Saturday, January 21, 2012

fluorescent peptide| What isfluorescent peptide |Papers on fluorescent peptide|Research on fluorescent peptide | Publications on fluorescent peptide

    Results: 1 to 20 of 53040

    1.
    Wei Sheng Wu Xue Bao. 2011 Nov 4;51(11):1502-9.

    [Construction and toxicity the recombinant SpltMNPV expressing the scorpion toxin gene].

    [Article in Chinese]

    Source

    School of Biology and Basic Medical Science, Medical College of Soochow University, Suzhou 215123, China. alicetangsz@yahoo.com.cn

    Abstract

    OBJECTIVE:

    To develop a high toxic recombinant Spodoptera litura multicapsid nucleopolyhedroviruse (SpltMNPV) insecticide.

    METHODS:

    We constructed a recombinant transfer vector that was characterized by disrupting of ecdysterioid UDP-glucosyltransferase (egt) gene and expressing the mature peptide of the Chinese scorpion, B. martensi Karsch (BmK ITal) gene at the control of ie-1 promoter. The transfer vector and the SpltMNPV II DNA cotransfected the SpLi cells. Recombinant viruses were purified by the end point dilution and fluorescent spot purification.

    RESULTS:

    We successfully screened the recombinant SpltMNPV-deltaegt-Pph-egfp-ie-1-BmK ITal of which the egt gene was knocked out and expressed the mature peptide of the BmK ITal gene at the control of ie-1 promoter. Bioassays showed that, compared to the wide-type SpltMNPV, the speed of the recombinant virus killing the S. litura (LT50) increased by 0.7-0.8 days.

    CONCLUSION:

    The insecticidal effect of SpltNPV could be increased by inserting the foreign gene, which provided a further opportunity to develop the SpltNPV into commercially viable products to control the S. litura.

    PMID:
    22260048
    [PubMed - in process]
    2.
    J Virol. 2012 Jan 18. [Epub ahead of print]

    INHIBITION OF THE LATENT MEMBRANE PROTEIN-1 IMPAIRS THE GROWTH AND TUMORIGENESIS OF AN EBV-LATENCY II TRANSFORMED T-CELL.

    Source

    CNRS UMR8161, Institut de Biologie de Lille, IFR 142, Université Lille-Nord de France, 1 rue du Pr Calmette, 59021 Lille Cedex.

    Abstract

    The Epstein-Barr virus (EBV) is a common human herpes virus. Its infection is associated with several human malignancies where it expresses a set of latent proteins among which is the latent membrane protein LMP1. LMP1 is able to transform numerous cell types and is considered the main oncogenic protein of EBV. The mechanism of action is based on mimicry to activated members of the TNF receptor superfamily, through its ability to bind similar adapters and to activate signaling pathways. We previously generated two unique models: a monocytic and lymphocytic (NC5) cell line immortalized by EBV which expresses the type II latency program.Here, we generated LMP1 dominant negatives (DNs), based on fusion between green fluorescent protein (GFP) and TES1 or TES2 (Transformation Effectors Site) of LMP1. Then, we generated cell lines conditionally expressing these DNs. These DNs inhibit NFkB and Akt pathways resulting in impairment of survival processes and increased apoptosis in these cell lines. This pro-apoptotic effect is due to reduced interaction of LMP1 with specific adapters and the recruitment of these adapters to DNs which enable generation of an apoptotic complex involving TRADD, FADD and caspase-8. Similar results were obtained with cell lines displaying a latency III program in which LMP1-DNs decrease cell viability. Finally, we prove that syntheticpeptides display similar inhibitory effects in EBV infected cells. DNs derived from LMP1 could be used to develop therapeutic approaches in malignant diseases associated with EBV.

    PMID:
    22258264
    [PubMed - as supplied by publisher]
    Click here to read
    3.
    PLoS One. 2012;7(1):e30076. Epub 2012 Jan 10.

    Cell Wall Antibiotics Provoke Accumulation of Anchored mCherry in the Cross Wall of Staphylococcus aureus.

    Source

    Microbial Genetics, University of Tübingen, Tübingen, Germany.

    Abstract

    A fluorescence microscopy method to directly follow the localization of defined proteins in Staphylococcus was hampered by the unstable fluorescence of fluorescent proteins. Here, we constructed plasmid (pCX) encoded red fluorescence (RF) mCherry (mCh) hybrids, namely mCh-cyto (no signal peptide and no sorting sequence), mCh-sec (with signal peptide), and mCh-cw (with signal peptide and cell wall sorting sequence). The S. aureus clones targeted mCh-fusion proteins into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SP(lip) and the -YSIRK motif SP(sasF). mCh-hybrids supplied with the +YSIRK motif SP(lip) were always expressed higher than those with -YSIRK motif SP(sasF). To study the location of the anchoring process and also the influence of SP type, mCh-cw was supplied on the one hand with +YSIRK motif (mCh-cw1) and the other hand with -YSIRK motif (mCh-cw2). MCh-cw1 preferentially localized at the cross wall, while mCh-cw2 preferentially localized at the peripheral wall. Interestingly, when treated with sub-lethal concentrations of penicillin or moenomycin, both mCh-cw1 and mCh-cw2 were concentrated at the cross wall. The shift from the peripheral wall to the cross wall required Sortase A (SrtA), as in the srtA mutant this effect was blunted. The effect is most likely due to antibiotic mediated increase of free anchoring sites (Lipid II) at the cross wall, the substrate of SrtA, leading to a preferential incorporation of anchored proteins at the cross wall.

    PMID:
    22253886
    [PubMed - in process]
    PMCID: PMC3254641
    Free PMC Article
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    4.
    Microb Cell Fact. 2012 Jan 18;11(1):10. [Epub ahead of print]

    Small surfactant-like peptides can drive soluble proteins into active aggregates.

    Abstract

    ABSTRACT:

    BACKGROUND:

    Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli) cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos) when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF) and a short beta structure peptide ELK16 (LELELKLKLELELKLK) have a similar property.

    RESULTS:

    In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6) were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used), Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same), Bacillus pumilus xylosidase (XynB), and green fluorescent protein (GFP), and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM) and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red) to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core.

    CONCLUSIONS:

    This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in vivo, and can be explored for production of functional biopolymers with detergent or other interfacial activities.

    PMID:
    22251949
    [PubMed - as supplied by publisher]
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    5.
    J Biol Chem. 2012 Jan 13. [Epub ahead of print]

    DHHC PROTEIN S-ACYLTRANSFERASES USE A SIMILAR PING-PONG KINETIC MECHANISM BUT DISPLAY DIFFERENT ACYL-COA SPECIFICITIES.

    Source

    Cornell University, United States.

    Abstract

    DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known about the kinetic mechanism of DHHC proteins. A high performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation (PAT) activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of Vmax and Km values for both the peptide N-myristoylated-GCG and palmitoyl-Coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl-enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl-chains 14-carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl-chain transfer to protein substrate, were reduced with stearoyl-CoA compared to palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteins and may account for the differential S-acylation of proteins observed in cells.

    PMID:
    22247542
    [PubMed - as supplied by publisher]
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    6.
    Biophys Chem. 2011 Dec 24. [Epub ahead of print]

    Characterization of channel-forming peptide nanostructures.

    Abstract

    We have prepared fluorescent analogs of known ion-channel-forming synthetic peptide nanostructures. These analogs were designed as probes to gain insight about the mechanism by which self-assembling amphiphilic peptides interact with lipid membranes. Conformational studies demonstrated that the labeled analogs retain their propensity to adopt a strong helical conformation in 2,2,2-trifluoroethanol and lipid bilayers. Attenuated total reflectance results indicated that the fluorescent peptide nanostructures are under an incorporation equilibrium between two forms, adsorbed at the surface or incorporated within the bilayer, similar to their unlabeled counterparts. However, when using a HeLa mimicking membrane, the proportion of peptide nanostructures in the transmembrane orientation decreases significantly. Finally, we were able to show by confocal microscopy studies that fluorescent analogs internalized into HeLa cells and localized into both the membranes of inner organelles and the cell membrane.

    Copyright © 2011 Elsevier B.V. All rights reserved.

    PMID:
    22245249
    [PubMed - as supplied by publisher]
    Click here to read
    7.
    J Mol Biol. 2012 Jan 5. [Epub ahead of print]

    Arginine Changes the Conformation of the Arginine Attenuator Peptide Relative to the Ribosome Tunnel.

    Source

    Department of Biology, Texas A&M University, College Station, TX 77843, USA.

    Abstract

    The fungal arginine attenuator peptide (AAP) is a regulatory peptide that controls ribosome function. As a nascentpeptide within the ribosome exit tunnel, it acts to stall ribosomes in response to arginine (Arg). We used three approaches to probe the molecular basis for stalling. First, PEGylation assays revealed that the AAP did not undergo overall compaction in the tunnel in response to Arg. Second, site-specific photocross-linking showed that Arg altered the conformation of the wild-type AAP, but not of nonfunctional mutants, with respect to the tunnel. Third, using time-resolved spectral measurements with a fluorescent probe placed in the nascent AAP, we detected sequence-specific changes in the disposition of the AAP near the peptidyltransferase center in response to Arg. These data provide evidence that an Arg-induced change in AAP conformation and/or environment in the ribosome tunnel is important for stalling.

    Copyright © 2011. Published by Elsevier Ltd.

    PMID:
    22244852
    [PubMed - as supplied by publisher]
    Click here to read
    8.
    Mol Biosyst. 2012 Jan 13. [Epub ahead of print]

    A fluorescent amino acid probe to monitor efficiency of peptide conjugation to glass surfaces for high density microarrays.

    Source

    Department of Bioengineering and Center for Bioengineering Research, Bourns College of Engineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA. jiayu.liao@ucr.edu.

    Abstract

    Using a fluorescent NBD amino acid, new protease substrates were developed that are attractive because of the excellent chemical stability and long wavelength of excitation (480 nm) of the NBD fluorophore. The fluorescent peptidesare synthesized by Fmoc solid-phase peptide synthesis. An example peptide was efficiently immobilized onto a microarray surface using click chemistry, and its proteolysis was monitored by fluorescence imaging. Excellent site specificity was achieved for the protease. Fluorescent peptides are also used to monitor the conjugation efficiency onto a surface using a standard microarray scanner.

    PMID:
    22241083
    [PubMed - as supplied by publisher]
    9.
    Bioconjug Chem. 2012 Jan 13. [Epub ahead of print]

    Fluorescent Peptide-PNA Chimeras for Imaging Monoamine Oxidase A mRNA in Neuronal Cells.

    Abstract

    Monoamine oxidases (MAO) catalyze the oxidative deamination of many biogenic amines and are integral proteins found in the mitochondrial outer membrane. Changes in MAO-A levels are associated with depression, trait aggression and addiction. Here we report the synthesis, characterization and in vitro evaluation of novel fluorescent peptide-peptidenucleic acid (PNA) chimeras for MAOA mRNA imaging in live neuronal cells. The probes were designed to include MAOA-specific PNA dodecamers, separated by an N-terminal spacer to a m-opioid receptor targeting peptide(DAMGO), with a spacer and a fluorophore on the C-terminus. The probe was successfully delivered into human SH-SY5Y neuroblastoma cells through mu-opioid receptor-mediated endocytosis. The Kd by flow cytometry was 11.6 ± 0.8 nM. Uptake studies by fluorescence microscopy showed ~5-fold higher signal in human SH-SY5Y neuroblastoma cells than in negative control CHO-K1 cells that lack mu-opioid receptors. Moreover, a peptide-mismatch control sequence showed no significant uptake in SH-SY5Y cells. Such fluorescent mRNA imaging agents might enable real time imaging and quantitation of neuronal mRNAs in live animal models.

    PMID:
    22239616
    [PubMed - as supplied by publisher]
    Click here to read
    10.

    Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-(TDOPA)3-flower-like gold–Fe3O4 optical nanoparticles.

    Authors

    Leung K.

    Source

    Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2011.
    2011 Oct 06 [updated 2012 Jan 05].

    Excerpt

    Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (1). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). Tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degrading the basement membrane and ECM (3). Gold has not been used as an X-ray contrast agent in vivo. Gold has a higher atomic number and a higher absorption coefficient than iodine, providing 2.7-fold greater contrast/weight than iodine (4). Furthermore, imaging gold at 80–100 keV reduces interference from bone absorption and provides lower soft tissue absorption, which would reduce radiation to patients. Hainfeld et al. (4) used gold nanoparticles (AuNPs; 1.9 nm in diameter, ~50 kDa) as a computed tomography (CT) contrast agent in mice; these experiments showed enhanced CT contrast of the vasculature, kidneys, and tumors in mice. However, plasma proteins in blood adsorb onto the surface of bare AuNPs, which produces large aggregates (5) that may result in altered pharmacokinetics and biodistribution of AuNPs (6). Polyethylene glycol (PEG) is found to minimize nonspecific adsorption of proteins onto NPs and to reduce their uptake by the liver (6). PEG-AuNPs have been being studied as cancer CT imaging and photothermal agents (7). Several families of MMPs are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastases (8-11). MMP expression is highly regulated in normal cells, such as trophoblasts, osteoclasts, neutrophils, and macrophages. Elevated levels of MMPs have been found in tumors associated with a poor prognosis for cancer patients (12). The peptide Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-NH2 was found to be a MMP substrate and is cleaved between the Leu and Gly residues. Lee et al. (13) used this sequence with a Cy5.5 NIR dye molecule to attach to AuNPs to form fluorescence-quenched nanoparticles (Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-AuNPs (Cy5.5-MMP-AuNPs or GANPs). The Cy5.5 molecules are in close proximity, resulting in fluorescence quenching because of efficient fluorescence resonance energy transfer to Au. NIR fluorescence signal will increase when the Leu-Gly bond is cleaved by MMPs, releasing Cy5.5-containing fragments. Cy5.5 is a NIR fluorescentdye with an absorbance maximum at 675 nm, an emission maximum at 694 nm, and a high extinction coefficient of 250,000 M−1cm−1. Cy5.5-MMP-AuNPs are being developed for NIR fluorescence imaging of MMPs expressed in tumors, atherosclerosis, myocardial infarction, and other diseases. Xie et al. (14) replaced the AuNP with Au-Fe3O4, a composite NP shaped like a flower, to induce a fluorescently quenched state to the overall nanostructure. There were three iron oxide “petals” on each AuNP. The MMP peptide was covalently linked to an anchoring unit, Lys-tridihydrophenylalanine (Lys-TDOPA) on the surface of the iron oxide NP. The gold surface was passivated with a thiolated PEG (SH-PEG5000). Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-(Lys-TDOPA)3-flower-like gold–Fe3O4 optical NPs (FANPs) have been evaluated for imaging protease expression in vivo.

    11.
    Macromol Rapid Commun. 2012 Jan 10. doi: 10.1002/marc.201100729. [Epub ahead of print]

    Live Monitoring of Cargo Release From Peptide-Based Hybrid Nanocapsules Induced by Enzyme Cleavage.

    Source

    Max-Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany.

    Abstract

    The miniemulsion process is used as a new route for the preparation of enzyme-responsive nanocapsules with payload-release properties. Peptide-based hybrid nanocapsules are prepared via interfacial polyaddition containing a water-soluble dye that is efficiently encapsulated inside. The influence of the synthetic parameters as the functionality of thepeptide and the nature of the dispersed phase on the structure of the nanocapsules were investigated. After redispersion in water, the enzymatic cleavage of the peptide sequence and the release of the fluorescent dye are both monitored in real time. This is evidenced because of the quenching FRET system framing the recognition site in the peptidesequence, and the fluorescence recovery of the self-quenched encapsulated dye respectively.

    Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

    PMID:
    22231909
    [PubMed - as supplied by publisher]
    12.

    [Calcitonin gene-related peptide promoting migration of rat bone marrow mesenchymal stem cells and stimulating expression of vascular endothelial growth factor].

    [Article in Chinese]

    Source

    Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou Guangdong 510515, PR China.

    Abstract

    OBJECTIVE:

    To explore the effects of calcitonin gene-related peptide (CGRP) on the migration of bone marrow mesenchymal stem cells (BMSCs) and vascular endothelial growth factor (VEGF) expression in vitro.

    METHODS:

    The BMSCs were isolated from Sprague Dawley rats using whole bone marrow adherence method. At 1, 2, and 3 weeks after culture, the expressions of CGRP receptor (CGRPR) was detected by Western blot. The BMSCs were treated with CGRP at concentration 1 x 10(-8) mol/L (experimental group) and did not treated (control group), and the efficacy of BMSCs migration was analyzed by Transwell chamber assay after 72 hours; at 1, 3, 5, and 7 days, the mRNA expressions of vascular cell adhesion molecule 1 (VCAM-1) were detected by real-time fluorescent quantitative PCR; the protein expressions of VEGF were examined using immunohistochemistry and Western blot.

    RESULTS:

    CGRPR expressed stably in the cultured BMSCs and reached the peak at 2 weeks. CGRP had a significantly enhanced role in promoting cell migration. The number of cell migration was (3.20 +/- 1.77) cells/HP in experimental group and (1.11 +/- 0.49) cells/HP in control group, showing significant difference (t = 4.230, P = 0.001). In experimental group, the expressions of VCAM-1 mRNA increased with time and reached the peak at 7 days. There were significant differences in the expressions of VCAM-1 mRNA between control group and experimental group at 3, 5, and 7 days (P < 0.05). Immunocytochemistry results showed positive DAB staining for VEGF at 5 and 7 days in experimental group. Western blot results showed that the protein expressions of VEGF increased significantly at 5 and 7 days in experimental group when compared with control group (P < 0.05), which was signfiantly higher at 5 days than at 7 days in experimental group (P < 0.05).

    CONCLUSION:

    CGRP can promote the migration of BMSCs and stimulate the protein expression of VEGF, which may plays an important role in regulating bone metabolism by increasing angiogenesis.

    PMID:
    22229198
    [PubMed - in process]
    13.
    Exp Physiol. 2012 Jan 6. [Epub ahead of print]

    Na transport across ruminal epithelium of hay-fed sheepis acutely stimulated by the peptide IGF-1 in vitro.

    Source

    1 Nanjing Agricultural University, Lab of Animal Physiology and Biochemistry, China;

    Abstract

    An energy-rich diet led to enhance ruminal Na absorption, which is associated with elevated plasma IGF-1 levels and increased number of IGF-1 receptors in rumen papillae. This study examined the in vitro effect of IGF-1 on Na transport across the ruminal epithelium of hay-fed sheep. At a concentration ranging from 20 to 100 μg/l, serosal LR3-IGF-1 rapidly (within 30 minutes) stimulated the mucosal to serosal Na flux (JmsNa) and consequently the net Na flux (JnetNa). Compared with controls, JnetNa increased by about 60% (p<0.05) through the serosal application of LR3-IGF-1 (20 μg/l). The IGF-1-induced increment of JmsNa and JnetNa was inhibited by mucosal amiloride (1 mmol/l). Neither IGF-1 nor amiloride altered Gt or Isc of the isolated ruminal epithelium. These data support the assumption that the stimulating effect of serosal-added IGF-1 on Na transport across the rumen epithelium is mediated by Na+/H+ exchange (NHE). A further study performed with cultured ruminal epithelial cells (REC) and a fluorescent probe (BCECF) to estimate the rate of pHi recovery after acid-loading. The pHi of isolated REC was 6.43 ± 0.15 after butyrate loading and recovered by 0.26 ± 0.02 pH units/15 min. Application of LR3-IGF-1 (20 μg/l) significantly increased the rate of pHi recovery to 0.33 ± 0.02 pH units/15 min. Amiloride administration reduced the recovery rate in both control and IGF-1-stimulated cells. These results show, for the first time, that an acute effect of IGF-1 on Na absorption across ruminal epithelium results from increased NHE activity. IGF-1 is thus important also for the fast functional adaptation of the ruminal Na transport via NHE.

    PMID:
    22227200
    [PubMed - as supplied by publisher]
    Click here to read
    14.
    Eur J Pharm Sci. 2011 Dec 30. [Epub ahead of print]

    Potential of the gastric motility drug lorglumide in prostate cancer imaging.

    Source

    Department of Neuroradiology, University of Tübingen, Germany; Peptide Synthesis Laboratory, Interfaculty Institute of Biochemistry, University of Tübingen, Germany.

    Abstract

    The use of tissue-specific receptor ligands is a promising approach for cancer diagnostics and therapy. Lorglumide, a highly effective competitive ligand for the cholecystokinine-A receptor (CCKRA) was conjugated to a fluorescent dye and a magnetic resonance imaging (MRI) contrast agent to obtain a bifunctional marker for tissue with high CCKRA expression. An intermediate conjugate containing only lorglumide and a fluorescent dye was also produced. By performing CCKRA mRNA expression analysis on carcinoma cell lines we found that CCKRA is highly expressed in PC3 prostate carcinoma cells compared to U373 glioma and U2OS osteosarcoma cells. Uptake, specificity and detection sensitivity of both lorglumide conjugates was evaluated by confocal laser scanning microscopy, fluorescence activated cell sorting (FACS) and magnetic resonance relaxometry. While the conjugate containing only lorglumide and rhodamine isothiocyanate as fluorescent dye showed clearly higher uptake than the bifunctional conjugate in FACS analysis, both conjugates clearly showed preferential staining of the PC3 prostate carcinoma cells. Magnetic resonance relaxometry experiments with the bifunctional conjugate containing the MRI contrast agent gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid confirmed the higher PC3-affinity of the lorglumide ligand. Confocal laser scanning microscopy images of PC3/U2OS mixed cell cultures incubated with the bifunctional conjugate also clearly showed PC3 preference and cytoplasmic dot-like staining concurring with uptake by receptor binding and subsequent receptor internalization. Considering these results, CCKRA ligands like lorglumide could play a role in the future design of prostate-cancer-specific markers.

    Copyright © 2012. Published by Elsevier B.V.

    PMID:
    22226647
    [PubMed - as supplied by publisher]
    Click here to read
    15.
    J Am Chem Soc. 2012 Jan 5. [Epub ahead of print]

    Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Förster Resonance Energy Transfer Relays: Characterization and Biosensing.

    Source

    Center for Bio/Molecular Science and Engineering, Code 6900, ‡Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory , Washington DC 20375, United States.

    Abstract

    The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.

    PMID:
    22220737
    [PubMed - as supplied by publisher]
    Click here to read
    16.
    Eur J Cardiothorac Surg. 2011 Dec 21. [Epub ahead of print]

    CD26/DPP-4 inhibition recruits regenerative stem cells via stromal cell-derived factor-1 and beneficially influences ischaemia-reperfusion injury in mouse lung transplantation.

    Source

    Division of Thoracic Surgery, University Hospital Zurich, Zurich, Switzerland.

    Abstract

    OBJECTIVESThe CD26 antigen is a transmembrane glycoprotein that is constitutively expressed on activated lymphocytes and in pulmonary parenchyma. This molecule is also identified as dipeptidyl peptidase-4 (DPP-4) that cleaves a host of biologically active peptides. Here, we aimed to identify an important substrate of CD26/DPP-4-stromal cell-derived factor-1 (SDF-1/CXCL12)-as a key modulator for stem-cell homing together with its receptor CXCR4 in response to ischaemic injury of the lung.METHODSOrthotopic single lung transplantation (Tx) was performed between syngeneic C57BL/6 mice. Inhibition of CD26/DPP-4 activity in recipients was achieved using vildagliptin (10 mg/kg, every 12 h) subcutaneously, and 6 h ischaemia time was applied prior to implantation. Forty-eight hours after Tx, lung histology, SDF-1 levels (enzyme-linked immunosorbent assay) in lung, spleen and plasma, and expression of the SDF-1 receptor CXCR4 in blood and lung were assessed. Homing of regenerative progenitor cells to the transplanted lung was evaluated using fluorescent-activated cell sorting.RESULTSCompared with untreated lung transplanted mice, systemic DPP-4 inhibition of Tx recipients resulted in an increase in protein concentration of SDF-1 in plasma, spleen and lung. Concordantly, the frequency of cells bearing the SDF-1 receptor CXCR4 rose significantly in the circulation and also in the lungs of DPP-4-inhibited recipients. We found co-expression of CXCR4/CD34 in the grafts of animals treated with vildagliptin, and the stem-cell markers Flt-3 and c-kit were present on a significantly increased number of cells. The morphology of grafts from DPP-4 inhibitor-treated recipients revealed less alveolar oedema when compared with untreated recipients.CONCLUSIONSTargeting the SDF-1-CXCR4 axis through CD26/DPP-4 inhibition increased the intragraft number of progenitor cells contributing to the recovery from ischaemia-reperfusion lung injury. Stabilization of endogenous SDF-1 is achievable and may be a promising strategy to intensify sequestration of regenerative stem cells and thus emerges as a novel therapeutic concept.

    PMID:
    22219460
    [PubMed - as supplied by publisher]
    17.
    PLoS One. 2011;6(12):e28885. Epub 2011 Dec 21.

    Comparison of IRES and F2A-Based Locus-Specific Multicistronic Expression in Stable Mouse Lines.

    Source

    Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore, Singapore.

    Abstract

    Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9(IRES-EGFP)) or the F2A (Sox9(F2A-EGFP)) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome 'skipping' mechanism. To investigate if the discrepancy in the 'skipping' process was locus-dependent, we further analyzed the FLAG(3)-Bapx1(F2A-EGFP) mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the 'skipping' mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP (Bapx1(F2A-Cre-F2A-EGFP)). While the 'skipping' was highly efficient between Bapx1 and Cre, the 'skipping' between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the 'skipping' at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides.

    PMID:
    22216134
    [PubMed - in process]
    PMCID: PMC3244433
    Free PMC Article
    Click here to readClick here to read
    18.
    PLoS Pathog. 2011 Dec;7(12):e1002454. Epub 2011 Dec 22.

    Helicobacter pylori versus the Host: Remodeling of the Bacterial Outer Membrane Is Required for Survival in the Gastric Mucosa.

    Source

    Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas, United States of America.

    Abstract

    Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response. Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4'-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4'-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4'-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4'-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, β-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host.

    PMID:
    22216004
    [PubMed - in process]
    PMCID: PMC3245313
    Free PMC Article
    Click here to readClick here to read
    19.
    Anal Biochem. 2011 Dec 2. [Epub ahead of print]

    A fluorescent method to determine vitamin K-dependent gamma-glutamyl carboxylase activity.

    Source

    Division of Nephrology and Clinical Immunology, University Hospital of the RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany.

    Abstract

    The gamma (γ)-glutamyl carboxylase is a key enzyme in vitamin K-dependent carboxylation of proteins involved in hemostasis and inflammation. It is an endoplasmic enzyme posttranslationally converting glutamic acid residues into γ-carboxyglutamic acid residues in proteins. The activity of tissue derived γ-glutamyl carboxylase is commonly assayed by incorporation of H(14)CO(3)(-) into synthetic peptides and subsequent quantification using liquid scintillation counting. We present a nonradioactive assay using a fluorescein isothiocyanate-labeled short peptide that can be readily detected in its unmodified and γ-glutamyl carboxylated form by reversed-phase HPLC. This method offers a convenient alternative to the established radioactive labeling techniques.

    Copyright © 2011 Elsevier Inc. All rights reserved.

    PMID:
    22210513
    [PubMed - as supplied by publisher]
    Click here to read
    20.
    Food Microbiol. 2012 Apr;29(2):205-14. Epub 2011 Jul 28.

    Comparison of different IlvE aminotransferases in Lactobacillus sakei and investigation of their contribution to aroma formation from branched chain amino acids.

    Source

    Technische Universität München, Lehrstuhl für Technische Mikrobiologie, Weihenstephaner Steig 16, D-85350 Freising, Germany.

    Abstract

    Branched chain aminotransferases (IlvE/BcaT), specific for leucine, isoleucine, and valine initialize the formation of methyl-branched volatiles, which are strongly linked to the typical aroma of cured meat products. Lactobacillus sakei, one of the dominating lactic acid bacteria species for meat fermentations and commonly used as starter lacks this enzyme, whereas the presence of IlvE has been reported for Lactobacillus paracasei, a non-starter lactic acid bacterium occurring in meat products and with probiotic properties, in Staphylococcus carnosus, a catalase positive cocci also used as starter for meat products, and in Enterococcus faecalis belonging to the natural microbiota of meat and that may impact on the aroma of fermented meat products. The genes for branched-chain aminotransferases of these three bacterial species were used to complement the IlvE negative strain L. sakei TMW1.1322. For that purpose, ilvE genes were heterologously expressed in L. sakei TMW1.1322 under the control of the constitutive L. sakei promotor pldhL via replicative plasmids and chromosomal integration. To examine effective expression the constructs were transcriptionally coupled to mCherry, a red fluorescent protein. Aminotransferase activities and formation of volatile compounds were compared. Activities of L. sakei ArcT and AspD purified enzymes were also measured. Conversion of branched chain amino acids to the corresponding α-keto-acids was significantly increased in all transformants expressing the ilvE genes. The activity of IlvE obtained from L. paracasei was highest. Substrate specificities of IlvEs towards leucine, isoleucine and valine were similar. However, enhanced transaminase activities did not increase formation of the respective methyl-branched volatiles by recombinant L. sakei strains. This indicates that presence of ilvE cannot be the only bottleneck in aroma formation from amino acids. Amino acid or peptide uptake into the cell via specific transport systems and the conversion of α-keto acids to the corresponding aldehydes, alcohols and carboxylic acids must be considered as further limiting steps.

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      Fluorescent Peptide Synthesis - Genosphere Biotech

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