocolor over reflectance, video-rate imaging. The application of the SFE for CSFR detection was assessed in a cadaver study. Briefly, 10μM (1×10-5M) fluorescein, 100X-1000X less than the standard clinical dose, was injected intra-cranially into the epidural space through an orbital roof puncture. The resulting rhinorrhea was assessed with a conventional, rigid ENT scope and second with the SFE in both video reflectance and multimodal fluorescent imaging modes. Neither system could visualize the 10μM ITF during white light imaging however the nanomolar sensitive SFE visualized the rhinorrhea during fluorescent imaging. Despite the low concentration used, a target-to-background ratio of 5.6 +/- 2.7 was achieved. To demonstrate SFE guidance of CSFR detection and repair, de-identified patient computed tomography (CT) scans were used to generate 3D printed phantoms. Cases were selected for unique anatomical features and overall clinical difficulty as determined by an experienced ENT clinician (GED). The sensitivity and minimally invasive nature of the SFE provide a unique platform for enhancing diagnosis and monitoring interventions in surgical endoscopic approaches into the sinuses.

  17. Improved facial nerve identification during parotidectomy with fluorescently labeled peptide.

    PubMed

    Hussain, Timon; Nguyen, Linda T; Whitney, Michael; Hasselmann, Jonathan; Nguyen, Quyen T

    2016-12-01

    Additional intraoperative guidance could reduce the risk of iatrogenic injury during parotid gland cancer surgery. We evaluated the intraoperative use of fluorescently labeled nerve binding peptide NP41 to aid facial nerve identification and preservation during parotidectomy in an orthotopic model of murine parotid gland cancer. We also quantified the accuracy of intraoperative nerve detection for surface and buried nerves in the head and neck with NP41 versus white light (WL) alone. Twenty-eight mice underwent parotid gland cancer surgeries with additional fluorescence (FL) guidance versus WL reflectance (WLR) alone. Eight mice were used for additional nerve-imaging experiments. Twenty-eight parotid tumor-bearing mice underwent parotidectomy. Eight mice underwent imaging of both sides of the face after skin removal. Postoperative assessment of facial nerve function measured by automated whisker tracking were compared between FL guidance (n = 13) versus WL alone (n=15). In eight mice, nerve to surrounding tissue contrast was measured under FL versus WLR for all nerve branches detectable in the field of view. Postoperative facial nerve function after parotid gland cancer surgery tended to be better with additional FL guidance. Fluorescent labeling significantly improved nerve to surrounding tissue contrast for both large and smaller buried nerve branches compared to WLR visualization and improved detection sensitivity and specificity. NP41 FL imaging significantly aids the intraoperative identification of nerve braches otherwise nearly invisible to the naked eye. Its application in a murine model of parotid gland cancer surgery tended to improve functional preservation of the facial nerve. NA Laryngoscope, 126:2711-2717, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

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

    PubMed Central

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

    2013-01-01

    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-(Asp)n (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-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with 67Ga, resulting in 67Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of 67Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, 67Ga-DOTA-(Asp)8, 67Ga-DOTA-(Asp)11, and 67Ga-DOTA-(Asp)14 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-(Asp)n was lower than that of 67Ga-DOTA-Bn-SCN-HBP, blood clearance of 67Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of 67Ga-DOTA-(Asp)11 and 67Ga-DOTA-(Asp)14 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. PMID:24391942

  19. Detection of disseminated peritoneal tumors by fluorescein diacrylate in mice

    NASA Astrophysics Data System (ADS)

    Harada, Yoshinori; Furuta, Hirokazu; Murayama, Yasutoshi; Dai, Ping; Fujikawa, Yuta; Urano, Yasuteru; Nagano, Tetsuo; Morishita, Koki; Hasegawa, Akira; Takamatsu, Tetsuro

    2009-02-01

    Tumor invasion to the peritoneum is a poor prognostic factor in cancer patients. Accurate diagnosis of disseminated peritoneal tumors is essential to accurate cancer staging. To date, peritoneal washing cytology during laparotomy has been used for diagnosis of peritoneal dissemination of gastrointestinal cancer, but its sensitivity has not been satisfactory. Thus, a more direct approach is indispensable to detect peritoneal dissemination in vivo. Fluorescein diacrylate (FDAcr) is an esterase-sensitive fluorescent probe derived from fluorescein. In cancer cells, fluorescent fluorescein generated by exogenous application of FDAcr selectively deposits owing to its stronger hydrolytic enzyme activity and its lower leakage rate. We examined whether FDAcr can specifically detect disseminated peritoneal tumors in athymic nude mouse models. Intraperitoneally administered FDAcr revealed disseminated peritoneal microscopic tumors not readily recognized on white-light imaging. These results suggest that FDAcr is a useful probe for detecting disseminated peritoneal tumors.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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.

  1. Direct and indirect antioxidant activity of polyphenol- and isothiocyanate-enriched fractions from Moringa oleifera.

    PubMed

    Tumer, Tugba Boyunegmez; Rojas-Silva, Patricio; Poulev, Alexander; Raskin, Ilya; Waterman, Carrie

    2015-02-11

    Moringa oleifera Lam. is a fast-growing, tropical tree with various edible parts used as nutritious food and traditional medicine. This study describes an efficient preparatory strategy to extract and fractionate moringa leaves by fast centrifugal partition chromatography (FCPC) to produce polyphenol and isothiocyanate (ITC) rich fractions. Characterization and further purification of these fractions showed that moringa polyphenols were potent direct antioxidants assayed by oxygen radical absorbance capacity (ORAC), whereas moringa ITCs were effective indirect antioxidants assayed by induction of NAD(P)H quinone oxidoreductase 1 (NQO1) activity in Hepa1c1c7 cells. In addition, purified 4-[(α-l-rhamnosyloxy)benzyl]isothiocyanate and 4-[(4'-O-acetyl-α-l-rhamnosyloxy)benzyl]isothiocyanate were further evaluated for their ORAC and NQO1 inducer potency in comparison with sulforaphane (SF). Both ITCs were as potent as SF in inducing NQO1 activity. These findings suggest that moringa leaves contain a potent mixture of direct and indirect antioxidants that can explain its various health-promoting effects.

  2. Dietary isothiocyanates, glutathione S-transferase M1 (GSTM1), and lung cancer risk in African Americans and Caucasians from Los Angeles County, California.

    PubMed

    Carpenter, Catherine L; Yu, Mimi C; London, Stephanie J

    2009-01-01

    Isothiocyanates, found in cruciferous vegetables, are anticarcinogenic. Racial differences in smoking do not fully account for the African-American excess lung cancer incidence. African Americans consume more cruciferous vegetables than U.S. Whites. Impact on lung cancer risk is unknown. The glutathione S transferase M1 (GSTM1) gene promotes urinary isothiocyanate excretion. We evaluated dietary isothiocyanates and lung cancer using a population-based case-control study of 933 African Americans and Caucasians (non-Hispanic U.S. White) from Los Angeles County, California (311 cases; 622 controls). Broccoli, cauliflower, greens, and cabbage food-frequency variables represented isothiocyanates. Isothiocyanates were protective for lung cancer risk. Adjusted odds ratio (OR) for the uppermost quartile > 80 micro mol isothiocyanates/wk, compared to lowest, was 0.65 [95% confidence interval (CI) = 0.41-1.00, trend P = 0.02]. Association was stronger among subjects with homozygous deletion of GSTM1 (OR = 0.52, 95% CI = 0.31-0.86) than subjects with at least one GSTM1 copy (OR = 0.77, 95% CI = 0.49-1.21). The difference was not statistically significant (P = 0.16). Despite African Americans consuming more cruciferous vegetables, the isothiocyanate association did not vary by race (P = 0.52). Reduced lung cancer risk with higher isothiocyanate intake may be slightly stronger among subjects with deletion of GSTM1.

  3. Dietary Isothiocyanates, Glutathione S-Transferase M1 (GSTM1), and Lung Cancer Risk in African Americans and Caucasians from Los Angeles County, California

    PubMed Central

    Carpenter, Catherine L.; Yu, Mimi C.; London, Stephanie J.

    2013-01-01

    Isothiocyanates, found in cruciferous vegetables, are anti-carcinogenic. Racial differences in smoking do not fully account for the African American excess lung cancer incidence. African Americans consume more cruciferous vegetables than US Whites. Impact on lung cancer risk is unknown. Glutathione S transferase M1 (GSTM1) gene promotes urinary isothiocyanate excretion. We evaluated dietary isothiocyanates and lung cancer using a population-based case-control study of 933 African Americans and Caucasians (non-Hispanic US White) from Los Angeles County, California (311 cases; 622 controls). Broccoli, cauliflower, greens and cabbage food-frequency variables represented isothiocyanates. Isothiocyanates were protective for lung cancer risk. Adjusted odds ratio (OR) for the uppermost quartile, > 80 μMol isothiocyanates/week, compared to lowest, was 0.65 (95% confidence interval (CL) = 0.41 – 1.00, trend p = 0.02). Association was stronger among subjects with homozygous deletion of GSTM1 (OR=0.52; 95% CL = 0.31 – 0.86), than subjects with at least one GSTM1 copy (OR = 0.77; 95% CL = 0.49 – 1.21). Difference was not statistically significant (p = 0.16). Despite African Americans consuming more cruciferous vegetables, the isothiocyanate association did not vary by race (p=0.52). Reduced lung cancer risk with higher isothiocyanate intake may be slightly stronger among subjects with deletion of GSTM1. PMID:19838921

  4. Antitrypanosomal isothiocyanate and thiocarbamate glycosides from Moringa peregrina.

    PubMed

    Ayyari, Mahdi; Salehi, Peyman; Ebrahimi, Samad Nejad; Zimmermann, Stefanie; Portmann, Lena; Krauth-Siegel, R Luise; Kaiser, Marcel; Brun, Reto; Rezadoost, Hassan; Rezazadeh, Shamsali; Hamburger, Matthias

    2014-01-01

    O-Methyl (1), O-ethyl (2), and O-butyl (3) 4-[(α-L-rhamnosyloxy) benzyl] thiocarbamate (E), along with 4-(α-L-rhamnosyloxy) benzyl isothiocyanate (4) have been isolated from the aerial parts of Moringa peregrina. The compounds were tested for in vitro activity against Trypanosoma brucei rhodesiense and cytotoxicity in rat skeletal myoblasts (L6 cells). The most potent compound was 4 with an IC50 of 0.10 µM against T.b. rhodesiense and a selectivity index of 73, while the thiocarbamate glycosides 1, 2, and 3 showed only moderate activity. Intraperitoneal administration of 50 mg/kg body weight/day of 4 in the T.b. rhodesiense STIB 900 acute mouse model revealed significant in vivo toxicity. Administration of 10 mg/kg body weight/day resulted in a 95% reduction of parasitemia on day 7 postinfection, but did not cure the animals. Because of its high in vitro activity and its ability to irreversibly inhibit trypanothione reductase, an attractive parasite-specific target enzyme, 4-[(α-L-rhamnosyloxy) benzyl] isothiocyanate (4), can be considered as a lead structure for the development and characterization of novel antitrypanosomal drugs. Georg Thieme Verlag KG Stuttgart · New York.

  5. Labelling Polymers and Micellar Nanoparticles via Initiation, Propagation and Termination with ROMP

    PubMed Central

    Thompson, Matthew P.; Randolph, Lyndsay M.; James, Carrie R.; Davalos, Ashley N.; Hahn, Michael E.

    2014-01-01

    In this paper we compare and contrast three approaches for labelling polymers with functional groups via ring-opening metathesis polymerization (ROMP). We explored the incorporation of functionality via initiation, termination and propagation employing an array of novel initiators, termination agents and monomers. The goal was to allow the generation of selectively labelled and well-defined polymers that would in turn lead to the formation of labelled nanomaterials. Norbornene analogues, prepared as functionalized monomers for ROMP, included fluorescent dyes (rhodamine, fluorescein, EDANS, and coumarin), quenchers (DABCYL), conjugatable moieties (NHS esters, pentafluorophenyl esters), and protected amines. In addition, a set of symmetrical olefins for terminally labelling polymers, and for the generation of initiators in situ is described. PMID:24855496

  6. 16 CFR 1615.5 - Labeling requirements.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Labeling requirements. 1615.5 Section 1615.5 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FLAMMABLE FABRICS ACT REGULATIONS STANDARD FOR THE... to protect the items from agents or treatments which are known to cause deterioration of their flame...

  7. 16 CFR 1615.5 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 2 2012-01-01 2012-01-01 false Labeling requirements. 1615.5 Section 1615.5 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FLAMMABLE FABRICS ACT REGULATIONS STANDARD FOR THE... to protect the items from agents or treatments which are known to cause deterioration of their flame...

  8. 16 CFR 1615.5 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 2 2014-01-01 2014-01-01 false Labeling requirements. 1615.5 Section 1615.5 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FLAMMABLE FABRICS ACT REGULATIONS STANDARD FOR THE... to protect the items from agents or treatments which are known to cause deterioration of their flame...

  9. DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells

    PubMed Central

    Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

    2016-01-01

    6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects. PMID:27547033

  10. DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells.

    PubMed

    Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

    2016-01-01

    6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects.

  11. Novel Peptide Sequence (“IQ-tag”) with High Affinity for NIR Fluorochromes Allows Protein and Cell Specific Labeling for In Vivo Imaging

    PubMed Central

    McCarthy, Jason R.; Weissleder, Ralph

    2007-01-01

    Background Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes. Methods Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR) (benz)indolium fluorochromes. The developed peptide sequence (“IQ-tag”) allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging. Significance The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development. PMID:17653285

  12. Novel alpha-MSH peptide analogs for melanoma targeting

    NASA Astrophysics Data System (ADS)

    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

  13. Lysozyme oxidation by singlet molecular oxygen: Peptide characterization using [18 O]-labeling oxygen and nLC-MS/MS.

    PubMed

    Marques, Emerson Finco; Medeiros, Marisa H G; Di Mascio, Paolo

    2017-11-01

    Singlet molecular oxygen ( 1 O 2 ) is generated in biological systems and reacts with different biomolecules. Proteins are a major target for 1 O 2 , and His, Tyr, Met, Cys, and Trp are oxidized at physiological pH. In the present study, the modification of lysozyme protein by 1 O 2 was investigated using mass spectrometry approaches. The experimental findings showed methionine, histidine, and tryptophan oxidation. The experiments were achieved using [ 18 O]-labeled 1 O 2 released from thermolabile endoperoxides in association with nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry. The structural characterization by nLC-MS/MS of the amino acids in the tryptic peptides of the proteins showed addition of [ 18 O]-labeling atoms in different amino acids. Copyright © 2017 John Wiley & Sons, Ltd.

  14. 9 CFR 112.5 - Review and approval of labeling.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

  15. 9 CFR 112.5 - Review and approval of labeling.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

  16. 9 CFR 112.5 - Review and approval of labeling.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

  17. 9 CFR 112.5 - Review and approval of labeling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

  18. 9 CFR 112.5 - Review and approval of labeling.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

  19. Design and in vivo evaluation of a patch system based on thiolated polymers.

    PubMed

    Hoyer, Herbert; Greindl, Melanie; Bernkop-Schnürch, Andreas

    2009-02-01

    A new oral patch delivery system has been designed to increase the overall oral bioavailability of drugs within the gastrointestinal tract. The patch system consists of four layered films: a mucoadhesive matrix layer, a water insoluble backing layer, a middle layer and an enteric surface layer. The separation layer between the two matrix layers contained lactose, starch and confectioners' sugar. The matrix layer, exhibiting a diameter of 2.5 mm and a weight of 5 mg, comprised Polycarbophil-cysteine conjugate (49%), fluoresceine isothiocyanate-dextran (26%), glutathione (5%), and mannitol (20%). A standard tablet formulation consisting of the same matrix served as control. Entire fluoresceine isothiocyanate-dextran (FD(4)) was released from the delivery system within 2 h. For in vivo studies patch systems were administered orally to male Sprague-Dawley rats. Maximum FD(4) concentration in blood of the patch system was 46.1 +/- 8.9 ng/mL and was reached 3 h after administration. In contrast c(max) of control tablets displayed 50.5 +/- 14.9 ng/mL after 2 h and the absorption of FD(4) after administration in oral solution was negligible. The absolute bioavailability of orally administered patch systems and control tablets was 0.54% and 0.32% respectively. Results of this study indicate that a prolonged and higher oral bioavailability of FD(4) is obtained with patches than with tablets.

  20. Microbial- and isothiocyanate-mediated control of Phytophthora and Pythium species

    Treesearch

    M.F. Cohen; E. Yamamoto; E. Condeso; B.L. Anacker; N. Rank; M. Mazzola

    2008-01-01

    Plant pathogens of the oomycete lineage share common susceptibilities to many biotic and abiotic stresses. We are investigating the potential of antagonistic bacteria, isothiocyanates, and mycophagous amoebae to control diseases caused by Phytophthora spp., including the etiologic agent of sudden oak death, Phytophthora ramorum (...

  1. Method and compositions for detecting of bloodstains using fluorescin-fluorescein reaction

    DOEpatents

    Di Benedetto, John; Kyle, Kevin; Boan, Terry; Marie, Charlene

    2004-02-17

    A method, compositions and kit are set forth for detecting blood stains. A reactant solution includes fluorescin solubilized (reduced) in acetic acid in ethanol. The solution may be buffered to a pH of approximately 9. After spraying the reactant solution on the suspected area an oxidizer is applied to promote the fluorescin to fluorescein reaction with the blood. The reacted fluorescein is then detected through luminescence for capture by photography.

  2. C-type natriuretic peptide and atrial natriuretic peptide receptors of rat brain.

    PubMed

    Brown, J; Zuo, Z

    1993-03-01

    Natriuretic peptide receptors in rat brain were mapped by in vitro autoradiography using 125I-labeled [Tyr0]CNP-(1-22) to bind atrial natriuretic peptide receptor (ANPR)-B and ANPR-C receptors selectively, and 125I-labeled alpha-ANP to select ANPR-A and ANPR-C receptors. Des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4- 23)-amide (C-ANP) was used for its selectivity for ANPR-C over ANPR-A. Specific binding of 125I-[Tyr0]CNP-(1-22) with a dissociation constant (Kd) approximately 1 nM occurred in olfactory bulb, cerebral cortex, lateral septal nucleus, choroid plexus, and arachnoid mater. This binding was abolished by C-type natriuretic peptide [CNP-(1-22)], alpha-ANP and C-ANP, and conformed to ANPR-C. 125I-alpha-ANP bound to all structures that bound 125I-[Tyr0]CNP-(1-22). This binding was also inhibited by both CNP-(1-22) and C-ANP, confirming the presence of ANPR-C-like binding sites. However, ANPR-C-like binding sites were heterogenous because only some had high affinities for 125I-[Tyr0]CNP-(1-22) and CNP-(1-22). 125I-alpha-ANP also bound sites without affinities for C-ANP or CNP-(1-22). These sites were consistent with ANPR-A. They occurred mainly on the olfactory bulb, the choroid plexus, and the subfornical organ. Guanosine 3',5'-cyclic monophosphate production was strongly stimulated by alpha-ANP but not by CNP-(1-22) in olfactory bulb. Neither ligand stimulated it in cortical tissue. Thus the natriuretic peptide binding sites of rat brain conformed to ANPR-A and to heterogenous ANPR-C-like sites. No ANPR-B were detected.

  3. Micro-instillation of fluorescein with an inoculation loop for ocular surface staining in dry eye syndrome.

    PubMed

    Courrier, Emilie; Renault, Didier; Kaspi, Mathilde; Marcon, Agathe; Lambert, Victor; Garcin, Thibaud; Chiambaretta, Frederic; Garhofer, Gerhard; Thuret, Gilles; Gain, Philippe

    2018-03-01

    To describe and validate the micro-instillation of fluorescein on the ocular surface by a disposable calibrated inoculation loop to improve corneal and conjunctival staining quality. Accuracy and precision of the volume of 0.5% sodium fluorescein collected by a single use 1 μl-calibrated inoculation loop were measured using a precision balance. Twenty patients (40 eyes) suffering from dry eye syndrome were enrolled in a prospective interventional nonrandomized study. Fluorescein was instilled with the loop, and slit-lamp images were taken within 30 seconds using cobalt blue light with and without a yellow barrier filter. For comparison, after a washout period, the same images were retaken after instillation of one drop of fluorescein from a single-dose unit. The main outcome measure was the staining quality assessed by three experts, blind to the instillation method. Patient discomfort (tolerance, by a questionnaire) was also compared. The mean volume collected by the loop was 1.18 ± 0.12 μl, compared with 33.70 ± 6.10 μl using the single-dose unit. The loop avoided excess dye responsible for unpleasant tearing, masking of lesions and rapid diffusion into the stroma. Micro-instillation greatly improved image quality without losing information. The yellow filter further improved image contrast. Tolerance was excellent. The 1 μl-calibrated inoculation loop is a safe, convenient, inexpensive, disposable, sterile, well-tolerated tool for reproducible micro-instillation of commercial fluorescein. By greatly improving staining quality, it will help standardize assessment of dry eye severity. © 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  4. 10 % fluorescein sodium vs 1 % isosulfan blue in breast sentinel lymph node biopsy.

    PubMed

    Ren, Lidong; Liu, Zhao; Liang, Mengdi; Wang, Li; Song, Xingli; Wang, Shui

    2016-11-03

    Sentinel lymph node biopsy (SLNB) is well accepted to be a standard procedure in breast cancer surgery with clinically negative lymph nodes. Isosulfan blue is the first dye approved by the USA Food and Drug Administration for the localization of the lymphatic system. Few alternative tracers have been investigated. In this study, we aimed to compare the differences between 10 % fluorescein sodium and 1 % isosulfan blue in breast sentinel lymph node biopsy and to investigate the feasibility of using 10 % fluorescein sodium as a new dye for breast sentinel lymph node biopsy. A total of 30 New Zealand rabbits were randomly divided into the fluorescein sodium group and the isosulfan blue group (15 rabbits per group). Fluorescein sodium or isosulfan blue was injected subcutaneously into the second pair of mammary areolas. The average fading time of the second lymph nodes in the isosulfan blue group was significantly shorter than that in the fluorescein sodium group. Moreover, the detection rates of SLNs were higher in the fluorescein sodium group than in the isosulfan blue group. No significant differences between the fluorescein sodium group and isosulfan blue group were observed regarding the distances between the detected sentinel lymph nodes and second pair of mammary areolas, the distances between the second lymph nodes and second pair of mammary areolas, the number of detected sentinel lymph nodes and second lymph nodes, the average dyeing time of the sentinel and the second lymph nodes, and the average fading time of the second lymph nodes. In summary, we first reported that fluorescein sodium is a potential new tracer for breast sentinel lymph node biopsy.

  5. A Peptide-Based Method for 13C Metabolic Flux Analysis in Microbial Communities

    PubMed Central

    Ghosh, Amit; Nilmeier, Jerome; Weaver, Daniel; Adams, Paul D.; Keasling, Jay D.; Mukhopadhyay, Aindrila; Petzold, Christopher J.; Martín, Héctor García

    2014-01-01

    The study of intracellular metabolic fluxes and inter-species metabolite exchange for microbial communities is of crucial importance to understand and predict their behaviour. The most authoritative method of measuring intracellular fluxes, 13C Metabolic Flux Analysis (13C MFA), uses the labeling pattern obtained from metabolites (typically amino acids) during 13C labeling experiments to derive intracellular fluxes. However, these metabolite labeling patterns cannot easily be obtained for each of the members of the community. Here we propose a new type of 13C MFA that infers fluxes based on peptide labeling, instead of amino acid labeling. The advantage of this method resides in the fact that the peptide sequence can be used to identify the microbial species it originates from and, simultaneously, the peptide labeling can be used to infer intracellular metabolic fluxes. Peptide identity and labeling patterns can be obtained in a high-throughput manner from modern proteomics techniques. We show that, using this method, it is theoretically possible to recover intracellular metabolic fluxes in the same way as through the standard amino acid based 13C MFA, and quantify the amount of information lost as a consequence of using peptides instead of amino acids. We show that by using a relatively small number of peptides we can counter this information loss. We computationally tested this method with a well-characterized simple microbial community consisting of two species. PMID:25188426

  6. Fluorescein angiography and retinal vascular development in premature infants.

    PubMed

    Purcaro, Velia; Velia, Purcaro; Baldascino, Antonio; Antonio, Baldascino; Papacci, Patrizia; Patrizia, Papacci; Giannantonio, Carmen; Carmen, Giannantonio; Molisso, Anna; Anna, Molisso; Molle, Fernando; Fernando, Molle; Lepore, Domenico; Domenico, Lepore; Romagnoli, Costantino; Costantino, Romagnoli

    2012-10-01

    To investigate the role of fluorescein angiography (FA) in the management of retinopathy of prematurity (ROP) in preterm newborns. An observational case series of 13 extremely low birth weight infants. From September 2009 to March 2010, 13 newborn infants with a gestational age <29 weeks end/or birth weight <1000 g underwent serial fluorescein angiography with RetCam (Clarity, Pleasanton, CA) every 2 weeks. The fluorescein angiograms were examined to optimize the timing of diagnosis of ROP and to investigate development of retinal and choroidal vascularization. There were no side effects related to FA. Variable features of retinal and choroidal circulation in preterm infants with a high risk of developing ROP were noted. FA allows vessels branching at the junction between vascular and avascular retina (V-Av junction) to be viewed easily and shows the ROP findings that sometimes cannot be seen by indirect ophthalmoscopy. Dye leakage is the most significant sign of progression to severe ROP or the need for surgery in newborn babies with ROP. RetCam-assisted intravenous FA is safe and allows a more objective assessment of the ROP stage and zone.

  7. Cell uptake, intracellular distribution, fate and reactive oxygen species generation of polymer brush engineered CeO2-x NPs

    NASA Astrophysics Data System (ADS)

    Qiu, Yuan; Rojas, Elena; Murray, Richard A.; Irigoyen, Joseba; Gregurec, Danijela; Castro-Hartmann, Pablo; Fledderman, Jana; Estrela-Lopis, Irina; Donath, Edwin; Moya, Sergio E.

    2015-04-01

    Cerium Oxide nanoparticles (CeO2-x NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO2-x NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO2-x NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO2-x NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell endosomes and lysosomes after 24 h of incubation. They also show higher co-localisation with lipid bodies when compared to unmodified CeO2-x NPs. The brush coating does not prevent CeO2-x NPs from displaying antioxidant properties.Cerium Oxide nanoparticles (CeO2-x NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO2-x NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO2-x NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO2-x NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell

  8. New stroboscopic light source and technique for intraoperative retinal fluorescein angiography during penetrating keratoplasty

    NASA Astrophysics Data System (ADS)

    Krueger, Ronald R.; Morales, Ronald B.; Chong, Lawrence P.; Smith, Ronald E.

    1994-06-01

    We report the development of a new stroboscopic light source system and technique for performing intraoperative fluorescein angiography during penetrating keratoplasty for aphakic or pseudophakic bullous keratopathy. A controllable pulse xenon light source system with a fiber optic endoilluminator probe is used to perform high-quality intraoperative fluorescein angiography during penetrating keratoplasty in pigmented rabbits and human subjects. Following corneal trephination and extraction of the intraocular lens, a temporary Cobo keratoprosthesis is secured while a 20-gauge endoilluminator is inserted into the vitreous cavity through a limbal incision. The endoilluminator is advanced to a retinal illumination area of approximately 3 DD and 10% fluorescein is injected intravenously. A microscope camera coupled to a 50:50 beamsplitter photographs the passage of fluorescein dye while the surgeon maintains an unaltered view through the operating microscope. Angiograms through a keratoprosthesis show excellent contrast and resolution, comparable to standard fluorescein angiography. Fine peripapillary vessels are seen reproducibly and with great detail in the rabbits. All the phases of retinal angiography can be seen, including arteriolar constriction and capillary nonperfusion in one of four human subjects examined. High quality intraoperative fluorescein angiography can be performed in patients undergoing penetrating keratoplasty for aphakic/ pseudophakic bullous keratopathy. With this technology, preexisting retinal disorders such as cystoid macular edema might be identified in the perioperative setting allowing for important management decisions to be made intraoperatively.

  9. Human corneal epithelial cell shedding and fluorescein staining in response to silicone hydrogel lenses and contact lens disinfecting solutions.

    PubMed

    Gorbet, Maud; Peterson, Rachael; McCanna, David; Woods, Craig; Jones, Lyndon; Fonn, Desmond

    2014-03-01

    A pilot study was conducted to evaluate human corneal epithelial cell shedding in response to wearing a silicone hydrogel contact lens/solution combination inducing corneal staining. The nature of ex vivo collected cells staining with fluorescein was also examined. A contralateral eye study was conducted in which up to eight participants were unilaterally exposed to a multipurpose contact lens solution/silicone hydrogel lens combination previously shown to induce corneal staining (renu® fresh™ and balafilcon A; test eye), with the other eye using a combination of balafilcon A soaked in a hydrogen peroxide care system (Clear Care®; control eye). Lenses were worn for 2, 4 or 6 hours. Corneal staining was graded after lens removal. The Ocular Surface Cell Collection Apparatus was used to collect cells from the cornea and the contact lens. In the test eye, maximum solution-induced corneal staining (SICS) was observed after 2 hours of lens wear (reducing significantly by 4 hours; p < 0.001). There were significantly more cells collected from the test eye after 4 hours of lens wear when compared to the control eye and the collection from the test eye after 2 hours (for both; n = 5; p < 0.001). The total cell yield at 4 hours was 813 ± 333 and 455 ± 218 for the test and control eyes, respectively (N = 5, triplicate, p = 0.003). A number of cells were observed to have taken up the fluorescein dye from the initial fluorescein instillation. Confocal microscopy of fluorescein-stained cells revealed that fluorescein was present throughout the cell cytoplasm and was retained in the cells for many hours after recovery from the corneal surface. This pilot study indicates that increased epithelial cell shedding was associated with a lens-solution combination which induces SICS. Our data provides insight into the transient nature of the SICS reaction and the nature of fluorescein staining observed in SICS.

  10. Photoclickable dendritic molecular glue: noncovalent-to-covalent photochemical transformation of protein hybrids.

    PubMed

    Uchida, Noriyuki; Okuro, Kou; Niitani, Yamato; Ling, Xiao; Ariga, Takayuki; Tomishige, Michio; Aida, Takuzo

    2013-03-27

    A water-soluble dendron with a fluorescein isothiocyanate (FITC) fluorescent label and bearing nine pendant guanidinium ion (Gu(+))/benzophenone (BP) pairs at its periphery (Glue(BP)-FITC) serves as a "photoclickable molecular glue". By multivalent salt-bridge formation between Gu(+) ions and oxyanions, Glue(BP)-FITC temporarily adheres to a kinesin/microtubule hybrid. Upon subsequent exposure to UV light, this noncovalent binding is made permanent via a cross-linking reaction mediated by carbon radicals derived from the photoexcited BP units. This temporal-to-permanent transformation by light occurs quickly and efficiently in this preorganized state, allowing the movements of microtubules on a kinesin-coated glass plate to be photochemically controlled. A fundamental difference between such temporal and permanent bindings was visualized by the use of "optical tweezers".

  11. A general approach for chemical labeling and rapid, spatially controlled protein inactivation

    PubMed Central

    Marks, Kevin M.; Braun, Patrick D.; Nolan, Garry P.

    2004-01-01

    Chemical labeling of proteins inside of living cells can enable studies of the location, movement, and function of proteins in vivo. Here we demonstrate an approach for chemical labeling of proteins that uses the high-affinity interaction between an FKBP12 mutant (F36V) and a synthetic, engineered ligand (SLF′). A fluorescein conjugate to the engineered ligand (FL-SLF′) retained binding to FKBP12(F36V) and possessed similar fluorescence properties as parental fluorescein. FL-SLF′ labeled FKBP12(F36V) fusion proteins in live mammalian cells, and was used to monitor the subcellular localization of a membrane targeted FKBP12(F36V) construct. Chemical labeling of FKBP12(F36V) fusion proteins with FL-SLF′ was readily detectable at low expression levels of the FKBP12(F36V) fusion, and the level of fluorescent staining with FL-SLF′ was proportional to the FKBP12(F36V) expression level. This FL-SLF′-FKBP12(F36V) labeling technique was tested in fluorophore assisted laser inactivation (FALI), a light-mediated technique to rapidly inactivate fluorophore-labeled target proteins. FL-SLF′ mediated FALI of a β-galactosidase-FKBP12(F36V) fusion protein, causing rapid inactivation of >90% of enzyme activity upon irradiation in vitro. FL-SLF′ also mediated FALI of a β-galactosidase fusion expressed in living NIH 3T3 cells, where β-galactosidase activity was reduced in 15 s. Thus, FL-SLF′ can be used to monitor proteins in vivo and to target rapid, spatially and temporally defined inactivation of target proteins in living cells in a process that we call FK-FALI. PMID:15218100

  12. Lacosamide Isothiocyanate-based Agents: Novel Agents to Target and Identify Lacosamide Receptors

    PubMed Central

    Park, Ki Duk; Morieux, Pierre; Salomé, Christophe; Cotten, Steven W.; Reamtong, Onrapak; Eyers, Claire; Gaskell, Simon J.; Stables, James P.; Liu, Rihe; Kohn, Harold

    2009-01-01

    (R)-Lacosamide ((R)-2, (R)-N-benzyl 2-acetamido-3-methoxypropionamide), has recently gained regulatory approval for the treatment of partial-onset seizures in adults. Whole animal pharmacological studies have documented that (R)-2 function is unique. A robust strategy is advanced for the discovery of interacting proteins associated with function and toxicity of (R)-2 through the use of (R)-2 analogs, 3, that contain “affinity bait (AB)” and “chemical reporter (CR)” functional groups. In 3, covalent modification of the interacting proteins proceeds at the AB moiety, and detection or isolation of the selectively captured protein occurs through the bioorthogonal CR group upon reaction with an appropriate probe. We report the synthesis, pharmacological evaluation, and interrogation of the mouse soluble brain proteome using 3 where the AB group is an isothiocyanate moiety. One compound, (R)-N-(4-isothiocyanato)benzyl 2-acetamido-3-(prop-2-ynyloxy)propionamide ((R)-9), exhibited excellent seizure protection in mice and, like (R)-2, anticonvulsant activity principally resided in the (R)-stereoisomer. Several proteins were preferentially labeled by (R)-9 compared with (S)-9, including collapsin response mediator protein 2. PMID:19795888

  13. Isothiocyanates Reduce Mercury Accumulation via an Nrf2-Dependent Mechanism during Exposure of Mice to Methylmercury

    PubMed Central

    Toyama, Takashi; Shinkai, Yasuhiro; Yasutake, Akira; Uchida, Koji; Yamamoto, Masayuki

    2011-01-01

    Background: Methylmercury (MeHg) exhibits neurotoxicity through accumulation in the brain. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) plays an important role in reducing the cellular accumulation of MeHg. Objectives: We investigated the protective effect of isothiocyanates, which are known to activate Nrf2, on the accumulation of mercury after exposure to MeHg in vitro and in vivo. Methods: We used primary mouse hepatocytes in in vitro experiments and mice as an in vivo model. We used Western blotting, luciferase assays, atomic absorption spectrometry assays, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays, and we identified toxicity in mice based on hind-limb flaccidity and mortality. Results: The isothiocyanates 6-methylsulfinylhexyl isothiocyanate (6-HITC) and sulforaphane (SFN) activated Nrf2 and up-regulated downstream proteins associated with MeHg excretion, such as glutamate-cysteine ligase, glutathione S-transferase, and multidrug resistance–associated protein, in primary mouse hepatocytes. Under these conditions, intracellular glutathione levels increased in wild-type but not Nrf2-deficient primary mouse hepatocytes. Pretreatment with 6-HITC and SFN before MeHg exposure suppressed cellular accumulation of mercury and cytotoxicity in wild-type but not Nrf2-deficient primary mouse hepatocytes. In comparison, in vivo administration of MeHg to Nrf2-deficient mice resulted in increased sensitivity to mercury concomitant with an increase in mercury accumulation in the brain and liver. Injection of SFN before administration of MeHg resulted in a decrease in mercury accumulation in the brain and liver of wild-type, but not Nrf2-deficient, mice. Conclusions: Through activation of Nrf2, 6-HITC and SFN can suppress mercury accumulation and intoxication caused by MeHg intake. PMID:21382770

  14. CRYPTOSPORIDIUM OOCYST RECOVERY IN WATER BY EPA METHOD 1623: EVALUATION OF A MODIFIED IMS DISSOCIATION

    EPA Science Inventory

    EPA Methods 1622 and 1623 are the benchmarks for detection of Cryptosporidium spp. oocysts in water. These methods consist of filtration, elution, purification by immunomagnetic separation (IMS), and microscopic analysis after staining with a fluorescein isothiocyanate conjugate...

  15. Direct and Indirect Antioxidant Activity of Polyphenol- and Isothiocyanate-Enriched Fractions from Moringa oleifera

    PubMed Central

    Boyunegmez Tumer, Tugba; Rojas-Silva, Patricio; Poulev, Alexander; Raskin, Ilya; Waterman, Carrie

    2016-01-01

    Moringa oleifera Lam. is a fast-growing, tropical tree with various edible parts used as nutritious food and traditional medicine. This study describes an efficient preparatory strategy to extract and fractionate moringa leaves by fast centrifugal partition chromatography (FCPC) to produce polyphenol and isothiocyanate (ITC) rich fractions. Characterization and further purification of these fractions showed that moringa polyphenols were potent direct antioxidants assayed by oxygen radical absorbance capacity (ORAC), whereas moringa ITCs were effective indirect antioxidants assayed by induction of NAD(P)H quinone oxidoreductase 1 (NQO1) activity in Hepa1c1c7 cells. In addition, purified 4-[(α-l-rhamnosyloxy)benzyl]-isothiocyanate and 4-[(4′-O-acetyl-α-l-rhamnosyloxy)benzyl]isothiocyanate were further evaluated for their ORAC and NQO1 inducer potency in comparison with sulforaphane (SF). Both ITCs were as potent as SF in inducing NQO1 activity. These findings suggest that moringa leaves contain a potent mixture of direct and indirect antioxidants that can explain its various health-promoting effects. PMID:25605589

  16. Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

    PubMed

    Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

    2015-10-21

    Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.

  17. Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in a human lymphoblastic leukemia cell line

    PubMed Central

    Rahman, Heshu Sulaiman; Rasedee, Abdullah; Abdul, Ahmad Bustamam; Zeenathul, Nazariah Allaudin; Othman, Hemn Hassan; Yeap, Swee Keong; How, Chee Wun; Hafiza, Wan Abd Ghani Wan Nor

    2014-01-01

    This investigation evaluated the antileukemia properties of a zerumbone (ZER)-loaded nanostructured lipid carrier (NLC) prepared by hot high-pressure homogenization techniques in an acute human lymphoblastic leukemia (Jurkat) cell line in vitro. The apoptogenic effect of the ZER-NLC on Jurkat cells was determined by fluorescent and electron microscopy, Annexin V-fluorescein isothiocyanate, Tdt-mediated dUTP nick-end labeling assay, cell cycle analysis, and caspase activity. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay showed that ZER-NLC did not have adverse effects on normal human peripheral blood mononuclear cells. ZER-NLC arrested the Jurkat cells at G2/M phase with inactivation of cyclin B1 protein. The study also showed that the antiproliferative effect of ZER-NLC on Jurkat cells is through the intrinsic apoptotic pathway via activation of caspase-3 and caspase-9, release of cytochrome c from the mitochondria into the cytosol, and subsequent cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP). These findings show that the ZER-NLC is a potentially useful treatment for acute lymphoblastic leukemia in humans. PMID:24549090

  18. Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

  19. Precise correlation of histopathological and fluorescein angiographic morphology using retinal vascular casting.

    PubMed

    Bek, T; Prause, J U

    1996-12-01

    The histopathology of three eyes obtained post mortem from 2 patients with age-related macular degeneration was correlated with the pre mortem fluorescein angiographic morphology. A precise point-by-point correlation between histopathology and the corresponding angiographic appearance was ensured by using the cast retinal vascular system as a pattern of reference. The study showed that both the photoreceptors, the pigment epithelium, and substances accumulated between the retinal and the choroidal vascular systems, may have a blocking effect on choroidal background fluorescence as seen on fluorescein angiograms. Furthermore, it is confirmed that fluorescein angiographic hyperfluorescence may be due to a lack of blocking of the choroidal fluorescence because of a window defect in the retinal photoreceptor layer and/or the pigment epithelium.

  20. Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C, 15N-labeled peptides and proteins

    NASA Astrophysics Data System (ADS)

    Janik, Rafal; Ritz, Emily; Gravelle, Andrew; Shi, Lichi; Peng, Xiaohu; Ladizhansky, Vladimir

    2010-03-01

    In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly 13C, 15N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22 kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600 MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]-CO[i - 2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed.

  1. Fluorescence Intensity- and Lifetime-Based Glucose Sensing Using Glucose/Galactose-Binding Protein

    PubMed Central

    Pickup, John C.; Khan, Faaizah; Zhi, Zheng-Liang; Coulter, Jonathan; Birch, David J. S.

    2013-01-01

    We review progress in our laboratories toward developing in vivo glucose sensors for diabetes that are based on fluorescence labeling of glucose/galactose-binding protein. Measurement strategies have included both monitoring glucose-induced changes in fluorescence resonance energy transfer and labeling with the environmentally sensitive fluorophore, badan. Measuring fluorescence lifetime rather than intensity has particular potential advantages for in vivo sensing. A prototype fiber-optic-based glucose sensor using this technology is being tested.Fluorescence technique is one of the major solutions for achieving the continuous and noninvasive glucose sensor for diabetes. In this article, a highly sensitive nanostructured sensor is developed to detect extremely small amounts of aqueous glucose by applying fluorescence energy transfer (FRET). A one-pot method is applied to produce the dextran-fluorescein isothiocyanate (FITC)-conjugating mesoporous silica nanoparticles (MSNs), which afterward interact with the tetramethylrhodamine isothiocyanate (TRITC)-labeled concanavalin A (Con A) to form the FRET nanoparticles (FITC-dextran-Con A-TRITC@MSNs). The nanostructured glucose sensor is then formed via the self-assembly of the FRET nanoparticles on a transparent, flexible, and biocompatible substrate, e.g., poly(dimethylsiloxane). Our results indicate the diameter of the MSNs is 60 ± 5 nm. The difference in the images before and after adding 20 μl of glucose (0.10 mmol/liter) on the FRET sensor can be detected in less than 2 min by the laser confocal laser scanning microscope. The correlation between the ratio of fluorescence intensity, I(donor)/I(acceptor), of the FRET sensor and the concentration of aqueous glucose in the range of 0.04–4 mmol/liter has been investigated; a linear relationship is found. Furthermore, the durability of the nanostructured FRET sensor is evaluated for 5 days. In addition, the recorded images can be converted to digital images by

  2. Assessing topology and surface orientation of an antimicrobial peptide magainin 2 using mechanically aligned bilayers and electron paramagnetic resonance spectroscopy.

    PubMed

    Mayo, Daniel J; Sahu, Indra D; Lorigan, Gary A

    2018-07-01

    Aligned CW-EPR membrane protein samples provide additional topology interactions that are absent from conventional randomly dispersed samples. These samples are aptly suited to studying antimicrobial peptides because of their dynamic peripheral topology. In this study, four consecutive substitutions of the model antimicrobial peptide magainin 2 were synthesized and labeled with the rigid TOAC spin label. The results revealed the helical tilts to be 66° ± 5°, 76° ± 5°, 70° ± 5°, and 72° ± 5° for the TOAC substitutions H7, S8, A9, and K10 respectively. These results are consistent with previously published literature. Using the EPR (electron paramagnetic resonance) mechanical alignment technique, these substitutions were used to critically assess the topology and surface orientation of the peptide with respect to the membrane. This methodology offers a rapid and simple approach to investigate the structural topology of antimicrobial peptides. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Use of deuterium labeling by high-temperature solid-state hydrogen-exchange reaction for mass spectrometric analysis of bradykinin biotransformation.

    PubMed

    Kopylov, Arthur T; Myasoedov, Nikolay F; Dadayan, Alexander K; Zgoda, Victor G; Medvedev, Alexei E; Zolotarev, Yurii A

    2016-06-15

    Studies of molecular biodegradation by mass spectrometry often require synthetic compounds labeled with stable isotopes as internal standards. However, labeling is very expensive especially when a large number of compounds are needed for analysis of biotransformation. Here we describe an approach for qualitative and quantitative analysis using bradykinin (BK) and its in vitro degradation metabolites as an example. Its novelty lies in the use of deuterated peptides which are obtained by a high-temperature solid-state exchange (HSCIE) reaction. Deuterated and native BK were analyzed by positive electrospray ionization high-resolution mass spectrometry (ESI-HRMS) using an Orbitrap Fusion mass spectrometer. High-energy collision-induced dissociation (HCD) experiments were performed on [M+H](+) and [M+2H](2+) ions in targeted-MS(2) mode with adjusted normalized HCD value. After the HSCIE reaction, each amino acid residue of the deuterated peptide contained deuterium atoms and the average degree of substitution was 5.5 atoms per the peptide molecule. The deuterated peptide demonstrated the same chromatographic mobility as the unlabeled counterpart, and lack of racemization during substitution with deuterium. Deuterium-labeled and unlabeled BKs were incubated with human plasma and their corresponding fragments BK(1-5) and BK(1-7), well known as the major metabolites, were detected. Quantitative assays demonstrated applicability of the heavy peptide for both sequencing and quantification of generated fragments. Applicability of the HSCIE deuterated peptide for analysis of routes of its degradation has been shown in in vitro experiments. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  4. IMPROVED METHODS FOR HEPATITIS A VIRUS AND ROTAVIRUS CONCENTRATION AND DETECTION IN RECREATIONAL, RAW POTABLE, AND FINISHED WATERS

    EPA Science Inventory

    The report contains procedures for detecting rotaviruses based upon an immunofluorescence test using a monoclonal antibody and fluorescein-isothiocyanate-conjugated antibody staining method to visualize virus-infected cells. Also contained in the report are test methods for detec...

  5. Fluorescein-guided intraoperative endoscopy in patients with hereditary hemorrhagic telangiectasia: first impressions.

    PubMed

    Pagella, Fabio; Pusateri, Alessandro; Zaccari, Dario; Bongetta, Daniele; Zoia, Cesare; Spinozzi, Giuseppe; Olivieri, Carla; Matti, Elina

    2017-03-01

    Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease that results in mucocutaneous telangiectasias and arteriovenous visceral malformations. Nasal telangiectasias lead to recurrent epistaxis, which affects up to 96% of patients. Different morphologic classifications and methods of visualization of nasal lesions have been described in the literature. We developed a new method of intraoperative endoscopy based on the intravenous administration of fluorescein. Preliminary data of this technique are reported. After the intravenous administration of sodium fluorescein, an intraoperative fluorescein-guided endoscopy was carried out using photographic customized yellow filters on top of a 0-degree, 4-mm endoscope. In 2015, 65 HHT patients underwent surgery for their epistaxis in our institution, and in 7 patients (3 males, 4 females; mean age, 54 years) an intraoperative fluorescein-guided intraoperative nasal endoscopy was performed. No adverse events or complications were observed. First impressions regarding the usage of this technique in HHT patients seem to be promising and positive in terms of efficacy and safety. However, further studies with larger cohorts of patients should be performed in order to better investigate the use of this method for diagnostic and surgical purposes in HHT. © 2016 ARS-AAOA, LLC.

  6. Ability of spermatozoa to bind to the zona pellucida during oligozoospermia induced with testosterone during a male contraceptive trial.

    PubMed

    Liu, D Y; Johnston, R; Baker, H W

    1995-06-01

    To determine the ability of spermatozoa to bind to the zona pellucida (ZP) in testosterone-induced oligozoospermia, previously fertile men participating in the World Heath Organization (WHO) male contraceptive trial in Melbourne were studied while oligozoospermic to various degrees. Semen analysis were performed according to WHO methods. One or two ejaculates from each subject were cryopreserved before commencing weekly intramuscular injections of 200 mg testosterone enanthate. The frozen spermatozoa were used as controls for ZP-binding tests of spermatozoa obtained during testosterone-induced oligozoospermia (< 10 x 10(6)/ml) in either the suppression or efficacy (n = 6) and recovery (n = 3) phases. Two other subjects in the recovery phase with normozoospermia were also tested. Human oocytes that failed to fertilized in vitro from infertile patients were used for the sperm-ZP binding test. Control (frozen) spermatozoa were labelled with fluorescein isothiocyanate and test (oligozoospermic semen) spermatozoa were labelled with tetramethylrhodamine B isothiocyanate. A mixture of equal numbers of labelled motile control and test spermatozoa were incubated with 4-6 ZP. There was a significantly (p < 0.01) lower number of spermatozoa bound per ZP in oligozoospermic samples (65 +/- 7, mean +/- SEM) than in controls (80 +/- 7). However, there were still large numbers of spermatozoa bound to the ZP for all the oligozoospermic samples. Five subjects had similar numbers of spermatozoa bound to the ZP for both control and oligozoospermic samples. Overall, the ZP-binding ratio of test and control spermatozoa averaged 0.82 (range 0.51-1.13).(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Reversible Opening of Intercellular Junctions of Intestinal Epithelial and Brain Endothelial Cells With Tight Junction Modulator Peptides.

    PubMed

    Bocsik, Alexandra; Walter, Fruzsina R; Gyebrovszki, Andrea; Fülöp, Lívia; Blasig, Ingolf; Dabrowski, Sebastian; Ötvös, Ferenc; Tóth, András; Rákhely, Gábor; Veszelka, Szilvia; Vastag, Monika; Szabó-Révész, Piroska; Deli, Mária A

    2016-02-01

    The intercellular junctions restrict the free passage of hydrophilic compounds through the paracellular clefts. Reversible opening of the tight junctions of biological barriers is investigated as one of the ways to increase drug delivery to the systemic circulation or the central nervous system. Six peptides, ADT-6, HAV-6, C-CPE, 7-mer (FDFWITP, PN-78), AT-1002, and PN-159, acting on different integral membrane and linker junctional proteins were tested on Caco-2 intestinal epithelial cell line and a coculture model of the blood-brain barrier. All peptides tested in nontoxic concentrations showed a reversible tight junctions modulating effect and were effective to open the paracellular pathway for the marker molecules fluorescein and albumin. The change in the structure of cell-cell junctions was verified by immunostaining for occludin, claudin-4,-5, ZO-1, β-catenin, and E-cadherin. Expression levels of occludin and claudins were measured in both models. We could demonstrate a selectivity of C-CPE, ADT-6, and HAV-6 peptides for epithelial cells and 7-mer and AT-1002 peptides for brain endothelial cells. PN-159 was the most effective modulator of junctional permeability in both models possibly acting via claudin-1 and -5. Our results indicate that these peptides can be effectively and selectively used as potential pharmaceutical excipients to improve drug delivery across biological barriers. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  8. Thiolated polycarbophil/glutathione: defining its potential as a permeation enhancer for oral drug administration in comparison to sodium caprate.

    PubMed

    Perera, Glen; Barthelmes, Jan; Vetter, Anja; Krieg, Christof; Uhlschmied, Cindy; Bonn, Günther K; Bernkop-Schnürch, Andreas

    2011-08-01

    Thiolated polyacrylates were shown to be permeation enhancers with notable potential. The aim of this study was to evaluate the permeation enhancing properties of a thiolated polycarbophil/glutathione (PCP-Cys/GSH) system for oral drug application in comparison to a well-established permeation enhancer, namely sodium caprate. In vitro permeation studies were conducted in Ussing-type chambers with sodium fluoresceine (NaFlu) and fluoresceine isothiocyanate labeled dextran (molecular mass 4 kDa; FD4) as model compounds. Bioavailability studies were carried out in Sprague Dawley rats with various formulations. Moreover, cytotoxic effects of both permeation enhancers were compared. Permeation enhancement ratios of 1% sodium caprate were found to be 3.0 (FD4) and 2.3 (NaFlu), whereas 1% PCP-Cys/0.5% GSH displayed enhancement ratios of 2.4 and 2.2. Both excipients performed at a similar level in vivo. Sodium caprate solutions increased oral bioavailability 2.2-fold (FD4) and 2.3-fold (NaFlu), while PCP-Cys hydrogels led to a 3.2-fold and 2.2-fold enhancement. Cell viability experiments revealed a significantly higher tolerance of Caco-2 cells towards 0.5% PCP-Cys (81% survival) compared to 0.5% sodium caprate (5%). As PCP-Cys is not absorbed from mucosal membranes due to its comparatively high molecular mass, systemic side-effects can be excluded. In conclusion, both systems displayed a similar potency for permeation enhancement of hydrophilic compounds. However, PCP-Cys seems to be less harmful to cultured cells.

  9. Evaluation of empirical rule of linearly correlated peptide selection (ERLPS) for proteotypic peptide-based quantitative proteomics.

    PubMed

    Liu, Kehui; Zhang, Jiyang; Fu, Bin; Xie, Hongwei; Wang, Yingchun; Qian, Xiaohong

    2014-07-01

    Precise protein quantification is essential in comparative proteomics. Currently, quantification bias is inevitable when using proteotypic peptide-based quantitative proteomics strategy for the differences in peptides measurability. To improve quantification accuracy, we proposed an "empirical rule for linearly correlated peptide selection (ERLPS)" in quantitative proteomics in our previous work. However, a systematic evaluation on general application of ERLPS in quantitative proteomics under diverse experimental conditions needs to be conducted. In this study, the practice workflow of ERLPS was explicitly illustrated; different experimental variables, such as, different MS systems, sample complexities, sample preparations, elution gradients, matrix effects, loading amounts, and other factors were comprehensively investigated to evaluate the applicability, reproducibility, and transferability of ERPLS. The results demonstrated that ERLPS was highly reproducible and transferable within appropriate loading amounts and linearly correlated response peptides should be selected for each specific experiment. ERLPS was used to proteome samples from yeast to mouse and human, and in quantitative methods from label-free to O18/O16-labeled and SILAC analysis, and enabled accurate measurements for all proteotypic peptide-based quantitative proteomics over a large dynamic range. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. A Study into the Collision-induced Dissociation (CID) Behavior of Cross-Linked Peptides*

    PubMed Central

    Giese, Sven H.; Fischer, Lutz; Rappsilber, Juri

    2016-01-01

    Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers. PMID:26719564

  11. Effects of natural and synthetic isothiocyanate-based H2S-releasers against chemotherapy-induced neuropathic pain: Role of Kv7 potassium channels.

    PubMed

    Di Cesare Mannelli, Lorenzo; Lucarini, Elena; Micheli, Laura; Mosca, Ilaria; Ambrosino, Paolo; Soldovieri, Maria Virginia; Martelli, Alma; Testai, Lara; Taglialatela, Maurizio; Calderone, Vincenzo; Ghelardini, Carla

    2017-07-15

    Hydrogen sulfide (H 2 S) is a crucial signaling molecule involved in several physiological and pathological processes. Nonetheless, the role of this gasotransmitter in the pathogenesis and treatment of neuropathic pain is controversial. The aim of the present study was to investigate the pain relieving profile of a series of slow releasing H 2 S donors (the natural allyl-isothiocyanate and the synthetics phenyl- and carboxyphenyl-isothiocyanate) in animal models of neuropathic pain induced by paclitaxel or oxaliplatin, anticancer drugs characterized by a dose-limiting neurotoxicity. The potential contribution of Kv7 potassium channels modulation was also studied. Mice were treated with paclitaxel (2.0 mg kg -1 ) i.p. on days 1, 3, 5 and 7; oxaliplatin (2.4 mg kg -1 ) was administered i.p. on days 1-2, 5-9, 12-14. Behavioral tests were performed on day 15. In both models, single subcutaneous administrations of H 2 S donors (1.33, 4.43, 13.31 μmol kg -1 ) reduced the hypersensitivity to cold non-noxious stimuli (allodynia-related measurement). The prototypical H 2 S donor NaHS was also effective. Activity was maintained after i.c.v. administrations. On the contrary, the S-lacking molecule allyl-isocyanate did not increase pain threshold; the H 2 S-binding molecule hemoglobin abolished the pain-relieving effects of isothiocyanates and NaHS. The anti-neuropathic properties of H 2 S donors were reverted by the Kv7 potassium channel blocker XE991. Currents carried by Kv7.2 homomers and Kv7.2/Kv7.3 heteromers expressed in CHO cells were potentiated by H 2 S donors. Sistemically- or centrally-administered isothiocyanates reduced chemotherapy-induced neuropathic pain by releasing H 2 S. Activation of Kv7 channels largely mediate the anti-neuropathic effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. On the barrier properties of the cornea: a microscopy study of the penetration of fluorescently labeled nanoparticles, polymers, and sodium fluorescein.

    PubMed

    Mun, Ellina A; Morrison, Peter W J; Williams, Adrian C; Khutoryanskiy, Vitaliy V

    2014-10-06

    Overcoming the natural defensive barrier functions of the eye remains one of the greatest challenges of ocular drug delivery. Cornea is a chemical and mechanical barrier preventing the passage of any foreign bodies including drugs into the eye, but the factors limiting penetration of permeants and nanoparticulate drug delivery systems through the cornea are still not fully understood. In this study, we investigate these barrier properties of the cornea using thiolated and PEGylated (750 and 5000 Da) nanoparticles, sodium fluorescein, and two linear polymers (dextran and polyethylene glycol). Experiments used intact bovine cornea in addition to bovine cornea de-epithelialized or tissues pretreated with cyclodextrin. It was shown that corneal epithelium is the major barrier for permeation; pretreatment of the cornea with β-cyclodextrin provides higher permeation of low molecular weight compounds, such as sodium fluorescein, but does not enhance penetration of nanoparticles and larger molecules. Studying penetration of thiolated and PEGylated (750 and 5000 Da) nanoparticles into the de-epithelialized ocular tissue revealed that interactions between corneal surface and thiol groups of nanoparticles were more significant determinants of penetration than particle size (for the sizes used here). PEGylation with polyethylene glycol of a higher molecular weight (5000 Da) allows penetration of nanoparticles into the stroma, which proceeds gradually, after an initial 1 h lag phase.

  13. Isothiocyanate-enriched moringa seed extract alleviates ulcerative colitis symptoms in mice

    PubMed Central

    Wu, Alex G.; Jaja-Chimedza, Asha; Graf, Brittany L.; Waterman, Carrie; Verzi, Michael P.; Raskin, Ilya

    2017-01-01

    Moringa (Moringa oleifera Lam.) seed extract (MSE) has anti-inflammatory and antioxidant activities. We investigated the effects of MSE enriched in moringa isothiocyanate-1 (MIC-1), its putative bioactive, on ulcerative colitis (UC) and its anti-inflammatory/antioxidant mechanism likely mediated through Nrf2-signaling pathway. Dextran sulfate sodium (DSS)-induced acute (n = 8/group; 3% DSS for 5 d) and chronic (n = 6/group; cyclic rotations of 2.5% DSS/water for 30 d) UC was induced in mice that were assigned to 4 experimental groups: healthy control (water/vehicle), disease control (DSS/vehicle), MSE treatment (DSS/MSE), or 5-aminosalicyic acid (5-ASA) treatment (positive control; DSS/5-ASA). Following UC induction, water (vehicle), 150 mg/kg MSE, or 50 mg/kg 5-ASA were orally administered for 1 or 2 wks. Disease activity index (DAI), spleen/colon sizes, and colonic histopathology were measured. From colon and/or fecal samples, pro-inflammatory biomarkers, tight-junction proteins, and Nrf2-mediated enzymes were analyzed at protein and/or gene expression levels. Compared to disease control, MSE decreased DAI scores, and showed an increase in colon lengths and decrease in colon weight/length ratios in both UC models. MSE also reduced colonic inflammation/damage and histopathological scores (modestly) in acute UC. MSE decreased colonic secretions of pro-inflammatory keratinocyte-derived cytokine (KC), tumor necrosis factor (TNF)-α, nitric oxide (NO), and myeloperoxidase (MPO) in acute and chronic UC; reduced fecal lipocalin-2 in acute UC; downregulated gene expression of pro-inflammatory interleukin (IL)-1, IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) in acute UC; upregulated expression of claudin-1 and ZO-1 in acute and chronic UC; and upregulated GSTP1, an Nrf2-mediated phase II detoxifying enzyme, in chronic UC. MSE was effective in mitigating UC symptoms and reducing UC-induced colonic pathologies, likely by suppressing pro-inflammatory biomarkers

  14. Isothiocyanate-enriched moringa seed extract alleviates ulcerative colitis symptoms in mice.

    PubMed

    Kim, Youjin; Wu, Alex G; Jaja-Chimedza, Asha; Graf, Brittany L; Waterman, Carrie; Verzi, Michael P; Raskin, Ilya

    2017-01-01

    Moringa (Moringa oleifera Lam.) seed extract (MSE) has anti-inflammatory and antioxidant activities. We investigated the effects of MSE enriched in moringa isothiocyanate-1 (MIC-1), its putative bioactive, on ulcerative colitis (UC) and its anti-inflammatory/antioxidant mechanism likely mediated through Nrf2-signaling pathway. Dextran sulfate sodium (DSS)-induced acute (n = 8/group; 3% DSS for 5 d) and chronic (n = 6/group; cyclic rotations of 2.5% DSS/water for 30 d) UC was induced in mice that were assigned to 4 experimental groups: healthy control (water/vehicle), disease control (DSS/vehicle), MSE treatment (DSS/MSE), or 5-aminosalicyic acid (5-ASA) treatment (positive control; DSS/5-ASA). Following UC induction, water (vehicle), 150 mg/kg MSE, or 50 mg/kg 5-ASA were orally administered for 1 or 2 wks. Disease activity index (DAI), spleen/colon sizes, and colonic histopathology were measured. From colon and/or fecal samples, pro-inflammatory biomarkers, tight-junction proteins, and Nrf2-mediated enzymes were analyzed at protein and/or gene expression levels. Compared to disease control, MSE decreased DAI scores, and showed an increase in colon lengths and decrease in colon weight/length ratios in both UC models. MSE also reduced colonic inflammation/damage and histopathological scores (modestly) in acute UC. MSE decreased colonic secretions of pro-inflammatory keratinocyte-derived cytokine (KC), tumor necrosis factor (TNF)-α, nitric oxide (NO), and myeloperoxidase (MPO) in acute and chronic UC; reduced fecal lipocalin-2 in acute UC; downregulated gene expression of pro-inflammatory interleukin (IL)-1, IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) in acute UC; upregulated expression of claudin-1 and ZO-1 in acute and chronic UC; and upregulated GSTP1, an Nrf2-mediated phase II detoxifying enzyme, in chronic UC. MSE was effective in mitigating UC symptoms and reducing UC-induced colonic pathologies, likely by suppressing pro-inflammatory biomarkers

  15. In vivo imaging of human retinal microvasculature using adaptive optics scanning light ophthalmoscope fluorescein angiography

    PubMed Central

    Pinhas, Alexander; Dubow, Michael; Shah, Nishit; Chui, Toco Y.; Scoles, Drew; Sulai, Yusufu N.; Weitz, Rishard; Walsh, Joseph B.; Carroll, Joseph; Dubra, Alfredo; Rosen, Richard B.

    2013-01-01

    The adaptive optics scanning light ophthalmoscope (AOSLO) allows visualization of microscopic structures of the human retina in vivo. In this work, we demonstrate its application in combination with oral and intravenous (IV) fluorescein angiography (FA) to the in vivo visualization of the human retinal microvasculature. Ten healthy subjects ages 20 to 38 years were imaged using oral (7 and/or 20 mg/kg) and/or IV (500 mg) fluorescein. In agreement with current literature, there were no adverse effects among the patients receiving oral fluorescein while one patient receiving IV fluorescein experienced some nausea and heaving. We determined that all retinal capillary beds can be imaged using clinically accepted fluorescein dosages and safe light levels according to the ANSI Z136.1-2000 maximum permissible exposure. As expected, the 20 mg/kg oral dose showed higher image intensity for a longer period of time than did the 7 mg/kg oral and the 500 mg IV doses. The increased resolution of AOSLO FA, compared to conventional FA, offers great opportunity for studying physiological and pathological vascular processes. PMID:24009994

  16. Aggressive resection at the infiltrative margins of glioblastoma facilitated by intraoperative fluorescein guidance.

    PubMed

    Neira, Justin A; Ung, Timothy H; Sims, Jennifer S; Malone, Hani R; Chow, Daniel S; Samanamud, Jorge L; Zanazzi, George J; Guo, Xiaotao; Bowden, Stephen G; Zhao, Binsheng; Sheth, Sameer A; McKhann, Guy M; Sisti, Michael B; Canoll, Peter; D'Amico, Randy S; Bruce, Jeffrey N

    2017-07-01

    OBJECTIVE Extent of resection is an important prognostic factor in patients undergoing surgery for glioblastoma (GBM). Recent evidence suggests that intravenously administered fluorescein sodium associates with tumor tissue, facilitating safe maximal resection of GBM. In this study, the authors evaluate the safety and utility of intraoperative fluorescein guidance for the prediction of histopathological alteration both in the contrast-enhancing (CE) regions, where this relationship has been established, and into the non-CE (NCE), diffusely infiltrated margins. METHODS Thirty-two patients received fluorescein sodium (3 mg/kg) intravenously prior to resection. Fluorescence was intraoperatively visualized using a Zeiss Pentero surgical microscope equipped with a YELLOW 560 filter. Stereotactically localized biopsy specimens were acquired from CE and NCE regions based on preoperative MRI in conjunction with neuronavigation. The fluorescence intensity of these specimens was subjectively classified in real time with subsequent quantitative image analysis, histopathological evaluation of localized biopsy specimens, and radiological volumetric assessment of the extent of resection. RESULTS Bright fluorescence was observed in all GBMs and localized to the CE regions and portions of the NCE margins of the tumors, thus serving as a visual guide during resection. Gross-total resection (GTR) was achieved in 84% of the patients with an average resected volume of 95%, and this rate was higher among patients for whom GTR was the surgical goal (GTR achieved in 93.1% of patients, average resected volume of 99.7%). Intraoperative fluorescein staining correlated with histopathological alteration in both CE and NCE regions, with positive predictive values by subjective fluorescence evaluation greater than 96% in NCE regions. CONCLUSIONS Intraoperative administration of fluorescein provides an easily visualized marker for glioma pathology in both CE and NCE regions of GBM. These

  17. 27 CFR 5.32 - Mandatory label information.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Mandatory label information. 5.32 Section 5.32 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU....D. 7020, 34 FR 20337, Dec. 30, 1969] Editorial Note: For Federal Register citations affecting § 5.32...

  18. 27 CFR 5.32 - Mandatory label information.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Mandatory label information. 5.32 Section 5.32 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU....D. 7020, 34 FR 20337, Dec. 30, 1969] Editorial Note: For Federal Register citations affecting § 5.32...

  19. Dietary Broccoli Alters Rat Cecal Microbiota to Improve Glucoraphanin Hydrolysis to Bioactive Isothiocyanates.

    PubMed

    Liu, Xiaoji; Wang, Yanling; Hoeflinger, Jennifer L; Neme, Bárbara P; Jeffery, Elizabeth H; Miller, Michael J

    2017-03-10

    Broccoli consumption brings many health benefits, including reducing the risk of cancer and inflammatory diseases. The objectives of this study were to identify global alterations in the cecal microbiota composition using 16S rRNA sequencing analysis and glucoraphanin (GRP) hydrolysis to isothiocyanates ex vivo by the cecal microbiota, following different broccoli diets. Rats were randomized to consume AIN93G (control) or different broccoli diets; AIN93G plus cooked broccoli, a GRP-rich powder, raw broccoli, or myrosinase-treated cooked broccoli. Feeding raw or cooked broccoli for four days or longer both changed the cecal microbiota composition and caused a greater production of isothiocyanates ex vivo. A more than two-fold increase in NAD(P)H: quinone oxidoreductase 1 activity of the host colon mucosa after feeding cooked broccoli for seven days confirmed the positive health benefits. Further studies revealed that dietary GRP was specifically responsible for the increased microbial GRP hydrolysis ex vivo, whereas changes in the cecal microbial communities were attributed to other broccoli components. Interestingly, a three-day withdrawal from a raw broccoli diet reversed the increased microbial GRP hydrolysis ex vivo. Findings suggest that enhanced conversion of GRP to bioactive isothiocyanates by the cecal microbiota requires four or more days of broccoli consumption and is reversible.

  20. A Targeted Nanoprobe Based on Carbon Nanotubes-Natural Biopolymer Chitosan Composites

    PubMed Central

    Wu, Baoyan; Zhao, Na

    2016-01-01

    A novel targeting theranostic nanoprobe based on single-walled carbon nanotubes (SWCNTs)-natural biopolymer chitosan composites was developed for cancer cell targeting imaging and fluorescence imaging-guided photodynamic therapy. First, chitosan was respectively conjugated with a tumor-homing molecule folic acid, or a photosensitizing drug pyropheophorbide a using a water-soluble carbodiimide coupling chemistry. Chitosan was fluorescently labeled by fluorescein isothiocyanate via the covalently linkage of the isothiocyanate group with the amino group. Second, SWCNTs were sonicated in the functional chitosan aqueous solution for 6 h at room temperature in order to obtain the nanoprobe (PPa/FITC-SWCNT-FA). The as-prepared nanoprobe has been characterized with transmission electron microscope, confocal microscopy, and cell cytotoxicity tests. Chitosan was decorated onto SWCNTs resulting in the water-dispersible PPa/FITC-SWCNT-FA, and can be selectively transported inside folate receptor-positive tumor cell with good targeting imaging. PPa/FITC-SWCNT-FA exhibited low dark toxicity about 7%–13%, and high phototoxicity about 60%–74% against HeLa cells upon a 635 nm laser irradiation, indicating satisfying biocompatibility and antitumor activity. These results suggest the study could offer a feasible alternative to presently available nanoparticle-based theranostic agents. PMID:28335344

  1. Monitoring of protease catalyzed reactions by quantitative MALDI MS using metal labeling.

    PubMed

    Gregorius, Barbara; Jakoby, Thomas; Schaumlöffel, Dirk; Tholey, Andreas

    2013-05-21

    Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.

  2. Noninvasive positron emission tomography imaging of cell death using a novel small-molecule probe, (18)F labeled bis(zinc(II)-dipicolylamine) complex.

    PubMed

    Wang, Hongliang; Tang, Xiaolan; Tang, Ganghua; Huang, Tingting; Liang, Xiang; Hu, Kongzhen; Deng, Huaifu; Yi, Chang; Shi, Xinchong; Wu, Kening

    2013-08-01

    The synthetic bis(zinc(II)-dipicolylamine) (DPAZn2) coordination complexes are known to have a high specific and selective affinity to target the exposed phosphatidylserine (PS) on the surface of dead and dying cells. An (18)F-labeled DPAZn2 complex (4-(18)F-Fluoro-benzoyl-bis(zinc(II)-dipicolylamine), (18)F-FB-DPAZn2) as positron emission tomography (PET) tracer was developed and evaluated for in vivo imaging of tumor treated with a chemical agent. The in vitro cell stain studies revealed that fluorescent DPAZn2 complexes (Dansyl-DPAZn2) stained the same cells (apoptotic and necrotic cells) as fluorescein isothiocyanate (FITC) labeled Annexin V (FITC-Annexin V). The radiosynthesis of (18)F-FB-DPAZn2 was achieved through the amidation the precursor bis(2,2'-dipicolylamine) derivative (DPA2) with the prosthetic group N-succinimidyl-4-[(18)F]-fluorobenzoate ((18)F-SFB) and chelation with zinc nitrate. In the biodistribution study, the fast clearance of (18)F-FB-DPAZn2 from blood and kidney was observed and high uptake in liver and intestine within 90 min postinjection was also found. For the PET imaging, significantly higher tumor uptake of (18)F-FB-DPAZn2 was observed in the adriamycin (ADM)-treated Hepa1-6 hepatocellular carcinoma-bearing mice than that in the untreated tumor-model mice, while a slightly decreased tumor uptake of (18)F-FDG was found in the ADM-treated tumor-bearing mice. The results indicate that (18)F-FB-DPAZn2 has the similar capability of apoptosis detection as FITC-Annexin V and seems to be a potential PET tracer for noninvasive evaluation and monitoring of anti-tumor chemotherapy. The high uptake of (18)F-FB-DPAZn2 in the abdomen needs to optimize the structure for improving its pharmacokinetics characteristics in the future work.

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

    PubMed

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Fluorescein for resection of high-grade gliomas: A safety study control in a single center and review of the literature

    PubMed Central

    Francaviglia, Natale; Iacopino, Domenico Gerardo; Costantino, Gabriele; Villa, Alessandro; Impallaria, Pietro; Meli, Francesco; Maugeri, Rosario

    2017-01-01

    Background: The importance of a complete resection of high-grade gliomas (HGGs) has been highlighted in scientific literature, in order to limit tumor recurrence and above all to improve disease-free survival rates. Several fluorescent biomarkers have been tested to improve intraoperative identification of residual tumor; 5-aminolevulinic acid (5-ALA) and fluorescein sodium (FS) are now starting to play a central role in glioma surgery. We performed a retrospective analysis on 47 patients operated for HGGs. Here we report our preliminary data. Methods: Data of 47 consecutive patients with HGG have been collected in our study (25 males, 22 females; mean age: 60.3 years, range: 27–86 years). Fluorescein (5 mg/kg of body weight) was injected intravenously right after the induction of general anesthesia. A YELLOW 560 filter was used on an OPMI Pentero 900 microscope (Carl Zeiss Meditec, Oberkochen, Germany) to complete a microsurgical tumor removal. Glioma resection and quality of life were evaluated preoperative and postoperatively. Results: Gross total resection (GTR) was achieved in 53.2% (n = 25) of patients. A subtotal resection (STR) (>95%) was achieved in 29.8% (n = 14), while a partial resection (PR) (<95%) was obtained in 17% (n = 8) of patients. Overall, in 83% (n = 39) of patients who underwent fluorescence-guided surgery the resection rate achieved was >95%. No adverse effects correlated to fluorescein have been recorded. Conclusion: Fluorescein seems to be safe and effective in the resection of HGGs, allowing a high rate of gross total removal of contrast enhanced areas. PMID:28781922

  5. Fluorescein for resection of high-grade gliomas: A safety study control in a single center and review of the literature.

    PubMed

    Francaviglia, Natale; Iacopino, Domenico Gerardo; Costantino, Gabriele; Villa, Alessandro; Impallaria, Pietro; Meli, Francesco; Maugeri, Rosario

    2017-01-01

    The importance of a complete resection of high-grade gliomas (HGGs) has been highlighted in scientific literature, in order to limit tumor recurrence and above all to improve disease-free survival rates. Several fluorescent biomarkers have been tested to improve intraoperative identification of residual tumor; 5-aminolevulinic acid (5-ALA) and fluorescein sodium (FS) are now starting to play a central role in glioma surgery. We performed a retrospective analysis on 47 patients operated for HGGs. Here we report our preliminary data. Data of 47 consecutive patients with HGG have been collected in our study (25 males, 22 females; mean age: 60.3 years, range: 27-86 years). Fluorescein (5 mg/kg of body weight) was injected intravenously right after the induction of general anesthesia. A YELLOW 560 filter was used on an OPMI Pentero 900 microscope (Carl Zeiss Meditec, Oberkochen, Germany) to complete a microsurgical tumor removal. Glioma resection and quality of life were evaluated preoperative and postoperatively. Gross total resection (GTR) was achieved in 53.2% ( n = 25) of patients. A subtotal resection (STR) (>95%) was achieved in 29.8% ( n = 14), while a partial resection (PR) (<95%) was obtained in 17% ( n = 8) of patients. Overall, in 83% ( n = 39) of patients who underwent fluorescence-guided surgery the resection rate achieved was >95%. No adverse effects correlated to fluorescein have been recorded. Fluorescein seems to be safe and effective in the resection of HGGs, allowing a high rate of gross total removal of contrast enhanced areas.

  6. Quantitative Spatial and Temporal Analysis of Fluorescein Angiography Dynamics in the Eye

    PubMed Central

    Hui, Flora; Nguyen, Christine T. O.; Bedggood, Phillip A.; He, Zheng; Fish, Rebecca L.; Gurrell, Rachel; Vingrys, Algis J.; Bui, Bang V.

    2014-01-01

    Purpose We describe a novel approach to analyze fluorescein angiography to investigate fluorescein flow dynamics in the rat posterior retina as well as identify abnormal areas following laser photocoagulation. Methods Experiments were undertaken in adult Long Evans rats. Using a rodent retinal camera, videos were acquired at 30 frames per second for 30 seconds following intravenous introduction of sodium fluorescein in a group of control animals (n = 14). Videos were image registered and analyzed using principle components analysis across all pixels in the field. This returns fluorescence intensity profiles from which, the half-rise (time to 50% brightness), half-fall (time for 50% decay) back to an offset (plateau level of fluorescence). We applied this analysis to video fluorescein angiography data collected 30 minutes following laser photocoagulation in a separate group of rats (n = 7). Results Pixel-by-pixel analysis of video angiography clearly delineates differences in the temporal profiles of arteries, veins and capillaries in the posterior retina. We find no difference in half-rise, half-fall or offset amongst the four quadrants (inferior, nasal, superior, temporal). We also found little difference with eccentricity. By expressing the parameters at each pixel as a function of the number of standard deviation from the average of the entire field, we could clearly identify the spatial extent of the laser injury. Conclusions This simple registration and analysis provides a way to monitor the size of vascular injury, to highlight areas of subtle vascular leakage and to quantify vascular dynamics not possible using current fluorescein angiography approaches. This can be applied in both laboratory and clinical settings for in vivo dynamic fluorescent imaging of vasculature. PMID:25365578

  7. Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation.

    PubMed

    Kofoed, Rikke H; Betzer, Cristine; Lykke-Andersen, Søren; Molska, Ewa; Jensen, Poul H

    2018-05-03

    When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as α-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

  8. Development of validated high-temperature reverse-phase UHPLC-PDA analytical method for simultaneous analysis of five natural isothiocyanates in cruciferous vegetables.

    PubMed

    Robin; Arora, Rohit; Arora, Saroj; Vig, Adarsh Pal

    2018-01-15

    In the present study reverse-phase UHPLC-PDA technique was developed at 60°C for simultaneous quantification of allyl, 3-butenyl, 4-(methylthio)butyl, benzyl and phenethyl isothiocyanates. The validation parameter showed a very good linearity, with a correlation coefficient of 1.00 for all detected standard analytes. Also, high precision and accuracy were observed with lowest obtained values of 1.39% and 99.1%, respectively. Different varieties of three plants, viz. Brassica rapa var. rapa L., Raphanus sativus L. var. oleiformis Pers. and Eruca sativa Mill., were analyzed with this method. After analysis, 4-(methylthio)butyl isothiocyanate was observed to be the major component in the varieties of arugula. Allyl, benzyl and phenethyl isothiocyanates were detected in turnip varieties and, in addition, 3-butenyl isothiocyanate was detected in radish varieties. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Peptide functionalized gold nanoparticles: the influence of pH on binding efficiency

    NASA Astrophysics Data System (ADS)

    Harrison, Emma; Hamilton, Jeremy W. J.; Macias-Montero, Manuel; Dixon, Dorian

    2017-07-01

    We report herein on the synthesis of mixed monolayer gold nanoparticles (AuNPs) capped with both polyethylene glycol (PEG) and one of three peptides. Either a receptor-mediated endocytosis peptide, an endosomal escape pathway (H5WYG) peptide or the Nrp-1 targeting RGD peptide (CRGDK) labeled with FITC. All three peptides have a thiol containing cysteine residue which can be used to bind the peptides to the AuNPs. In order to investigate the influence of pH on peptide attachment, PEGylated AuNPs were centrifuged, the supernatant removed, and the nanoparticles were then re-suspended in a range of pH buffer solutions above, below and at the respective isoelectric points of the peptides before co-functionalization. Peptide attachment was investigated using dynamic light scattering, Ultra-violet visible spectroscopy (UV/Vis), FTIR and photo luminescence spectroscopy. UV/Vis analysis coupled with protein assay results and photoluminescence of the FITC tagged RGD peptide concluded that a pH of ∼8 optimized the cysteine binding and stability, irrespective of the peptide used.

  10. Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp

    PubMed Central

    Han, Juhye; Jyoti, Md. Anirban; Song, Ho-Yeon; Jang, Woong Sik

    2016-01-01

    The candidacidal activity of histatin 5 is initiated through cell wall binding, followed by translocation and intracellular targeting, while the halocidin peptide exerts its activity by attacking the Candida cell membrane. To improve antimicrobial activities and to understand the killing mechanism of two peptides, six hybrid peptides were designed by conjugating histatin 5 and halocidin. A comparative approach was established to study the activity, salt tolerance, cell wall glucan binding assay, cytotoxicity, generation of ROS and killing kinetics. CD spectrometry was conducted to evaluate secondary structures of these hybrid peptides. Furthermore the cellular localization of hybrid peptides was investigated by confocal fluorescence microscopy. Of the six hybrid congeners, di-PH2, di-WP2 and HHP1 had stronger activities than other hybrid peptides against all tested Candida strains. The MIC values of these peptides were 1–2, 2–4 and 2–4 μg/ml, respectively. Moreover, none of the hybrid peptides was cytotoxic in the hemolytic assay and cell-based cytotoxicity assay. Confocal laser microscopy showed that di-PH2 and HHP1 were translocated into cytoplasm whereas di-WP2 was accumulated on surface of C. albicans to exert their candidacidal activity. All translocated peptides (Hst 5, P113, di-PH2) were capable of generating intracellular ROS except HHP1. Additionally, the KFH residues at C-terminal end of these peptides were assumed for core sequence for active translocation. PMID:26918792

  11. Extraction and identification of isothiocyanates from broccolini seeds.

    PubMed

    Zhang, Bochao; Wang, Xiaoqin; Yang, Yanjing; Zhang, Xuewu

    2011-01-01

    Broccolini (Brassica oleracea Italica x Alboglabra) is a cross between broccoli and kai-lan (Chinese broccoli), which contains abundant glucosinolates. The intact glucosinolates are believed to be inactive, while their hydrolysis products, such as isothiocyanates (ITCs), are found to have bacteriocidal and anticarcinogenic activities. So far, no report is available about generation of ITCs during the process of glucosinolate hydrolysis in broccolini. In this study, the hydrolysis of broccolini seed glucosinolates was investigated under controlled conditions of pH, time and temperature, and the ITCs produced were determined. The results showed that an optimum hydrolysis of glucosinolates could be achieved at a temperature of 250C, at pH 7.0, and a reaction time of eight hours. Furthermore, GC-MS analysis indicated that the extracted ITCs primarily were: 3-BITC (3-benzyl-ITC) (10.8%), 4-methylpentyl-ITC (0.5%), 1-isothiocyanato-butane (26.8%), PEITC (phenethyl-ITC) (22.6%) and SFN (sulforaphane) (19.2%).

  12. Comparative study between extraction techniques and column separation for the quantification of sinigrin and total isothiocyanates in mustard seed.

    PubMed

    Cools, Katherine; Terry, Leon A

    2012-07-15

    Glucosinolates are β-thioglycosides which are found naturally in Cruciferae including the genus Brassica. When enzymatically hydrolysed, glucosinolates yield isothiocyanates and give a pungent taste. Both glucosinolates and isothiocyanates have been linked with anticancer activity as well as antifungal and antibacterial properties and therefore the quantification of these compounds is scientifically important. A wide range of literature exists on glucosinolates, however the extraction and quantification procedures differ greatly resulting in discrepancies between studies. The aim of this study was therefore to compare the most popular extraction procedures to identify the most efficacious method and whether each extraction can also be used for the quantification of total isothiocyanates. Four extraction techniques were compared for the quantification of sinigrin from mustard cv. Centennial (Brassica juncea L.) seed; boiling water, boiling 50% (v/v) aqueous acetonitrile, boiling 100% methanol and 70% (v/v) aqueous methanol at 70 °C. Prior to injection into the HPLC, the extractions which involved solvents (acetonitrile or methanol) were freeze-dried and resuspended in water. To identify whether the same extract could be used to measure total isothiocyanates, a dichloromethane extraction was carried out on the sinigrin extracts. For the quantification of sinigrin alone, boiling 50% (v/v) acetonitrile was found to be the most efficacious extraction solvent of the four tested yielding 15% more sinigrin than the water extraction. However, the removal of the acetonitrile by freeze-drying had a negative impact on the isothiocyanate content. Quantification of both sinigrin and total isothiocyanates was possible when the sinigrin was extracted using boiling water. Two columns were compared for the quantification of sinigrin revealing the Zorbax Eclipse to be the best column using this particular method. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. In Vitro Investigation of Influences of Chitosan Nanoparticles on Fluorescein Permeation into Alveolar Macrophages.

    PubMed

    Chachuli, Siti Haziyah Mohd; Nawaz, Asif; Shah, Kifayatullah; Naharudin, Idanawati; Wong, Tin Wui

    2016-06-01

    Pulmonary infection namely tuberculosis is characterized by alveolar macrophages harboring a large microbe population. The chitosan nanoparticles exhibit fast extracellular drug release in aqueous biological milieu. This study investigated the matrix effects of chitosan nanoparticles on extracellular drug diffusion into macrophages. Oligo, low, medium and high molecular weight chitosan nanoparticles were prepared by nanospray drying technique. These nanoparticles were incubated with alveolar macrophages in vitro and had model drug sodium fluorescein added into the same cell culture. The diffusion characteristics of sodium fluorescein and nanoparticle behavior were investigated using fluorescence microscopy, scanning electron microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy techniques. The oligochitosan nanoparticles enabled macrophage membrane fluidization with the extent of sodium fluorescein entry into macrophages being directly governed by the nanoparticle loading. Using nanoparticles made of higher molecular weight chitosan, sodium fluorescein permeation into macrophages was delayed due to viscous chitosan diffusion barrier at membrane boundary. Macrophage-chitosan nanoparticle interaction at membrane interface dictates drug migration into cellular domains.

  14. Fluorescein-guided surgery for grade IV gliomas with a dedicated filter on the surgical microscope: preliminary results in 12 cases.

    PubMed

    Acerbi, Francesco; Broggi, Morgan; Eoli, Marica; Anghileri, Elena; Cuppini, Lucia; Pollo, Bianca; Schiariti, Marco; Visintini, Sergio; Orsi, Chiara; Franzini, Angelo; Broggi, Giovanni; Ferroli, Paolo

    2013-07-01

    Fluorescein is widely used as a fluorescent tracer for many applications. Its capability to accumulate in cerebral areas with blood-brain barrier damage makes it an ideal dye for intraoperative visualization of malignant gliomas (MG). We report our preliminary experience in fluorescein-guided removal of grade IV gliomas using a dedicated filter on the surgical microscope. In September 2011 we started a prospective phase II trial (FLUOGLIO) to evaluate the safety and obtain initial indications about the efficacy of fluorescein-guided surgery for MG. Patients with suspected MG amenable to complete resection of contrast-enhancing areas were eligible to participate in this study. This report is based on a preliminary analysis of the results of 12 patients with grade IV gliomas out of 15 consecutive cases (age range 48-72 years) enrolled since September 2011. Fluorescein was injected intravenously (i.v.) after intubation (5-10 mg/kg). The tumor was removed using a microsurgical technique and fluorescence visualization by BLU 400 or YELLOW 560 filters on a Pentero microscope (Carl Zeiss, Germany). The study was approved by our ethics committee and registered on the European Regulatory Authorities website (EudraCT no. 2011-002527-18). Histological analysis confirmed grade IV gliomas in 12/15 cases. Median preoperative tumor volume was 33.15 cm(3) (9.6-87.8 cm(3)). No adverse reaction related to the administration of fluorescein was registered. Contrast-enhanced tumor was completely removed in 75 % of the patients. This preliminary analysis suggested that the use of intravenous fluorescein during surgery on grade IV gliomas is safe and allows a high rate of complete resection of contrast-enhanced tumor at the early postoperative MRI.

  15. Where and When to Cut? Fluorescein Guidance for Brain Stem and Spinal Cord Tumor Surgery-Technical Note.

    PubMed

    Molina, Eric Suero; Stummer, Walter

    2017-12-29

    Spinal cord and brain stem lesions require a judicious approach with an optimized trajectory due to a clustering of functions on their surfaces. Intraoperative mapping helps locate function. To confidently locate such lesions, neuronavigation alone lacks the desired accuracy and is of limited use in the spinal cord. To evaluate the clinical value of fluoresceins for initial delineation of such critically located lesions. We evaluated fluorescein guidance in the surgical resection of lesions with blood-brain barrier disruption demonstrating contrast enhancement in magnet resonance imaging in the spinal cord and in the brain stem in 3 different patients. Two patients harbored a diffuse cervical and thoracic spinal cord lesion, respectively. Another patient suffered metastatic lesions in the brain stem and at the floor of the fourth ventricle. Low-dose fluorescein (4 mg/kg body weight) was applied after anesthesia induction and visualized using the Zeiss Pentero 900 Yellow560 filter (Carl Zeiss, Oberkochen, Germany). Fluorescein was helpful for locating lesions and for defining the best possible trajectory. During resection, however, we found unspecific propagation of fluorescein within the brain stem up to 6 mm within 3 h after application. As these lesions were otherwise distinguishable from surrounding tissue, monitoring resection was not an issue. Fluorescein guidance is a feasible tool for defining surgical entry zones when aiming for surgical removal of spinal cord and brain stem lesions. Unselective fluorescein extravasation cautions against using such methodology for monitoring completeness of resection. Providing the right timing, a window of pseudoselectivity could increase fluoresceins' clinical value in these cases. © Congress of Neurological Surgeons 2017.

  16. gamma. -Preprotachykinin-(72-92)-peptide amide: An endogenous preprotachykinin I gene-derived peptide that preferentially binds to neurokinin-2 receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dam, T.V.; Takeda, Y.; Krause, J.E.

    1990-01-01

    The presence of N-terminally extended forms of neurokinin A has recently been reported in the mammalian brain. Among them, gamma-preprotachykinin-(72-92)-peptide amide (gamma-PPT-(72-92)-NH2), a peptide derived by posttranslational processing of gamma-preprotachykinin, is most prominent. We report here that this peptide most likely acts on neurokinin-2 receptor sites since neurokinin A (a putative neurokinin-2 agonist) and gamma-PPT-(72-92)-NH2 are potent competitors of 125I-labeled gamma-PPT-(72-92)-NH2 binding whereas selective neurokinin-1 and -3 agonists are not. Moreover, the distribution of 125I-labeled gamma-PPT-(72-92)-NH2 and 125I-labeled neurokinin A binding sites are very similar in rat brain. On the other hand, 125I-labeled Bolton-Hunter-substance P (a neurokinin-1 ligand) and 125I-labeledmore » Bolton-Hunter-eledoisin (a neurokinin-3 ligand) binding sites are differentially located in this tissue. Thus, it appears that gamma-PPT-(72-92)-NH2 binds to neurokinin-2 receptors and should be considered as a putative endogenous ligand for this receptor class.« less

  17. Physical stability of arginine-glycine-aspartic acid peptide coated on anodized implants after installation.

    PubMed

    Huh, Jung-Bo; Lee, Jeong-Yeol; Jeon, Young-Chan; Shin, Sang-Wan; Ahn, Jin-Soo; Ryu, Jae-Jun

    2013-05-01

    The aim of this study was to evaluate the stability of arginine-glycine-aspartic acid (RGD) peptide coatings on implants by measuring the amount of peptide remaining after installation. Fluorescent isothiocyanate (FITC)-fixed RGD peptide was coated onto anodized titanium implants (width 4 mm, length 10 mm) using a physical adsorption method (P) or a chemical grafting method (C). Solid Rigid Polyurethane Foam (SRPF) was classified as either hard bone (H) or soft bone (S) according to its density. Two pieces of artificial bone were fixed in a customized jig, and coated implants were installed at the center of the boundary between two pieces of artificial bone. The test groups were classified as: P-H, P-S, C-H, or C-S. After each installation, implants were removed from the SRPF, and the residual amounts and rates of RGD peptide in implants were measured by fluorescence spectrometry. The Kruskal-Wallis test was used for the statistical analysis (α=0.05). Peptide-coating was identified by fluorescence microscopy and XPS. Total coating amount was higher for physical adsorption than chemical grafting. The residual rate of peptide was significantly larger in the P-S group than in the other three groups (P<.05). The result of this study suggests that coating doses depend on coating method. Residual amounts of RGD peptide were greater for the physical adsorption method than the chemical grafting method.

  18. 16 CFR § 1615.5 - Labeling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 2 2013-01-01 2013-01-01 false Labeling requirements. § 1615.5 Section § 1615.5 Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FLAMMABLE FABRICS ACT REGULATIONS... precautionary instructions to protect the items from agents or treatments which are known to cause deterioration...

  19. Evaluating Ga-68 Peptide Conjugates for Targeting VPAC Receptors: Stability and Pharmacokinetics.

    PubMed

    Kumar, Pardeep; Tripathi, Sushil K; Chen, C P; Wickstrom, Eric; Thakur, Mathew L

    2018-05-25

    In recent years, considerable progress has been made in the use of gallium-68 labeled receptor-specific peptides for imaging oncologic diseases. The objective was to examine the stability and pharmacokinetics of [ 68 Ga]NODAGA and DOTA-peptide conjugate targeting VPAC [combined for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP)] receptors on tumor cells. A VPAC receptor-specific peptide was chosen as a model peptide and conjugated to NODAGA and DOTA via solid-phase synthesis. The conjugates were characterized by HPLC and MALDI-TOF. Following Ga-68 chelation, the radiochemical purity of Ga-68 labeled peptide conjugate was determined by radio-HPLC. The stability was tested against transmetallation using 100 nM Fe 3+ /Zn 2+ /Ca 2+ ionic solution and against transchelation using 200 μM DTPA solution. The ex vivo and in vivo stability of the Ga-68 labeled peptide conjugate was tested in mouse plasma and urine. Receptor specificity was determined ex vivo by cell binding assays using human breast cancer BT474 cells. Positron emission tomography (PET)/X-ray computed tomography (CT) imaging, tissue distribution, and blocking studies were performed in mice bearing BT474 xenografts. The chemical and radiochemical purity was greater than 95 % and both conjugates were stable against transchelation and transmetallation. Ex vivo stability at 60 min showed that the NODAGA-peptide-bound Ga-68 reduced to 42.1 ± 3.7 % (in plasma) and 37.4 ± 2.9 % (in urine), whereas the DOTA-peptide-bound Ga-68 was reduced to 1.2 ± 0.3 % (in plasma) and 4.2 ± 0.4 % (in urine) at 60 min. Similarly, the in vivo stability for [ 68 Ga]NODAGA-peptide was decreased to 2.1 ± 0.2 % (in plasma) and 2.2 ± 0.4 % (in urine). For [ 68 Ga]DOTA-peptide, it was decreased to 1.4 ± 0.3 % (in plasma) and 1.2 ± 0.4 % (in urine) at 60 min. The specific BT474 cell binding was 53.9 ± 0.8 % for [ 68 Ga]NODAGA-peptide

  20. Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB.

    PubMed

    Smallwood, Chuck R; Marco, Amparo Gala; Xiao, Qiaobin; Trinh, Vy; Newton, Salete M C; Klebba, Phillip E

    2009-06-01

    We studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature-dependent in vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37 degrees C. At 0 degrees C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N- and C- domains of FepA. However, colicin B penetration into the cell at 37 degrees C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. FepA was expressed at lower levels in a tonB host strain, but when we accounted for this decrease its FM labelling was comparable whether TonB was present or absent. Thus we did not detect TonB-dependent structural changes in FepA, either alone or when it interacted with colicin B at 37 degrees C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N-domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.

  1. Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C,15N-labeled peptides and proteins.

    PubMed

    Janik, Rafal; Ritz, Emily; Gravelle, Andrew; Shi, Lichi; Peng, Xiaohu; Ladizhansky, Vladimir

    2010-03-01

    In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly (13)C,(15)N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]-CO[i-2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  2. In vivo analysis of intestinal permeability following hemorrhagic shock

    PubMed Central

    Alsaigh, Tom; Chang, Marisol; Richter, Michael; Mazor, Rafi; Kistler, Erik B

    2015-01-01

    AIM: To determine the time course of intestinal permeability changes to proteolytically-derived bowel peptides in experimental hemorrhagic shock. METHODS: We injected fluorescently-conjugated casein protein into the small bowel of anesthetized Wistar rats prior to induction of experimental hemorrhagic shock. These molecules, which fluoresce when proteolytically cleaved, were used as markers for the ability of proteolytically cleaved intestinal products to access the central circulation. Blood was serially sampled to quantify the relative change in concentration of proteolytically-cleaved particles in the systemic circulation. To provide spatial resolution of their location, particles in the mesenteric microvasculature were imaged using in vivo intravital fluorescent microscopy. The experiments were then repeated using an alternate measurement technique, fluorescein isothiocyanate (FITC)-labeled dextrans 20, to semi-quantitatively verify the ability of bowel-derived low-molecular weight molecules (< 20 kD) to access the central circulation. RESULTS: Results demonstrate a significant increase in systemic permeability to gut-derived peptides within 20 min after induction of hemorrhage (1.11 ± 0.19 vs 0.86 ± 0.07, P < 0.05) compared to control animals. Reperfusion resulted in a second, sustained increase in systemic permeability to gut-derived peptides in hemorrhaged animals compared to controls (1.2 ± 0.18 vs 0.97 ± 0.1, P < 0.05). Intravital microscopy of the mesentery also showed marked accumulation of fluorescent particles in the microcirculation of hemorrhaged animals compared to controls. These results were replicated using FITC dextrans 20 [10.85 ± 6.52 vs 3.38 ± 1.11 fluorescent intensity units (× 105, P < 0.05, hemorrhagic shock vs controls)], confirming that small bowel ischemia in response to experimental hemorrhagic shock results in marked and early increases in gut membrane permeability. CONCLUSION: Increased small bowel permeability in hemorrhagic

  3. Interpretation and use of the 5-level EQ-5D response labels varied with survey language among Asians in Singapore.

    PubMed

    Luo, Nan; Wang, Ye; How, Choon How; Tay, Ee Guan; Thumboo, Julian; Herdman, Michael

    2015-10-01

    This study aimed to investigate the comparability of the English, Chinese, and Malay versions of the 5-level EQ-5D (EQ-5D-5L) response labels in Singapore. Visitors to a primary care institution in Singapore (n = 743) were asked to complete two exercises: (1) rating the severity of the EQ-5D-5L response labels presented in English, Chinese, or Malay using a 0-100 numeric rating scale and (2) using the labels to describe various hypothetical health problems. Label ratings and choices between language versions were compared using regression analysis. Perceived severity of the English and Chinese labels was similar. Compared with their English counterparts, the Malay label "slight(ly)" was rated as more severe (adjusted mean difference: 10.5 to 14.5) and "unable"/"extreme(ly)" as less severe (adjusted mean difference: -13.3 to -11.0) (P < 0.001 for all). The Malay labels "no(t)" and "unable"/"extreme(ly)" and the less severe Chinese labels were more frequently used to describe hypothetical health problems than their English counterparts. Interpretation and use of the EQ-5D-5L response labels vary among Singaporeans using different language versions of the instrument. Future studies need to investigate ways to reduce the variations and increase the cross-cultural measurement equivalence of the instrument. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 2: Peptide Tags and Unnatural Amino Acids.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-04-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  5. Cell Uptake and Validation of Novel PECs for Biomedical Applications.

    PubMed

    Palamà, Ilaria E; Musarò, Mariarosaria; Coluccia, Addolorata M L; D'Amone, Stefania; Gigli, Giuseppe

    2011-01-01

    This pilot study provides the proof of principle for biomedical application of novel polyelectrolyte complexes (PECs) obtained via electrostatic interactions between dextran sulphate (DXS) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that DXS/PAH polyelectrolyte complexes were Monodispersed with regular rounded-shape features and average diameters of 250 nm at 2 : 1 weight ratios of DXS/PAH. Fluorescently labelled DXS and fluorescein-isothiocyanate- (FITC-)conjugate DXS were used to follow cell uptake efficiency of PECs and biodegradability of their enzymatically degradable DXS-layers by using confocal laser scanning microscopy (CLSM). Moreover, quantitative MTT and Trypan Blue assays were employed to validate PECs as feasible and safe nanoscaled carriers at single-cell level without adverse effects on metabolism and viability.

  6. Cell Uptake and Validation of Novel PECs for Biomedical Applications

    PubMed Central

    Palamà, Ilaria E.; Musarò, Mariarosaria; Coluccia, Addolorata M. L.; D'Amone, Stefania; Gigli, Giuseppe

    2011-01-01

    This pilot study provides the proof of principle for biomedical application of novel polyelectrolyte complexes (PECs) obtained via electrostatic interactions between dextran sulphate (DXS) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that DXS/PAH polyelectrolyte complexes were Monodispersed with regular rounded-shape features and average diameters of 250 nm at 2 : 1 weight ratios of DXS/PAH. Fluorescently labelled DXS and fluorescein-isothiocyanate- (FITC-)conjugate DXS were used to follow cell uptake efficiency of PECs and biodegradability of their enzymatically degradable DXS-layers by using confocal laser scanning microscopy (CLSM). Moreover, quantitative MTT and Trypan Blue assays were employed to validate PECs as feasible and safe nanoscaled carriers at single-cell level without adverse effects on metabolism and viability. PMID:21876815

  7. Preparation of poly(methyl methacrylate) microcapsules by in situ polymerization on the surface of calcium carbonate particles.

    PubMed

    Sato, Katsuhiko; Nakajima, Tatsuya; Anzai, Jun-ichi

    2012-12-01

    Poly(methyl methacrylate) (PMMA) microcapsules were prepared by the in situ polymerization of methyl methacrylate (MMA) and N,N'-methylenebisacrylamide on the surface of calcium carbonate (CaCO(3)) particles, followed by the dissolution of the CaCO(3) core in ethylenediaminetetraacetic acid solution. The microcapsules were characterized using fluorescence microscopy, atomic force microscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy. The average sizes of the CaCO(3) particles and PMMA capsules were 3.8±0.6 and 4.0±0.6 μm, respectively. A copolymer consisting of MMA and rhodamine B-bearing MMA was also used to prepare microcapsules for fluorescent microscopy observations. Fluorescein isothiocyanate-labeled bovine serum albumin was enclosed in the PMMA microcapsules and its release properties were studied. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. The use of fluorescein sodium in the biopsy and gross-total resection of a tectal plate glioma.

    PubMed

    Ung, Timothy H; Kellner, Christopher; Neira, Justin A; Wang, Shih-Hsiu J; D'Amico, Randy; Faust, Phyllis L; Canoll, Peter; Feldstein, Neil A; Bruce, Jeffrey N

    2015-12-01

    Intravenous administration of fluorescein sodium fluoresces glioma burden tissue and can be visualized using the surgical microscope with a specialized filter. Intraoperative guidance afforded through the use of fluorescein may enhance the fidelity of tissue sampling, and increase the ability to accomplish complete resection of tectal lesions. In this report the authors present the case of a 19-year-old man with a tectal anaplastic pilocytic astrocytoma in which the use of fluorescein sodium and a Zeiss Pentero surgical microscope equipped with a yellow 560 filter enabled safe complete resection. In conjunction with neurosurgical navigation, added intraoperative guidance provided by fluorescein may be beneficial in the resection of brainstem gliomas.

  9. In Response to Laovirojjanakul W, Acharya N and Gonzales JA's "Ultra-Widefield Fluorescein Angiography in Intermediate Uveitis".

    PubMed

    Babu, Kalpana; Kumaradas, Mrinalini

    2017-12-28

    We read with great interest the article by Laovirojjanakul et al. on ultra-widefield fluorescein angiography in intermediate uveitis. We would like to share a similar case of chronic intermediate uveitis highlighting a fern-like pattern of diffuse vascular leakage on fluorescein angiography, with good visual acuity, absence of clinically active inflammation, and a similar fluorescein angiography picture over a follow-up of 4 years.

  10. Covalent dye attachment influences the dynamics and conformational properties of flexible peptides

    PubMed Central

    Crevenna, Alvaro H.; Bomblies, Rainer; Lamb, Don C.

    2017-01-01

    Fluorescence spectroscopy techniques like Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for the in vitro and in vivo investigation of conformational dynamics in biomolecules. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore either by a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, as used in FCS with photoinduced electron transfer (PET). The attachment of fluorophores to the molecule of interest can potentially alter the molecular properties and may affect the relevant conformational states and dynamics especially of flexible biomolecules like intrinsically disordered proteins (IDP). Using the intrinsically disordered S-peptide as a model system, we investigate the impact of terminal fluorescence labeling on the molecular properties. We perform extensive molecular dynamics simulations on the labeled and unlabeled peptide and compare the results with in vitro PET-FCS measurements. Experimental and simulated timescales of end-to-end fluctuations were found in excellent agreement. Comparison between simulations with and without labels reveal that the π-stacking interaction between the fluorophore labels traps the conformation of S-peptide in a single dominant state, while the unlabeled peptide undergoes continuous conformational rearrangements. Furthermore, we find that the open to closed transition rate of S-peptide is decreased by at least one order of magnitude by the fluorophore attachment. Our approach combining experimental and in silico methods provides a benchmark for the simulations and reveals the significant effect that fluorescence labeling can have on the conformational dynamics of small biomolecules, at least for inherently flexible short peptides. The presented protocol is not only useful for comparing PET-FCS experiments with simulation results but provides a strategy to minimize the influence on

  11. A strategy to modulate the electrophoretic behavior in plastic microchips using sodium polystyrene sulfonate.

    PubMed

    Guo, Jinxiu; Chen, Yu; Zhao, Lizhi; Sun, Ping; Li, Hongli; Zhou, Lei; Wang, Xiayan; Pu, Qiaosheng

    2016-12-16

    Plastic microchips have been broadly used as disposable microfluidic devices, but the poorly defined surface properties limit their application. Herein, we proved that an anionic polymer could be used as the background electrolyte (BGE) to provide a strong and stable cathodic electroosmotic flow (EOF) and modulate the electrophoretic behavior for efficient separation in relative thicker microchannels (∼75μm id). A cathodic EOF of ∼3.3×10 -4 cm 2 V -1 s -1 was maintained using sodium polystyrene sulfonate (PSSNa) with a molecular weight of 5×10 5 as the BGE, which ensured fluorescein isothiocyanate labeled biogenic amines (BAs) appeared ahead of other components in the electropherograms obtained with microchips of cyclic olefin copolymer. Four selected BAs appeared within 50s and theoretical plate numbers of 8.0×10 5 /m were achieved. The role of PSSNa was evaluated with streaming potential, dynamic light scattering, contact angle and atomic force microscopy. Its functionalities as surface modifier, viscosity regulator and pseudostationary phase were also confirmed. The proposed electrophoretic method was applied in the fast determination of BAs in fish meat samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. pH-regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments.

    PubMed

    Levi Mortera, Stefano; Dioni, Ilaria; Greco, Viviana; Neri, Cristina; Rovero, Paolo; Urbani, Andrea

    2014-05-01

    Among the most common stable-isotope labeling strategies, the reaction of formaldehyde with peptides in the presence of NaCNBH₃ features many attractive aspects that are conducive to its employment in quantitation experiments in proteomics. Reductive amination, with formaldehyde and d(2)-formaldehyde, is reported to be a fast, easy, and specific reaction, undoubtedly inexpensive if compared with commercially available kits for differential isotope coding. Acetaldehyde and d(4)-acetaldehyde could be employed as well without a substantial increase in terms of cost, and should provide a wider spacing between the differentially tagged peptides in the mass spectrum. Nevertheless, only a single paper reports about a diethylation approach for quantitation. We undertook a systematic analytical investigation on the reductive amination of some standard peptides pointing out the occasional occurrence of side reactions in dependence of pH or reagents order of addition, particularly observing the formation of cyclic adducts ascribable to rearrangements involving the generated Schiff-base and all the nucleophilic sites of its chemical environment. We also tried to evaluate how much this side-products amount may impair isotope coded relative quantitation.

  13. Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry *

    PubMed Central

    Lin, Shu; Wein, Samuel; Gonzales-Cope, Michelle; Otte, Gabriel L.; Yuan, Zuo-Fei; Afjehi-Sadat, Leila; Maile, Tobias; Berger, Shelley L.; Rush, John; Lill, Jennie R.; Arnott, David; Garcia, Benjamin A.

    2014-01-01

    To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua™ technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono- (me1), di- (me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the

  14. Integrated on-chip derivatization and electrophoresis for the rapid analysis of biogenic amines.

    PubMed

    Beard, Nigel P; Edel, Joshua B; deMello, Andrew J

    2004-07-01

    We demonstrate the monolithic integration of a chemical reactor with a capillary electrophoresis device for the rapid and sensitive analysis of biogenic amines. Fluorescein isothiocyanate (FITC) is widely employed for the analysis of amino-group containing analytes. However, the slow reaction kinetics hinders the use of this dye for on-chip labeling applications. Other alternatives are available such as o-phthaldehyde (OPA), however, the inferior photophysical properties and the UV lambdamax present difficulties when using common excitation sources leading to a disparity in sensitivity. Consequently, we present for the first time the use of dichlorotriazine fluorescein (DTAF) as a superior in situ derivatizing agent for biogenic amines in microfluidic devices. The developed microdevice employs both hydrodynamic and electroosmotic flow, facilitating the creation of a polymeric microchip to perform both precolumn derivatization and electrophoretic analysis. The favorable photophysical properties of the DTAF and its fast reaction kinetics provide detection limits down to 1 nM and total analysis times (including on-chip mixing and reaction) of <60 s. The detection limits are two orders of magnitude lower than current limits obtained with both FITC and OPA. The optimized microdevice is also employed to probe biogenic amines in real samples.

  15. Colorful Polyelectrolytes: An Atom Transfer Radical Polymerization Route to Fluorescent Polystyrene Sulfonate.

    PubMed

    Huberty, Wayne; Tong, Xiaowei; Balamurugan, Sreelatha; Deville, Kyle; Russo, Paul S; Zhang, Donghui

    2016-03-01

    A labeled green fluorescent polystyrene sulfonate (LNaPSS) has been synthesized using atom transfer radical polymerization of a styrene sulfonate monomer with a fluorescent co-monomer, fluorescein thiocyanate-vinyl aniline. As a result this 100 % sulfonated polymer contains no hydrophobic patches along the chain backbone besides the fluorescent marker itself. The concentration of the fluorescent monomer was kept low to maintain the characteristic properties of the anionic polyelectrolyte, LNaPSS. ATRP conditions facilitated the production of polymers spanning a range of molecular weights from 35,000 to 175,000 in gram-scale batches with polydispersity indices of 1.01-1.24. Molecular weight increased with the monomer to initiator ratio. Gel permeation chromatography results show a unimodal distribution, and the polymer structure was also confirmed by (1)H NMR and FT-IR spectroscopy. Fluorescence spectroscopy confirmed covalent bonding of fluorescein isothiocyanate to the polymer, indicating that the polymer is suitable as a probe in fluorescence microscopy. To demonstrate this ability, the polymer was used to locate structural features in salt crystals formed during drying, as in the evaporation of sea mist. A second application to probe diffusion studies is also demonstrated.

  16. ORGANOPHOSPHORUS HYDROLASE-BASED ASSAY FOR ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    We report a rapid and versatile Organophosphorus hydrolase (OPH)-based method for measurement of organophosphates. This assay is based on a substrate-dependent change in pH at the local vicinity of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC), ...

  17. Photonic-plasmonic hybrid single-molecule nanosensor measures the effect of fluorescent labels on DNA-protein dynamics

    PubMed Central

    Liang, Feng; Guo, Yuzheng; Hou, Shaocong; Quan, Qimin

    2017-01-01

    Current methods to study molecular interactions require labeling the subject molecules with fluorescent reporters. However, the effect of the fluorescent reporters on molecular dynamics has not been quantified because of a lack of alternative methods. We develop a hybrid photonic-plasmonic antenna-in-a-nanocavity single-molecule biosensor to study DNA-protein dynamics without using fluorescent labels. Our results indicate that the fluorescein and fluorescent protein labels decrease the interaction between a single DNA and a protein due to weakened electrostatic interaction. Although the study is performed on the DNA-XPA system, the conclusion has a general implication that the traditional fluorescent labeling methods might be misestimating the molecular interactions. PMID:28560341

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

    PubMed

    Persson, Morten; Madsen, Jacob; Østergaard, Søren; Ploug, Michael; Kjaer, Andreas

    2012-05-01

    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. Synthesis of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) was based on solid-phase peptide synthesis protocols using the Fmoc strategy. (68)GaCl(3) was eluted from a (68)Ge/(68)Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, 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. In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) with (68)Ga was done at 95°C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH(2) was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for (68)Ga-NODAGA-AE105-NH(2) and (68)Ga-DOTA-AE105-NH(2), respectively. A significantly higher tumor-to-muscle ratio (P<.05) was found for (68)Ga

  19. An SH2 domain model of STAT5 in complex with phospho-peptides define ``STAT5 Binding Signatures''

    NASA Astrophysics Data System (ADS)

    Gianti, Eleonora; Zauhar, Randy J.

    2015-05-01

    The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

  20. 46 CFR 160.023-5 - Labeling and marking.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Labeling and marking. 160.023-5 Section 160.023-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Hand Combination Flare and Smoke Distress Signals § 160.023-5...

  1. 46 CFR 160.023-5 - Labeling and marking.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Labeling and marking. 160.023-5 Section 160.023-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Hand Combination Flare and Smoke Distress Signals § 160.023-5...

  2. 46 CFR 160.023-5 - Labeling and marking.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Labeling and marking. 160.023-5 Section 160.023-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Hand Combination Flare and Smoke Distress Signals § 160.023-5...

  3. 46 CFR 160.023-5 - Labeling and marking.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Labeling and marking. 160.023-5 Section 160.023-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Hand Combination Flare and Smoke Distress Signals § 160.023-5...

  4. 46 CFR 160.058-5 - Labeling and marking.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Labeling and marking. 160.058-5 Section 160.058-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-5...

  5. 46 CFR 160.058-5 - Labeling and marking.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 6 2012-10-01 2012-10-01 false Labeling and marking. 160.058-5 Section 160.058-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-5...

  6. 46 CFR 160.058-5 - Labeling and marking.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 6 2014-10-01 2014-10-01 false Labeling and marking. 160.058-5 Section 160.058-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-5...

  7. 46 CFR 160.058-5 - Labeling and marking.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 6 2011-10-01 2011-10-01 false Labeling and marking. 160.058-5 Section 160.058-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-5...

  8. 46 CFR 160.058-5 - Labeling and marking.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 6 2010-10-01 2010-10-01 false Labeling and marking. 160.058-5 Section 160.058-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Desalter Kits, Sea Water, for Merchant Vessels § 160.058-5...

  9. 6-Methylsulfinylhexyl isothiocyanate and its homologues as food-originated compounds with antibacterial activity against Escherichia coli and Staphylococcus aureus.

    PubMed

    Ono, H; Tesaki, S; Tanabe, S; Watanabe, M

    1998-02-01

    Cruciferae plants, banana and coriander each showed antibacterial activity. The highest activity among the food-stuffs tested was found in the stems of wasabi. An ethereal extract from wasabi stems had potent antibacterial activity and we isolated the active compound from the extract. Instrumental analysis identified the compound as 6-methylsulfinylhexyl isothiocyanate. Some homologues of 6-methylsulfinylhexyl isothiocyanate were also active against Escherichia coli and Staphylococcus aureus.

  10. Metabolic Activity of Radish Sprouts Derived Isothiocyanates in Drosophila melanogaster

    PubMed Central

    Baenas, Nieves; Piegholdt, Stefanie; Schloesser, Anke; Moreno, Diego A.; García-Viguera, Cristina; Rimbach, Gerald; Wagner, Anika E.

    2016-01-01

    We used Drosophila melanogaster as a model system to study the absorption, metabolism and potential health benefits of plant bioactives derived from radish sprouts (Raphanus sativus cv. Rambo), a Brassicaceae species rich in glucosinolates and other phytochemicals. Flies were subjected to a diet supplemented with lyophilized radish sprouts (10.6 g/L) for 10 days, containing high amounts of glucoraphenin and glucoraphasatin, which can be hydrolyzed by myrosinase to the isothiocyanates sulforaphene and raphasatin, respectively. We demonstrate that Drosophila melanogaster takes up and metabolizes isothiocyanates from radish sprouts through the detection of the metabolite sulforaphane-cysteine in fly homogenates. Moreover, we report a decrease in the glucose content of flies, an upregulation of spargel expression, the Drosophila homolog of the mammalian PPARγ-coactivator 1 α, as well as the inhibition of α-amylase and α-glucosidase in vitro. Overall, we show that the consumption of radish sprouts affects energy metabolism in Drosophila melanogaster which is reflected by lower glucose levels and an increased expression of spargel, a central player in mitochondrial biogenesis. These processes are often affected in chronic diseases associated with aging, including type II diabetes mellitus. PMID:26901196

  11. Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

    PubMed Central

    Rigby, Susan; Procop, Gary W.; Haase, Gerhard; Wilson, Deborah; Hall, Geraldine; Kurtzman, Cletus; Oliveira, Kenneth; Von Oy, Sabina; Hyldig-Nielsen, Jens J.; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. PMID:12037084

  12. Identification of a hepatoprotective peptide in wheat gluten hydrolysate against D-galactosamine-induced acute hepatitis in rats.

    PubMed

    Sato, Kenji; Egashira, Yukari; Ono, Shin; Mochizuki, Satoshi; Shimmura, Yuki; Suzuki, Yoshio; Nagata, Megumi; Hashimoto, Kaori; Kiyono, Tamami; Park, Eun Young; Nakamura, Yasushi; Itabashi, Mariko; Sakata, Yuka; Furuta, Seigo; Sanada, Hiroo

    2013-07-03

    A hepatoprotective peptide, pyroglutamyl leucine (pyroGlu-Leu), was identified in wheat gluten hydrolysate through an in vivo activity-guided fractionation approach based on D-galactosamine-induced acute hepatitis in rats and fractionation of peptides with large-scale preparative ampholine-free isoelectric focusing. The active acidic fraction predominantly consisted of pyroglutamyl peptides and free pyroglutamic acid. Pyroglutamyl peptides were derivatized with phenyl isothiocyanate after removal of a pyroglutamyl residue by pyroglutamate aminopeptidase. The derivatives were purified by reversed-phase HPLC and subjected to sequence analysis. The active fraction contained pyroGlu-Ile, pyroGlu-Leu, pyroGlu-Gln, pyroGlu-Gln-Gln, and free pyroGlu. Ingestion of pyroGlu-Leu at 20 mg/kg body weight significantly decreased serum aspartate and alanine aminotransferases to approximately 30% and 20% of those values of the vehicle group, respectively, which were near the normal levels. Thirty minutes after ingestion of pyroGlu-Leu at 20 mg/kg, the concentration of pyroGlu-Leu in portal blood plasma increased to approximately 2 μM.

  13. Sulforaphane, a Natural Isothiocyanate Compound, Improves Cardiac Function and Remodeling by Inhibiting Oxidative Stress and Inflammation in a Rabbit Model of Chronic Heart Failure.

    PubMed

    Ma, Tongliang; Zhu, Decai; Chen, Duoxue; Zhang, Qiaoyun; Dong, Huifang; Wu, Wenwu; Lu, Huihe; Wu, Guangfu

    2018-03-12

    BACKGROUND The aim of this study was to investigate the effects of sulforaphane (SFN), a natural isothiocyanate compound, in a rabbit ascending aortic cerclage model of chronic heart failure (CHF). MATERIAL AND METHODS Thirty New Zealand White rabbits were divided into the sham operation group (n=10), the CHF group (n=10), and the CHF + SFN group (n=10) treated with subcutaneous SFN (0.5 mg/kg) for five days per week for 12 weeks. After 12 weeks, echocardiography and biometric analysis were performed, followed by the examination of the rabbit hearts. Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to detect levels of inflammatory cytokines, superoxide dismutase (SOD), and malondialdehyde (MDA). RESULTS In the CHF group, compared with the sham operation group, there was an increase in the heart weight to body weight ratio (HW/BW), the left ventricular weight to body weight ratio (LVW/BW), the left ventricular end diastolic diameter (LVEDD), the left ventricular end systolic diameter (LVESD), plasma brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) levels, the cardiac collagen volume fraction (CVF), apoptotic index, expression levels of collagen I, collagen III, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and malondialdehyde (MDA) in the myocardial tissue, and a decrease in the left ventricular shortening fraction (LVFS) and left ventricular ejection fraction (LVEF), and cardiac superoxide dismutase (SOD) activity. These changes were corrected in the SFN-treated group. CONCLUSIONS In a rabbit model of CHF, treatment with SFN improved cardiac function and remodeling by inhibiting oxidative stress and inflammation.

  14. Spectroscopic studies on the interaction of fluorescein and safranine T in PC liposomes

    NASA Astrophysics Data System (ADS)

    Bozkurt, Ebru; Bayraktutan, Tuğba; Acar, Murat; Toprak, Mahmut

    2013-01-01

    In this study, the fluorescence quenching of fluorescein by safranine T in liposome media had been investigated systematically by fluorescence spectroscopy, UV-vis absorption spectroscopy and fluorescence decay lifetime measurements. The spectroscopic data were analyzed using a Stern-Volmer equation to determine the quenching process. The experimental results showed that the intrinsic fluorescence of fluorescein was strongly quenched by safranine T, and that the quenching mechanism was considered as static quenching by forming a ground-complex. The Stern-Volmer quenching constant Ksv, and the bimolecular quenching constant Kq were estimated. The distances between the donor (fluorescein) and the acceptor (safranine T) were calculated according to the Förster non-radiation energy transfer theory. In addition, the partition coefficient of the safranine T (Kp) in the L-egg lecithin phosphatidylcholine liposomes was also calculated by utilizing the fluorescence quenching.

  15. Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

    NASA Astrophysics Data System (ADS)

    Jin, Dayong; Piper, James A.; Leif, Robert C.; Yang, Sean; Ferrari, Belinda C.; Yuan, Jingli; Wang, Guilan; Vallarino, Lidia M.; Williams, John W.

    2009-03-01

    A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (>1 μs) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-μm calibration beads in environmental water dirt samples. Europium-labeled 5-μm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria flow cytometer was used to sort accurately a very low number of beads (<100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%+/-30% and 91%+/-3% with 10 and 100 target beads, respectively, that were mixed with over one million nontarget autofluorescent background particles. Under the same conditions, a conventional FCM was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.

  16. 49 CFR 172.411 - EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false EXPLOSIVE 1.1, 1.2, 1.3, 1.4, 1.5 and 1.6 labels..., 1.2, 1.3, 1.4, 1.5 and 1.6 labels, and EXPLOSIVE Subsidiary label. (a) Except for size and color....5 and EXPLOSIVE 1.6 labels must be as follows: EXPLOSIVE 1.4: EC02MR91.016 EXPLOSIVE 1.5: EC02MR91...

  17. 99mTc-D(LPR): A novel retro-inverso peptide for VEGF receptor-1 targeted tumor imaging.

    PubMed

    Rezazadeh, Farzaneh; Sadeghzadeh, Nourollah; Abedi, Seyed Mohammad; Abediankenari, Saeid

    2018-05-31

    The aim of this study was to evaluate the ability of D (LPR), a novel retro-inverso peptidomimetic derivative for imaging colon cancer. Two different D (LPR) analogs were designed and compared based on conjugation of HYNIC at peptide's C or N terminal and then labeled with technetium-99m using tricine/EDDA as an exchange coligands. The radiolabeled conjugates were assessed for in vitro stability in saline and serum. The VEGFR-1 and NRP-1 receptors affinity, in vitro internalization and also dissociation Constance was evaluated. SPCET imaging and biodistribution studies were performed in nude mice bearing HT-29 xenograft tumors. Both peptides labeled with technetium-99m in high radiochemical yield (˃97%). Peptide stability studies indicated a high metabolic stability of the radiopeptides in solution and serum. In vitro blocking studies demonstrated specific binding and internalization of [ 99m Tc]Tc-HYNIC-peptides in cultured HUVEC cells. The K d value for 99m Tc-peptide 1 and 99m Tc-peptide 2 were found to be 56.8 ± 12.9 nM and 71.6 ± 17.9 nM respectively. The tumor to muscle ratio was significant at 0.5 and 1 h after injection (4.5 and 4 for 99m Tc-peptide 1 and 4.9 and 4.4 for 99m Tc-peptide 2 at 0.5 and 1 h p.i. respectively). SPECT imaging studies revealed that both radioconjugates had prominent activity accumulation in VEGFR-1 and NRP-1 expressing HT-29 tumors. This study is the first instance of using a radiolabeled retro-inverso peptide for tumor imaging which is a promising tool to improve the performance of fragile peptide probes in vivo as imaging agents and warrant further investigations in other peptide-target systems. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Label-free peptide aptamer based impedimetric biosensor for highly sensitive detection of TNT with a ternary assembly layer.

    PubMed

    Li, Yanyan; Zhao, Manru; Wang, Haiyan

    2017-11-01

    We report a label-free peptide aptamer based biosensor for highly sensitive detection of TNT which was designed with a ternary assembly layer consisting of anti-TNT peptide aptamer (peptamer), dithiothreitol (DTT), and 6-mercaptohexanol (MCH), forming Au/peptamer-DTT/MCH. A linear relationship between the change in electron transfer resistance and the logarithm of the TNT concentration from 0.44 to 18.92 pM, with a detection limit of 0.15 pM, was obtained. In comparison, the detection limit of the aptasensor with a common binary assembly layer (Au/peptamer/MCH) was 0.15 nM. The remarkable improvement in the detection limit could be ascribed to the crucial role of the ternary assembly layer, providing an OH-richer hydrophilic environment and a highly compact surface layer with minimal surface defects, reducing the non-covalent binding (physisorption) of the peptamer and non-specific adsorption of TNT onto the electrode surface, leading to high sensitivity, and which can serve as a general sensing platform for the fabrication of other biosensors.

  19. 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

    NASA Astrophysics Data System (ADS)

    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.

  20. 5-a-day fruit and vegetable food product labels: reduced fruit and vegetable consumption following an exaggerated compared to a modest label.

    PubMed

    Appleton, K M; Pidgeon, H J

    2018-05-15

    Food product labels based on the WHO 5-a-day fruit and vegetable (FV) message are becoming increasingly common, but these labels may impact negatively on complementary or subsequent FV consumption. This study aimed to investigate the impact of a '3 of your 5-a-day' versus a '1 of your 5-a-day' smoothie product label on subsequent FV consumption. Using an acute experimental design, 194 participants (90 males, 104 females) were randomised to consume a smoothie labelled as either '3 of your 5-a-day' (N = 97) or '1 of your 5-a-day' (N = 97) in full, following a usual breakfast. Subsequent FV consumption was measured for the rest of the day using 24-h recall. Usual FV consumption was also assessed via 24-h recall for the day before the study. Regression analyses revealed a significantly lower subsequent FV consumption following smoothies displaying the '3 of your 5-a-day' label compared to the '1 of your 5-a-day' label (Beta = - 0.15, p = 0.04). Secondary analyses revealed these effects to be driven mainly by changes to consumption in usual high FV consumers, in females and in vegetable as opposed to fruit consumption. These findings demonstrate a role for label information in food intake, and the potential negative impacts of an exaggerated food product label on healthy food consumption and healthy dietary profiles.

  1. A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

    PubMed Central

    Zheng, Ya-Li; Kesavapany, Sashi; Gravell, Maneth; Hamilton, Rebecca S; Schubert, Manfred; Amin, Niranjana; Albers, Wayne; Grant, Philip; Pant, Harish C

    2005-01-01

    The extracellular aggregation of amyloid β (Aβ) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Aβ peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Aβ1−42-induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin-dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting ‘normal' Cdk5 activity. PMID:15592431

  2. Bacterial Expression and Purification of the Amyloidogenic Peptide PAPf39 for Multidimensional NMR Spectroscopy

    PubMed Central

    Shanmuganathan, Aranganathan; Bishop, Anthony C.; French, Kinsley C.; McCallum, Scott A.; Makhatadze, George I.

    2013-01-01

    PAPf39 is a 39 residue peptide fragment from human prostatic acidic phosphatase that forms amyloid fibrils in semen. These fibrils have been implicated in facilitating HIV transmission. To enable structural studies of PAPf39 by NMR spectroscopy, efficient methods allowing the production of milligram quantities of isotopically labeled peptide are essential. Here, we report the high-yield expression, as a fusion to ubiquitin at the N-terminus and an intein at the C-terminus, and purification of uniformly labeled 13C- and 15N-labeled PAPf39 peptide. This allows the study of the PAPf39 monomer conformational ensemble by NMR spectroscopy. To this end, we performed the NMR chemical shift assignment of the PAPf39 peptide in the monomeric state at low pH. PMID:23314347

  3. Evaluation of changes in serum chemistry in association with feed withdrawal or high dose oral gavage with Dextran Sodium Sulfate (DSS) induced gut leakage in broiler chickens

    USDA-ARS?s Scientific Manuscript database

    Dextran sodium sulfate (DSS) has been shown to be effective at inducing enteric inflammation in broiler chickens, resulting in increased leakage of orally administered fluorescein isothiocyanate dextran to circulation. In a previous study, two doses of DSS (0.45g/dose) administered as oral gavage re...

  4. Fluoride-induced modulation of ionic transport in asymmetric nanopores functionalized with "caged" fluorescein moieties.

    PubMed

    Ali, Mubarak; Ahmed, Ishtiaq; Ramirez, Patricio; Nasir, Saima; Cervera, Javier; Niemeyer, Christof M; Ensinger, Wolfgang

    2016-04-28

    We demonstrate experimentally and theoretically a nanofluidic fluoride sensing device based on a single conical pore functionalized with "caged" fluorescein moieties. The nanopore functionalization is based on an amine-terminated fluorescein whose phenolic hydroxyl groups are protected with tert-butyldiphenylsilyl (TBDPS) moieties. The protected fluorescein (Fcn-TBDPS-NH2) molecules are then immobilized on the nanopore surface via carbodiimide coupling chemistry. Exposure to fluoride ions removes the uncharged TBDPS moieties due to the fluoride-promoted cleavage of the silicon-oxygen bond, leading to the generation of negatively charged groups on the fluorescein moieties immobilized onto the pore surface. The asymmetrical distribution of these groups along the conical nanopore leads to the electrical rectification observed in the current-voltage (I-V) curve. On the contrary, other halides and anions are not able to induce any significant ionic rectification in the asymmetric pore. In each case, the success of the chemical functionalization and deprotection reactions is monitored through the changes observed in the I-V curves before and after the specified reaction step. The theoretical results based on the Nernst-Planck and Poisson equations further demonstrate the validity of an experimental approach to fluoride-induced modulation of nanopore current rectification behaviour.

  5. Polymer microneedles fabricated from alginate and hyaluronate for transdermal delivery of insulin.

    PubMed

    Yu, Weijiang; Jiang, Guohua; Zhang, Yang; Liu, Depeng; Xu, Bin; Zhou, Junyi

    2017-11-01

    To reduce the inconvenient and painful of subcutaneous needle injection, the polymer microneedle patches that fabricated from modified alginate and hyaluronate were prepared for transdermal delivery of insulin. The as-prepared microneedles (MNs) exhibited excellent mechanical strength to penetrate the skin and good degradability to release loaded insulin. In vitro skin insertion capability was determined by staining with tissue-marking dye after insertion, and the real-time penetration depth was monitored using optical coherence tomography. Confocal microscopy images revealed that the rhodamine B and fluorescein isothiocyanate-labeled insulin (FITC-insulin) can gradually diffuse from the puncture sites to deeper tissue. In vivo and pharmacodynamic studies were then conducted to estimate the feasibility of the administration of insulin-loaded microneedle patches on diabetic mice for glucose regulation. The relative pharmacologic availability (RPA) and relative bioavailability (RBA) of insulin from microneedle patches were 90.5±6.8% and 92.9±7%, respectively. These results suggests the MNs developed in this study have a promising application in diabetes treatment via transdermal delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Low voltage electrophoresis chip with multi-segments synchronized scanning

    NASA Astrophysics Data System (ADS)

    Gu, Wenwen; Wen, Zhiyu; Xu, Yi

    2017-03-01

    For low voltage electrophoresis chip, there is always a problem that the samples are truncated and peaks are broadened, as well as longer time for separation. In this paper, a low voltage electrophoresis separation model was established, and the separation conditions were discussed. A new driving mode was proposed for applying low voltage, which was called multi-segments synchronized scanning. By using this driving mode, the reversed electric field that existed between the multi-segments can enrich samples and shorten the sample zone. The low voltage electrophoresis experiments using multi-segments synchronized scanning were carried out by home-made silicon-PDMS-based chip. The fluorescein isothiocyanate (FITC) labeled lysine and phenylalanine mixed samples with the concentration of 10-4 mol/L were successfully separated under the optimal conditions of 10 mmol/L borax buffer (pH = 10.0), 200 V/cm separation electric field and electrode switch time of 2.5 s. The separation was completed with a resolution of 2.0, and the peak time for lysine and phenylalanine was 4 min and 6 min, respectively.

  7. Fluoresceination of FepA during Colicin B Killing: Effects of Temperature, Toxin and TonB

    PubMed Central

    Smallwood, Chuck R.; Marco, Amparo Gala; Xiao, Qiaobin; Trinh, Vy; Newton, Salete M. C.; Klebba, Phillip E.

    2009-01-01

    We studied the reactivity of 35 genetically engineered Cys sulfhydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature-dependent in vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37 °C. At 0 °C colicin B binding impaired or blocked labeling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N - and C- domains of FepA. However, colicin B penetration into the cell at 37 °C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. FepA was expressed at lower levels in a tonB host strain, but when we accounted for this decrease its FM-labeling was comparable whether TonB was present or absent. Thus we did not detect TonB-dependent structural changes in FepA, either alone or when it interacted with colicin B at 37 °C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N-domain residues argues against the idea (Devanathan and Postle, Mol. Microbiol. 65: 441–453, 2007) that the colicin B polypeptide traverses the FepA channel. PMID:19432807

  8. Synthesis of a 68Ga-Labeled Peptoid−Peptide Hybrid for Imaging of Neurotensin Receptor Expression in Vivo

    PubMed Central

    2010-01-01

    The neurotensin receptor subtype 1 (NTS1) represents an attractive molecular target for imaging various tumors. Positron emission tomography (PET) gained widespread importance due to its sensitivity. We combined the design of a metabolically stable neurotensin analogue with a 68Ga-radiolabeling approach. The 68Ga-labeled peptoid−peptide hybrid [68Ga]3 revealed high stability, specific tumor uptake (0.7%ID/g, 65 min p.i.), and advantageous biokinetics in vivo using HT29 tumor-bearing nude mice. Because of the ability to internalize into NTS1-expressing tumor cells, [68Ga]3 proved to be highly suitable for a reliable and practical visualization of NTS1-expressing tumors in vivo by small animal PET. PMID:24900199

  9. Synthesis of a (68)ga-labeled peptoid-Peptide hybrid for imaging of neurotensin receptor expression in vivo.

    PubMed

    Maschauer, Simone; Einsiedel, Jürgen; Hocke, Carsten; Hübner, Harald; Kuwert, Torsten; Gmeiner, Peter; Prante, Olaf

    2010-08-12

    The neurotensin receptor subtype 1 (NTS1) represents an attractive molecular target for imaging various tumors. Positron emission tomography (PET) gained widespread importance due to its sensitivity. We combined the design of a metabolically stable neurotensin analogue with a (68)Ga-radiolabeling approach. The (68)Ga-labeled peptoid-peptide hybrid [(68)Ga]3 revealed high stability, specific tumor uptake (0.7%ID/g, 65 min p.i.), and advantageous biokinetics in vivo using HT29 tumor-bearing nude mice. Because of the ability to internalize into NTS1-expressing tumor cells, [(68)Ga]3 proved to be highly suitable for a reliable and practical visualization of NTS1-expressing tumors in vivo by small animal PET.

  10. Benzyl isothiocyanate induces protective autophagy in human lung cancer cells through an endoplasmic reticulum stress-mediated mechanism

    PubMed Central

    Zhang, Qi-cheng; Pan, Zhen-hua; Liu, Bo-ning; Meng, Zhao-wei; Wu, Xiang; Zhou, Qing-hua; Xu, Ke

    2017-01-01

    Isothiocyanates, such as allyl isothiocya¬nate (AITC), benzyl isothiocyanate (BITC), phenethyl isothio¬cyanate (PEITC) and sulforaphane (SFN), are natural compounds abundant in cruciferous vegetables, which have substantial chemopreventive activities against various human malignancies. However, the mechanisms underlying the inhibition of tumor cell growth by isothiocyanates are not fully understood. Since autophagy has dual functions in cancer, in the present study we investigated the effects of BITC on autophagy induction in human lung cancer cells in vitro and in vivo. BITC (1–100 μmol/L) dose-dependently inhibited the growth of 3 different human lung cancer cell lines A549 (adenocarcinoma), H661 (large cell carcinoma) and SK-MES-1 (squamous cell carcinoma) with IC50 values of 30.7±0.14, 15.9±0.22 and 23.4±0.11 μmol/L, respectively. BITC (10–40 μmol/L) induced autophagy in the lung cancer cells, evidenced by the formation of acidic vesicular organelles (AVOs), the accumulation of LC3-II, the punctate pattern of LC3, and the expression of Atg5. Pretreatment with the autophagy inhibitor 3-MA (5 mmol/L) significantly enhanced the BITC-caused growth inhibition in the lung cancer cells. Furthermore, BITC (20–40 μmol/L) activated ER stress, as shown by the increased cytosolic Ca2+ level and the phosphorylation of the ER stress marker proteins PERK and eIF2α in the lung cancer cells. Pretreatment with the ER stress inhibitor 4-PBA (5 mmol/L) attenuated the autophagy induction and potentiated the BITC-induced cell growth inhibition. In nude mice bearing A549 xenografts, administration of BITC (100 mg·kg-1·d-1, ip) for 8 weeks markedly suppressed the lung tumor growth, and significantly enhanced both autophagy and ER stress in the tumor tissues. Our results demonstrate that BITC inhibits human lung cancer cell growth in vitro and in vivo. In addition, BITC induces autophagy in the lung cancer cells, which protects the cancer cells against the inhibitory

  11. Approaches to N-Methylwelwitindolinone C Isothiocyanate: Facile Synthesis of the Tetracyclic Core

    PubMed Central

    Heidebrecht, Richard W.; Gulledge, Brian; Martin, Stephen F.

    2010-01-01

    The synthesis of a functionalized, tetracyclic core of N-methylwelwitindolinone C isothiocyanate is reported. The approach features a convergent coupling between an indole iminium ion and a highly functionalized vinylogous silyl ketene acetal followed by an intramolecular palladium-catalyzed cyclization that proceeds via an enolate arylation. PMID:20446675

  12. Natural isothiocyanates express antimicrobial activity against developing and mature biofilms of Pseudomonas aeruginosa.

    PubMed

    Kaiser, Stefan J; Mutters, Nico T; Blessing, Brigitte; Günther, Frank

    2017-06-01

    The antimicrobial properties of natural isothiocyanates (ITCs) found in plants such as nasturtium (Tropaeolum majus) and horseradish (Armoracia rusticana), and the need of new chemotherapeutic options for treatment of infections caused by multidrug-resistant and biofilm-forming Gram-negative bacteria such as Pseudomonas aeruginosa (Pa), led us to evaluate the effects of three major ITCs, allylisothiocyanate (AITC), benzylisothiocyanate (BITC), and phenylethyl-isothiocyanate (PEITC), and a mixture (ITCM) adapted to the ITC composition after release of active components out of natural sources. Out of 105Pa isolates 27 isolates with increased biofilm formation were selected for testing. The effects of ITCs on Pa were evaluated regarding (1) planktonic bacterial proliferation, (2) biofilm formation, (3) metabolic activity in mature biofilms, and (4) synergism of ITCs and antibiotics. (1) Each ITC had anti-Pa activity. Mean minimum inhibitory concentrations (MICs) were (μg/ml, mean±standard deviation): AITC 103±6.9; BITC, 2145±249; PEITC 29,423±1652; and ITCM, 140±5. (2) Treating bacteria with PEITC and ITCM in concentrations below the MIC significantly inhibited biofilm formation. Particularly, ITCM reduced biofilm mass and bacterial proliferation. (3) ITCs significantly inhibited metabolic activity in mature biofilms. (4) Combining ITCs with meropenem synergistically increased antimicrobial efficacy on Pa biofilms. ITCs represent a promising group of natural anti-infective compounds with activity against Pa biofilms. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Electrospun polyvinyl alcohol ultra-thin layer chromatography of amino acids.

    PubMed

    Lu, Tian; Olesik, Susan V

    2013-01-01

    Electrospun polyvinyl alcohol (PVA) ultrathin layer chromatographic (UTLC) plates were fabricated using in situ crosslinking electrospinning technique. The value of these ULTC plates were characterized using the separation of fluorescein isothiocyanate (FITC) labeled amino acids and the separation of amino acids followed visualization using ninhydrin. The in situ crosslinked electrospun PVA plates showed enhanced stability in water and were stable when used for the UTLC study. The selectivity of FITC labeled amino acids on PVA plate was compared with that on commercial Si-Gel plate. The efficiency of the separation varied with analyte concentration, size of capillary analyte applicator, analyte volume, and mat thickness. The concentration of 7mM or less, 50μm i.d. capillary applicator, minimum volume of analyte solution and three-layered mat provides the best efficiency of FITC-labeled amino acids on PVA UTLC plate. The efficiency on PVA plate was greatly improved compared to the efficiency on Si-Gel HPTLC plate. The hydrolysis products of aspartame in diet coke, aspartic acid and phenylalanine, were also successfully analyzed using PVA-UTLC plate. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA).

    PubMed

    Guo, Guangyu; Li, Ning

    2011-07-01

    In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. (15)N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of (15)N or (14)N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the (15)N supply, which led to a uniform incorporation of (15)N into the whole plant protein complement. The incorporation rate (97.43±0.11%) of (15)N into (15)N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of (15)N/(14)N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified (15)N/(14)N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed (15)N-coded and (14)N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  15. Fluorescent labeling of tetracysteine-tagged proteins in intact cells.

    PubMed

    Hoffmann, Carsten; Gaietta, Guido; Zürn, Alexander; Adams, Stephen R; Terrillon, Sonia; Ellisman, Mark H; Tsien, Roger Y; Lohse, Martin J

    2010-09-01

    In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder-ethanedithiol (FlAsH-EDT₂). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg⁻¹ of protein, such as G protein-coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT₂ as described takes 2-3 h, depending on the number of samples to be processed.

  16. Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    PubMed Central

    Hoffmann, Carsten; Gaietta, Guido; Zürn, Alexander; Adams, Stephen R; Terrillon, Sonia; Ellisman, Mark H; Tsien, Roger Y; Lohse, Martin J

    2011-01-01

    In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed. PMID:20885379

  17. Interaction study between synthetic glycoconjugate ligands and endocytic receptors using flow cytometry.

    PubMed

    Yura, Hirofumi; Ishihara, Masayuki; Kanatani, Yasuhiro; Takase, Bonpei; Hattori, Hidemi; Suzuki, Shinya; Kawakami, Mitsuyuki; Matsui, Takemi

    2006-04-01

    Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17alpha-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry.

  18. Kinetics of acrylodan-labelled cAMP-dependent protein kinase catalytic subunit denaturation.

    PubMed

    Kivi, Rait; Loog, Mart; Jemth, Per; Järv, Jaak

    2013-10-01

    Fluorescence spectroscopy was used to study denaturation of cAMP-dependent protein kinase catalytic subunit labeled with an acrylodan moiety. The dye was covalently bound to a cystein residue introduced into the enzyme by replacement of arginine in position 326 in the native sequence, located near the enzyme active center. This labeling had no effect on catalytic activity of the enzyme, but provided possibility to monitor changes in protein structure through measuring the fluorescence spectrum of the dye, which is sensitive to changes in its environment. This method was used to monitor denaturation of the protein kinase catalytic subunit and study the kinetics of this process as well as influence of specific ligands on stability of the protein. Stabilization of the enzyme structure was observed in the presence of adenosine triphosphate, peptide substrate RRYSV and inhibitor peptide PKI[5-24].

  19. Validation of an automated fluorescein method for determining bromide in water

    USGS Publications Warehouse

    Fishman, M. J.; Schroder, L.J.; Friedman, L.C.

    1985-01-01

    Surface, atmospheric precipitation and deionized water samples were spiked with ??g l-1 concentrations of bromide, and the solutions stored in polyethylene and polytetrafluoroethylene bottles. Bromide was determined periodically for 30 days. Automated fluorescein and ion chromatography methods were used to determine bromide in these prepared samples. Analysis of the data by the paired t-test indicates that the two methods are not significantly different at a probability of 95% for samples containing from 0.015 to 0.5 mg l-1 of bromide. The correlation coefficient for the same sets of paired data is 0.9987. Recovery data, except for the surface water samples to which 0.005 mg l-1 of bromide was added, range from 89 to 112%. There appears to be no loss of bromide from solution in either type of container.Surface, atmospheric precipitation and deionized water samples were spiked with mu g l** minus **1 concentrations of bromide, and the solutions stored in polyethylene and polytetrafluoroethylene bottles. Bromide was determined periodically for 30 days. Automated fluorescein and ion chromatography methods were used to determine bromide in these prepared samples. Analysis of the data by the paired t-test indicates that the two methods are not significantly different at a probability of 95% for samples containing from 0. 015 to 0. 5 mg l** minus **1 of bromide. The correlation coefficient for the same sets of paired data is 0. 9987. Recovery data, except for the surface water samples to which 0. 005 mg l** minus **1 of bromide was added, range from 89 to 112%. Refs.

  20. Polarization of fluorescein fluorescence in single cells.

    PubMed

    Udkoff, R; Norman, A

    1979-01-01

    Measurement of fluorescence polarization (P) gives information about the immediate environment of the fluorescent molecule. We used a flow polarimeter to investigate the factors influencing P of fluorescein in mammalian cells to determine whether such measurements are useful for characterizing heterogeneous cell populations. Fluorescein was introduced into cells by incubation with FDA. Measurements of the intensity of fluorescence (TI) and polarization (P) revealed an unexpected dependence: P decreased with increasing intensity of fluorescence. This may be accounted for by the classical model of the binding of small molecules to protein in which P is dependent on the ratio bound to unbound molecules. We have been able to estimate the quenching due to binding and construct a Scatchard plot. We estimated a wavelength shift from in vitro data consistent with the dependence of P on wavelength seen in our cell work. Generally, the distributions of P are symmetrical. Photon statistics broadens the P distribution of dim cells. However, structure does develop in the P distribution when the cells are deprived of calcium or incubated in the cold. This appears as a shoulder on the P distribution or resolves into two peaks. Calcium deprivation may differentially affect a subpopulation of cells whose significance remains to be explored in various cell types.