1.
Dendrimer D5 is a Vector for Peptide Transport to Brain Cells.
Source
B. P. Konstantinov Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Leningrad region, Gatchina, Russia. svesar1@yandex.ru.
Abstract
Dendrimers are a new class of nonviral vectors for gene or drug transport. Dendrimer capacity to penetrate through the blood-brain barrier remaines little studied. Biotinylated polylysine dendrimer D5, similarly to human growth hormonebiotinylated fragment covalently bound to D5 dendrimer, penetrates through the blood-brain barrier and accumulates in Drosophila brain after injection into the abdomen. Hence, D5 dendrimer can serve as a vector for peptide transport to brain cells.
- PMID:
- 22268035
- [PubMed - in process]
Inhibin α-Subunit N-terminus Interacts With Activin Type IB receptor To Disrupt Activin Signaling.
Source
Northwestern University, United States;
Abstract
Inhibin is a heterodimeric peptide hormone produced in the ovary that antagonizes activin signaling and FSH synthesis in the pituitary. The inhibin β-subunit interacts with the activin type II receptor (ActRII) to functionally antagonize activin. The inhibin α-subunit mature domain (N-terminus) arose relatively early during the evolution of the hormone, and inhibin function is decreased by an antibody directed against the α-subunit N-terminal extension region or by deletion of the N-terminal region. We hypothesized that the α-subunit N-terminal extension region interacts with the activin type I receptor (ALK4) to antagonize activin signaling in the pituitary. Human or chicken free α-subunit inhibited activin signaling in a pituitary gonadotrope derived cell line (LβT2) in a dose-dependent manner, whereas an N-terminal extension deletion mutant did not. An α-subunit N-terminal peptide, but not a control peptide, was able to inhibit activin A signaling and decrease activin-stimulated FSH synthesis. Biotinylated inhibin A, but not activin A, bound ALK4. Soluble ALK4-ECD bioneutralized human free α-subunit in LβT2 cells, but did not affect activin A function. Competitive binding ELISAs with N-terminal mutants and an N-terminal region peptide confirmed that this region is critical for direct interaction of the α-subunit with ALK4. These data expand our understanding of how endocrine inhibin achieves potent antagonism of local, constitutive activin action in the pituitary, through a combined mechanism of competitive binding of both ActRII and ALK4 by each subunit of the inhibin heterodimer, in conjunction with the co-receptor betaglycan, to block activin receptor-ligand binding, complex assembly, and downstream signaling.
Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography.
Source
Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120, Halle (Saale), Germany.
Abstract
A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylatedproteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith's surface are envisaged for the future development of monoliths with improved enrichment characteristics.
A High Throughput Scintillation Proximity Imaging Assay for Protein Methyltransferases.
Source
HTS Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY, USA. djaballh@mskcc.org.
Abstract
Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues ofbiotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z' values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 µM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.
- PMID:
- 22256970
- [PubMed - as supplied by publisher]
Exposure of the lysine in the gamma chain dodecapeptide of human fibrinogen is not enhanced by adsorption to poly(ethylene terephthalate) as measured by biotinylation and mass spectrometry.
Source
Department of Biomedical Engineering and Center for Materials Innovation, Washington University, St. Louis, Missouri; Department of Neurology, Hope Center for Neurological Disorders and Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, Missouri.
Abstract
Conformational changes in adsorbed fibrinogen may enhance the exposure of platelet adhesive sites that are inaccessible in solution. To test this hypothesis, mass spectrometric methods were developed to quantify chemical modification of lysine residues following adsorption of fibrinogen to biomaterials. The quantitative method used an internal standard consisting of isotope-labeled fibrinogen secreted by human HepG2 cells in culture. Lysine residues in the internal standard were partially reacted with NHS-biotin. For the experimental samples, normal human fibrinogen was adsorbed to poly(ethylene terephthalate) (PET) particles. The adsorbed fibrinogen was reacted with NHS-biotin and then eluted from the particles. Constant amounts of internal standard were added to sample fibrinogen and analyzed by liquid chromatography/tandem mass spectrometry. Biotinylation of the lysine residue in the platelet-adhesive gamma chain dodecapeptide (GCDP) was quantified by comparison with the internal standard. Approximately 80% of the GCDPpeptides were biotinylated when fibrinogen was reacted with NHS-biotin in solution or adsorbed onto PET. These results are generally consistent with previous antibody binding studies and suggest that other regions of fibrinogen may be crucial in promoting platelet adhesion to materials. The results do not directly address but are consistent with the hypothesis that only activated platelets adhere to adsorbed fibrinogen. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.
Copyright © 2011 Wiley Periodicals, Inc.
Development of a high-throughput screen targeting caspase-8-mediated cleavage of the amyloid precursor protein.
Source
Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245, USA; Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX 78245, USA.
Abstract
Caspases, effectors of apoptosis, are key mediators of neuronal death in several neurodegenerative diseases. Caspase-8 and caspase-6 have been implicated in the pathogenesis of amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, and Alzheimer's disease (AD). ß-Amyloid precursor protein (APP) is cleaved at Asp664 in its intracellular domain by caspase-8. We and other laboratories recently showed that obliteration of the caspase cleavage site on APP alleviates functional AD-like deficits in a mouse model. Therefore, caspase cleavage of APP constitutes a potential novel target for therapeutic intervention. To identify chemical inhibitors of caspase-8 cleavage, we screened a subset of the chemical library at the Harvard NeuroDiscovery Center's Laboratory for Drug Discovery in Neurodegeneration. We show that caspase-8, but not caspase-1, -3, or -9, cleaves a biotinylated peptide derived from APP at Asp664, and we report the development of a sensitive high-throughput assay for caspase-8 cleavage of APP and the use of that assay for the identification of specific small molecule "hit" compounds that potently inhibit Asp664 cleavage of APP. Furthermore, we demonstrate that one of these compounds (LDN-0021835) inhibits the cleavage of APP at Asp664 in cell-based assays.
Copyright © 2011 Elsevier Inc. All rights reserved.
Efficient and versatile one-step affinity purification of in vivo biotinylatedproteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II.
Source
Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 6, Czech Republic; Department of Biochemistry, Faculty of Natural Science, Charles University, Albertov 6, Prague 2, Czech Republic.
Abstract
Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptideis used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.
Copyright © 2011. Published by Elsevier Inc.
Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by sortase A-mediated protein ligation.
Source
CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria, 3052, Australia; telephone: +61-3-9662-7100; fax: +61-3-9662-7314.
Abstract
Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody-receptor interaction. Biotechnol. Bioeng. © 2011 Wiley Periodicals, Inc.
Copyright © 2011 Wiley Periodicals, Inc.
Detection of Edwardsiella tarda by fluorometric or biosensor methods using apeptide ligand.
Source
Department of Chemistry, Sejong University, Seoul 143-747, Republic of Korea.
Abstract
In this study, we identified a peptide ligand for Edwardsiella tarda from a phage peptide library and tested two approaches for sensitive detection of the bacteria with the peptide labeled with fluorescein or biotin. At first, the fluorescent peptide was proved to be advantageous in the fluorescence polarization (FP) assay because sensitivity of the assay is maximized when a fluorophore is linked to a small molecule. The FP assay using the fluorescent peptideenabled detection of E. tarda in a range from 5.2×10(3) to 2.1×10(5) cells. Second, we devised a new assay method using a quartz crystal microbalance (QCM) biosensor connected to a filter module. When a mixture of E. tarda and thebiotinylated peptide was injected into the filter module, the E. tarda-peptide complex was separated from the unboundpeptide by a filter and detected with a streptavidin-coated QCM sensor chip. On injection of samples containing thebiotinylated peptide and E. tarda, concentration-dependent frequency change was observed in a range from 8×10(2) to 8×10(6) cells. The two approaches are expected to facilitate development of assay methods using other bacteria-bindingpeptides.
Copyright © 2011 Elsevier Inc. All rights reserved.
Targeting of Histone Acetyltransferase p300 by Cyclopentenone Prostaglandin Δ(12)-PGJ(2) through Covalent Binding to Cys(1438).
Source
Center for Molecular Toxicology and Carcinogenesis and Center for Molecular Immunology and Infectious Disease, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
Abstract
Inhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys(1438) in the substrate binding site could be targeted by Δ(12)-prostaglandin J(2) (Δ(12)-PGJ(2)), a cyclopentenone prostaglandin (CyPG) derived from PGD(2). We demonstrate here the ability of CyPGs to inhibit p300 HAT-dependent acetylation of histone H3. A cell-based assay system clearly showed that the α,β-unsaturation in the cyclopentenone ring of Δ(12)-PGJ(2) was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy-Δ(12,14)-PGJ(2), which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that Δ(12)-PGJ(2) places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys(1438) of p300 to form a covalent Michael adduct. Site-directed mutagenesis of the p300 HAT domain, peptide competition assay involving p300 wild type and mutant peptides, followed by mass spectrometric analysis confirmed the covalent interaction of Δ(12)-PGJ(2) with Cys(1438). Using biotinylatedderivatives of Δ(12)-PGJ(2) and 9,10-dihydro-15-deoxy-Δ(12,14)-PGJ(2), we demonstrate the covalent interaction of Δ(12)-PGJ(2) with the p300 HAT domain, but not the latter. In agreement with the in vitro filter binding assay, CyPGs were also found to inhibit H3 histone acetylation in cell-based assays. In addition, Δ(12)-PGJ(2) also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in in vitro assays. This study demonstrates, for the first time, that Δ(12)-PGJ(2) inhibits p300 through Michael addition, where α,β-unsaturated carbonyl function is absolutely required for the inhibitory activity.
Y654 of β-catenin is essential for FLT3/ITD-related tyrosine phosphorylation and nuclear localization of β-catenin.
Source
Department of Infectious Diseases, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya Hematology and Oncology Branch, Tosei General Hospital, Seto Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya National Center for Geriatrics and Gerontology, Morioka-Machi, Obu, Japan.
Abstract
β-Catenin plays a dual role as a key effecter in the regulation of adherens junctions as well as a transcriptional co-activator. Tyrosine phosphorylation of β-catenin affects the cell adhesion, migration, and gene transcription in many types of human cancer cells, including acute myeloid leukemia cells with FLT3 internal tandem duplication (FLT3/ITD-AML). Here, we investigated the relationship between three tyrosine residues (Y86, Y142, and Y654) in β-catenin and oncogenic FLT3/ITD kinase. In the experiments using COS-7 cells expressing FLT3/ITD and Wt or mutant β-catenin, FLT3/ITD phosphorylated Y654, and this residue was essential for β-catenin's nuclear localization by FLT3/ITD. Promoter-reporter assays demonstrated that Y654 phosphorylation of β-catenin was closely related to TCF transcriptional activity. In vitro kinase assays, using recombinant FLT3 and biotinylated β-catenin peptide including Y654 showed that FLT3 directly phosphorylated Y654 of β-catenin. These results explain how FLT3/ITD affects the tyrosine phosphorylation, nuclear localization, and transcriptional activity of β-catenin. Targeting Y654 phosphorylation may lead to the development of novel approaches to therapy for FLT3/ITD-AML.
© 2012 John Wiley & Sons A/S.
Identification of host cell factors required for intoxication through use of modified cholera toxin.
Source
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Abstract
We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using abiotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ~12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.
Glutathionylation in the photosynthetic model organism Chlamydomonas reinhardtii: a proteomic survey.
Source
FRE3354 CNRS, Universite Pierre et Marie Curie, France;
Abstract
Protein glutathionylation is a redox post-translational modification occurring under oxidative stress conditions and playing a major role in cell regulation and signaling. This modification has been mainly studied in non-photosynthetic organisms while much less is known in photosynthetic organisms despite their important exposure to oxidative stress caused by changes in environmental conditions. We report a large scale proteomic analysis using biotinylatedglutathione and streptavidin affinity chromatography that allowed identification of 225 glutathionylated proteins in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. Moreover, 56 sites of glutathionylation were also identified after peptide affinity purification and tandem mass spectrometry. The targets identified belong to a wide range of biological processes and pathways among which the Calvin-Benson cycle appears to be a major target. The glutathionylation of four enzymes of this cycle, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, ribose-5-phosphate isomerase and phosphoglycerate kinase was confirmed in vitro by western blot and activity measurements. The results suggest that glutathionylation could constitute a major mechanism of regulation of the Calvin-Benson cycle under oxidative stress conditions.
Ubiquitination of an artificial RING finger without a substrate and a tag.
Source
Department of Pharmaceutical Health Care, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo, Japan. miyamoto@himeji-du.ac.jp.
Abstract
Alpha-helical region substitution was applied to the SIAH1 and EL5 RING fingers. The Williams-Beuren syndrome transcription factor (WSTF) PHD_SIAH1 and WSTF PHD_EL5 RING fingers were created as the artificial ubiquitin-ligating enzyme (E3). These fingers possess E3 activities of mono-ubiquitination and poly-ubiquitination, respectively, with ubiquitin-conjugating enzyme (E2)-binding capabilities. Artificial E3s bind two zinc atoms and adopt a zinc-dependent ordered structure and ubiquitinate upon themselves without a substrate and a tag. Ubiquitination experiments using biotinylated ubiquitin showed that the WSTF PHD_EL5 RING finger is poly-ubiquitinated via residue Lys(63) of ubiquitin. Substitution of alpha-helical region might be applicable to various RING fingers with mono-ubiquitination or poly-ubiquitination. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.
Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.
In-depth analysis of cysteine oxidation by the RBC proteome: Advantage of peroxiredoxin II knockout mice.
Source
Department of Oral Biochemistry, Dental Science Research Institute, The 2nd Stage of Brain Korea 21 for Dental School, Chonnam National University, Gwangju, Republic of Korea.
Abstract
Peroxiredoxin II (Prdx II, a typical 2-Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Mice lacking Prdx II proteins had heinz bodies in their peripheral blood, and morphologically abnormal cells were detected in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). In this study, a labeling experiment with the thiol-modifying reagent biotinylated iodoacetamide (BIAM) in Prdx II(-/-) mice revealed that a variety of RBC proteins were highly oxidized. To identify oxidation-sensitive proteins in Prdx II(-/-) mice, we performed RBC comparative proteome analysis in membrane and cytosolic fractions by nano-UPLC-MS(E) shotgun proteomics. We found oxidation-sensitive 54 proteins from 61 peptides containing cysteine oxidation, and analyzed comparative expression pattern in healthy RBCs of Prdx II(+/+) mice, healthy RBCs of Prdx II(-/-) mice, and abnormal RBCs of Prdx II(-/-) mice. These proteins belonged to cellular functions related with RBC lifespan maintain, such as cytoskeleton, stress-induced proteins, metabolic enzymes, signal transduction, and transporters. Furthermore, protein networks among identified oxidation-sensitive proteins were analyzed to associate with various diseases. Consequently, we expected that RBC proteome might provide clues to understand redox-imbalanced diseases.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Biotinylated probes for the analysis of protein modification by electrophiles.
Source
Vanderbilt University School of Medicine, Nashville, TN, USA.
Abstract
Formation of covalent protein adducts by lipid electrophiles contributes to diseases and toxicities linked to oxidative stress, but analysis of the adducts presents a challenging analytical problem. We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which form adducts that can be postlabeled with azidobiotin tags by Cu(+)-catalyzed cycloaddition (Click chemistry). To enhance the selectivity of adduct capture, we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions. This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts.
A novel small Odorranalectin-bearing cubosomes: Preparation, brain delivery and pharmacodynamic study on amyloid-β(25-35)-treated rats following intranasal administration.
Source
School of Pharmacy, Fudan University, Shanghai, China; School of Pharmacy, Shanghai Jiaotong University, Shanghai, China.
Abstract
Because of the immunogenicity and toxicity in vivo of large molecules such as lectins, the application of these molecules is remarkably restricted in drug delivery systems. In this study, to improve the brain drug delivery and reduce the immunogenicity of traditional lectin modified delivery system, Odorranalectin (OL, 1700Da), a novel non-immunogenic small peptide, was selected to establish an OL-modified cubosomes (Cubs) system. The streptavidin (SA)-conjugated Cubs were prepared by incorporating maleimide-PEG-oleate and taking advantage of its thiol group binding reactivity to conjugate with 2-iminothiolane thiolated SA; mono-biotinylated OL was then coupled with the SA-modified Cubs. The OL-decorated Cubs (OL-Cubs) devised via a non-covalent SA-biotin "bridge" made it easy to conjugate OL and determine the number of ligands on the surface of the Cubs using sensitive chemiluminescent detection. Retention of the bio-recognitive activity of OL after covalent coupling was verified by hemagglutination testing. Nose-to-brain delivery characteristic of OL-Cubs was investigated by in vivo fluorescent biodistribution using coumarin-6 as a marker. The relative uptake of coumarin carried by OL-Cubs was 1.66- to 3.46-fold in brain tissues compared to that incorporated in the Cubs. Besides, Gly14-Humanin (S14G-HN) as a model peptide drug was loaded into cubosomes and evaluated for its pharmacodynamics on Alzheimer's disease (AD) rats following intranasal administration by Morris water maze test and acetylcholinesterase activity determination. The results suggested that OL functionalization enhanced the therapeutic effects of S14G-HN-loaded cubosomes on AD. Thus, OL-Cubs might offer a novel effective and noninvasive system for brain drug delivery, especially for peptides and proteins.
Copyright © 2011 Elsevier B.V. All rights reserved.
Sialic acid and sialyl-lactose glyco-conjugates: design, synthesis and binding assays to lectins and swine influenza H1N1 virus.
Source
Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina, 45110, Ioannina, Greece. stella.zevgiti@gmail.com.
Abstract
The terminal parts of the influenza hemagglutinin (HA) receptors α2,6- and α2,3-sialyllactoses were conjugated to an artificial carrier, named sequential oligopeptide carrier (SOC(4) ), to formulate human and avian receptor mimics, respectively. SOC(4) , formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of biomolecules to the Lys-N(ε) H(2) groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC(4) -glyco-conjugate bearing two copies of the α2,6-sialyllactose is specifically recognized by the biotinylated Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an α2,6-linkage. SOC(4) -glyco-conjugate bearing two copies of the α2,3-sialyllactose was not recognized by thebiotinylated Maackia amurensis lectin, despite its well-known α2,3-sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both the SOC(4) -glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC(4) [(Ac)(2) ,(3'SL-Aoa)(2) ]-NH(2) 5 and Ac-SOC(4) [(Ac)(2) ,(6'SL-Aoa)(2) ]-NH(2) 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus-receptor interactions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.
Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.
A chelating-bond breaking and re-linking technique for rapid re-immobilization of immune micro-sensors.
Source
State Key Lab of Transducer Technology, and, Science and Technology on Microsystem Lab, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, 865 Changning Road, Shanghai, 200050, China.
Abstract
With high sensitivity and specificity to antigen, immune micro-sensors can be used in rapid detection of pathogenic microbial. This study proposes and develops a method for rapidly regeneration of antibody on a resonant micro-cantilever sensor. A nitrilotriacetic acid (NTA) derivative is synthesized with cystine and bromoacetic acid, then added with 2-mercaptoethanol to prepare a mixed self-assembled monolayer (SAM) on Au (111) surface of the cantilever. Ni(2+) ions are thereafter chelated on the mixed SAM to form a breakable and re-linkable chelating-bond layer. Repeatable cycles of antibody immobilization and erasing are experimentally validated with a detectable marker of synthesized biotinylatedpoly peptides harboring six histidine residues (named as His-Bio). Two distinguished pathogenic microbial, Escherichia. coli O157:H7 and Bacillus Anthracis, are detected with the rapidly regenerated sensor. The E. coli O157:H7 sensor exhibits a three-time repeated detection to the 10(3) CFU/ml concentration microbial. Then, an E. coli O157:H7 sensor is eluted with Tris-HCl (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH = 3.0) and rapidly reconstructed into a B. Anthracis sensor by changing the re-immobilized antibody. The cantilever sensor no longer responses to E. coli O157:H7 even in a high concentration of 10(7) CFU/ml. In contrast, the sensor is experimentally confirmed being resoluble to low concentration B. Anthracis at 10(3) spores/ml level. The proposed fast regeneration method is promising in repeatedly or multi-target detection applications of micro/nano immune-sensors, e.g. the resonant micro-cantilevers.
High resolution time-of-flight mass analysis of the entire range of intact singly-charged proteins.
Source
Washington State University, Department of Chemistry, Pullman, Washington 99164, United States.
Abstract
The proof of principle for high-resolution analysis of intact singly charged proteins of any size is presented. Singly charged protein ions were produced by electrospray ionization followed by surface-induced charge reduction at atmospheric pressure. The inlet and trapping system "stops" the forward momentum of the protein ions over a very broad range to be captured by the digitally produced electric fields of a large radius linear ion trap whereupon they are moved into a smaller radius linear ion trap and collected and concentrated in front of its exit end-cap electrode using digital waveform manipulation. The protein ions are then ejected on demand from the end of the small radius linear quadrupole in a tightly collimated ion beam with an instrumentally defined kinetic energy into the acceleration region of an orthogonal acceleration reflectron time-of-flight mass analyzer where their flight times were measured and detected with a Photonis BiPolar TOF detector. We present results that clearly prove that massive singly charged ions can yield high-resolution mass spectra with very low chemical noise and without loss of sensitivity with increasing mass across the entire spectrum. Analysis of noncovalently bound protein complexes was demonstrated with streptavidin-Cy5 bound with a biotinylated peptide mimic. Our results suggest proteins across the entire range can be directly quantified using our mass analysis technique. We present evidence that solvent molecules noncovalently adduct onto the proteins while yielding consistent flight time distributions. Finally, we provide a look into future that will result from the ability to rapidly measure and quantify protein distributions.
- PMID:
- 22047146
- [PubMed - in process]
- PMCID: PMC3237766
- [Available on 2012/12/15]
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