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Covalent labeling

Benno M Müller, Rudi Loth, Peter-Georg Hoffmeister, Friederike Zühl, Liv Kalbitzer, Michael C Hacker, Michaela Schulz-Siegmund
: The concept of macromers allows for a broad adjustment of biomaterial properties by macromer chemistry or copolymerization. Copolymerization strategies can also be used to introduce reactive sites for subsequent surface modification. Control over surface features enables adjustment of cellular reactions with regard to site and object of implantation. We designed macromer-derived polymer films which function as non-implantable analytical substrates for the investigation of surface properties of equally composed scaffolds for bone tissue engineering...
January 6, 2017: Acta Biomaterialia
Jean-Marc Zingg, Angelo Azzi, Mohsen Meydani
The CD36 scavenger receptor binds several ligands and mediates ligand uptake and ligand-dependent signal transduction and gene expression, events that may involve CD36 internalization. Here we show that CD36 internalization in THP-1 monocytes is triggered by α-tocopherol (αT) and more strongly by α-tocopheryl phosphate (αTP) and EPC-K1, a phosphate diester of αTP and L-ascorbic acid. αTP-triggered CD36 internalization is prevented by the specific covalent inhibitor of selective lipid transport by CD36, sulfo-N-succinimidyl oleate (SSO)...
January 6, 2017: Journal of Cellular Biochemistry
Theodoros Kelesidis, Ingrid Schmid
Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe...
January 5, 2017: Current Protocols in Cytometry
Qian Zhao, Xiaohu Ouyang, Xiaobo Wan, Ketan S Gajiwala, John C Kath, Lyn H Jones, Alma L Burlingame, Jack Taunton
Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase-inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallography and mass spectrometry confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site...
January 4, 2017: Journal of the American Chemical Society
Silke B Lohan, Siavash Saeidpour, Agnieszka Solik, Sabine Schanzer, Heike Richter, Pin Dong, Maxim E Darvin, Roland Bodmeier, Alexa Patzelt, Gaith Zoubari, Michael Unbehauen, Rainer Haag, Jürgen Lademann, Christian Teutloff, Robert Bittl, Martina C Meinke
An improvement of the penetration efficiency combined with the controlled release of actives in the skin can facilitate the medical treatment of skin diseases immensely. Dexamethasone (Dx), a synthetic glucocorticoid, is frequently used for the treatment of inflammatory skin diseases. To investigate the penetration of nano-sized lipid particles (NLP) loaded with Dx in comparison to a commercially available base cream, different techniques were applied. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the penetration of Dx, which was covalently labeled with the spin probe 3-(Carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA)...
December 30, 2016: European Journal of Pharmaceutics and Biopharmaceutics
Bin Zhou, Youyi Qiu, Qingqing Wen, Mingyao Zhu, Peihui Yang
A mutiplex cytosensor based on a dual electrochemiluminescence (ECL) signal system was fabricated for in situ and simultaneous detection of the expression levels of multiple cell-surface receptors, mannose and epidermal growth factor receptor (EGFR), using luminol-capped gold nanoparticles (Au@luminol) and CdS quantum dots (CdS QDs) as potential-resolved ECL nanoprobes. Two spatially resolved areas on indium tin oxide (ITO) electrodes were modified with polyaniline (PANI) by electropolymerization, on which gold nanoparticles (AuNPs) were attached to strengthen conductivity and stability of the sensing interface...
January 11, 2017: ACS Applied Materials & Interfaces
L Gwozdzinski, A Pieniazek, J Bernasinska, M Grabowski, E Kowalczyk, K Gwozdzinski
Varicose veins (VV) are enlarged veins of the subcutaneous tissue, usually caused by faulty or damaged venous valves leading to impaired blood flow. Blood stasis, excessive clotting disorder and alterations in the vein walls are symptoms of Virchow's triad which may affect the morphotic elements of blood, including erythrocytes. The aim of this study was to investigate alterations in the properties of the erythrocytes taken from varicose veins in comparison to those from antecubital vein of patients with chronic venous disease...
December 21, 2016: Microvascular Research
Md Asif Amin, Ritaban Halder, Catherine Ghosh, Biman Jana, Kankan Bhattacharyya
Effect of ethanol on the size and structure of a protein cytochrome C (Cyt C) is investigated using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. For FCS studies, Cyt C is covalently labeled with a fluorescent probe, alexa 488. FCS studies indicate that on addition of ethanol, the size of the protein varies non-monotonically. The size of Cyt C increases (i.e., the protein unfolds) on addition of alcohol (ethanol) up to a mole fraction of 0.2 (44.75% v/v) and decreases at higher alcohol concentration...
December 21, 2016: Journal of Chemical Physics
Geuncheol Gil, Pan Mao, Bharathi Avula, Zulfiqar Ali, Amar G Chittiboyina, Ikhlas A Khan, Larry A Walker, Daojing Wang
Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original bioactivity. Most proteins undergo posttranslational modification, and only certain proteoforms have the right conformation with accessible domains and available residues for small molecule binding. We developed a top-down mass spectrometry (MS) centric workflow for rapid evaluation of the bioactivity of crude botanical extracts after a one-step reaction...
December 21, 2016: ACS Chemical Biology
Jin-Sil Choi, Yazhen Zhu, Hongsheng Li, Parham Peyda, Thuy Tien Nguyen, Mo Yuan Shen, Yang Michael Yang, Jingyi Zhu, Mei Liu, Mandy M Lee, Shih-Sheng Sun, Yang Yang, Hsiao-Hua Yu, Kai Chen, Gary S Chuang, Hsian-Rong Tseng
Tattooing has been utilized by the medical community for precisely demarcating anatomic landmarks. This practice is especially important for identifying biopsy sites of nonmelanoma skin cancer (NMSC) due to the long interval (i.e., up to 3 months) between the initial diagnostic biopsy and surgical treatment. Commercially available tattoo pigments possess several issues, which include causing poor cosmesis, being mistaken for a melanocytic lesion, requiring additional removal procedures when no longer desired, and potentially inducing inflammatory responses...
December 20, 2016: ACS Nano
Benedikt M Beckmann
RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365 nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3 days to complete...
December 16, 2016: Methods: a Companion to Methods in Enzymology
Ksenia S Stankevich, Nadezhda V Danilenko, Ruslan M Gadirov, Semen I Goreninskii, Sergei I Tverdokhlebov, Victor D Filimonov
A new approach for the immobilization of poly(acrylic) acid (PAA) as a chemically reactive cross-linker on the surface of poly(lactic) acid-based (PLA) biomaterials is described. The proposed technique includes non-covalent attachment of a PAA layer to the surface of PLA-based biomaterial via biomaterial surface treatment with solvent/non-solvent mixture followed by the entrapment of PAA from its solution. Surface morphology and wettability of the obtained PLA-PAA composite materials were investigated by AFM and the sitting drop method respectively...
February 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
Yi Wang, Peter W Villalta, Lijuan Peng, Karen H Dingley, Michael A Malfatti, Kenneth W Turteltaub, Robert J Turesky
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed during the high temperature cooking of meats. The cytochrome P450-mediated N-hydroxylation of the exocyclic amine group of PhIP produces 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), an electrophilic metabolite which forms adducts with DNA and proteins. Previous studies conducted by our laboratory showed that the reaction of N-oxidized-PhIP metabolites with human albumin in vitro primarily occurs at the Cys34 residue, to produce an acid-labile linked sulfinamide adduct...
December 16, 2016: Chemical Research in Toxicology
Terence L Kirley, Kenneth D Greis, Andrew B Norman
Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab')2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab')2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric...
October 28, 2016: Biochemical and Biophysical Research Communications
Xiao-Mei Shi, Gao-Chao Fan, Qingming Shen, Jun-Jie Zhu
Sensitive and accurate analysis of DNA is crucial to better understanding of DNA functions and early diagnosis of fatal disease. Herein, an enhanced photoelectrochemical (PEC) DNA biosensor was proposed based on dual-signal amplification via coupling inorganic-organic nanocomposites sensitization with λ-exonuclease (λ-Exo)-assisted target recycling. The short DNA sequence about chronic myelogenous leukemia (CML, type b3a2) was selected as target DNA (tDNA). ZnO nanoplates were deposited with CdS nanocrystals to form ZnO/CdS hetero-nanostructure, and it was used as PEC substrate for immobilizing hairpin DNA (hDNA)...
December 28, 2016: ACS Applied Materials & Interfaces
Youssra K Al-Hilaly, Luca Biasetti, Ben J F Blakeman, Saskia J Pollack, Shahin Zibaee, Alaa Abdul-Sada, Julian R Thorpe, Wei-Feng Xue, Louise C Serpell
Parkinson's disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable α-synuclein (α-syn) amyloid fibrils. α-synuclein is an intrinsically disordered protein that has the capacity to assemble to form β-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD patients...
December 16, 2016: Scientific Reports
Christine Emnett, Hairong Li, Xiaoping Jiang, Ann Benz, Joseph Boggiano, Sara Conyers, David F Wozniak, Charles F Zorumski, David E Reichert, Steven Mennerick
Ketamine is a psychotomimetic and antidepressant drug. Although antagonism of cell-surface NMDA receptors (NMDARs) may trigger ketamine's psychoactive effects, ketamine or its major metabolite norketamine could act intracellularly to produce some behavioral effects. To explore the viability of this latter hypothesis, we examined intracellular accumulation of novel visualizable analogues of ketamine/norketamine. We introduced an alkyne "click" handle into norketamine (alkyne-norketamine, A-NK) at the key nitrogen atom...
December 16, 2016: Scientific Reports
M Braner, R Wieneke, R Tampé
High background originating from non-reacted, 'always-on' fluorescent probes remains a key unsolved problem in life science since washing procedures are not easily applicable. Covalent labeling approaches with simultaneous activation of fluorescence are advantageous to increase sensitivity and to reduce background signal. Here, we combined high-affinity protein trans-splicing with fluorophore/quencher pairs for online detection of covalent N-terminal 'traceless' protein labeling under physiological conditions in cellular environments...
January 3, 2017: Chemical Communications: Chem Comm
Cassandra M Joiner, Meghan E Breen, James Clayton, Anna K Mapp
In vivo covalent chemical capture by using photoactivatable unnatural amino acids (UAAs) is a powerful tool for the identification of transient protein-protein interactions (PPIs) in their native environment. However, the isolation and characterization of the crosslinked complexes can be challenging. Here, we report the first in vivo incorporation of the bifunctional UAA BPKyne for the capture and direct labeling of crosslinked protein complexes through post-crosslinking functionalization of a bioorthogonal alkyne handle...
December 14, 2016: Chembiochem: a European Journal of Chemical Biology
Lars Büter, Lisa M Frensemeier, Martin Vogel, Uwe Karst
A new hyphenated technique based on on-line dual (oxidative and reductive) electrochemistry coupled to liquid chromatography and high resolution electrospray mass spectrometry is presented. Two liquid streams are combined, with one containing a disulfide, which is reduced to the respective thiol in an electrochemical cell based on a titanium working electrode. The other stream contains phenol, which is electrochemically activated to benzoquinone on a boron-doped diamond working electrode. Upon combination of the two streams, a Michael addition takes places, leading to the covalent coupling of thiol to quinone...
January 6, 2017: Journal of Chromatography. A
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