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- Andersson, Margaretha, et al.
(författare)
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Characterization of Surface-Modified Nanoparticles for in Vivo Biointeraction. A Sedimentation Field Flow Fractionation Study
- 2005
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Ingår i: Analytical Chemistry. ; 77, s. 5488-5493
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Tidskriftsartikel (refereegranskat)abstract
- Sedimentation field flow fractionation (SdFFF) is an emerging high-performance analytical tool for separation and determination of size and adsorption characteristics of colloidal particles. This study demonstrates how SdFFF can be used to characterize nanoparticles prepared for in vivo applications including (1) the quantification of polymer uptake on nanoparticles where surface coverage is crucial and (2) the coupling of cell adhesive peptides containing the Arg-Gly-Asp motif (RGD). Quantitative information about polymer adhesion in order to prepare a bioinert surface and an accurate determination of ligand uptake are both of obvious importance for the understanding of, for example, relations between the number of attached molecules for biointeraction and an observed therapeutic effect. In addition, the present work highlights the necessity to perform careful characterization of commercially available particulate starting materials, in terms of size and polydispersity, prior to biological experimentation.
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| 3. |
- Artursson, Elisabet, et al.
(författare)
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Catalytic-site conformational equilibrium in nerve-agent adducts of acetylcholinesterase : Possible implications for the HI-6 antidote substrate specificity
- 2013
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Ingår i: Biochemical Pharmacology. - : Elsevier. - 0006-2952 .- 1356-1839. ; 85:9, s. 1389-1397
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Tidskriftsartikel (refereegranskat)abstract
- Nerve agents such as tabun, cyclosarin and Russian VX inhibit the essential enzyme acetylcholinesterase (AChE) by organophosphorylating the catalytic serine residue. Nucleophiles, such as oximes, are used as antidotes as they can reactivate and restore the function of the inhibited enzyme. The oxime HI-6 shows a notably low activity on tabun adducts but can effectively reactivate adducts of cyclosarin and Russian VX. To examine the structural basis for the pronounced substrate specificity of HI-6, we determined the binary crystal structures of Mus musculus AChE (mAChE) conjugated by cyclosarin and Russian VX and found a conformational mobility of the side chains of Phe338 and His447. The interaction between HI-6 and tabun-adducts of AChE were subsequently investigated using a combination of time resolved fluorescence spectroscopy and X-ray crystallography. Our findings show that HI-6 binds to tabun inhibited Homo sapiens AChE (hAChE) with an IC50 value of 300 μM and suggest that the reactive nucleophilic moiety of HI-6 is excluded from the phosphorus atom of tabun. We propose that a conformational mobility of the side-chains of Phe338 and His447 is a common feature in nerve-agent adducts of AChE. We also suggest that the conformational mobility allow HI-6 to reactivate conjugates of cyclosarin and Russian VX while a reduced mobility in tabun conjugated AChE results in steric hindrance that prevents efficient reactivation.
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| 4. |
- Berg, Lotta, et al.
(författare)
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Targeting Acetylcholinesterase : Identification of Chemical Leads by High Throughput Screening, Structure Determination and Molecular Modeling
- 2011
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Ingår i: PLOS ONE. - San Francisco : Public Library of Science. - 1932-6203. ; 6:11
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Tidskriftsartikel (refereegranskat)abstract
- Acetylcholinesterase (AChE) is an essential enzyme that terminates cholinergic transmission by rapid hydrolysis of the neurotransmitter acetylcholine. Compounds inhibiting this enzyme can be used (inter alia) to treat cholinergic deficiencies (e. g. in Alzheimer's disease), but may also act as dangerous toxins (e. g. nerve agents such as sarin). Treatment of nerve agent poisoning involves use of antidotes, small molecules capable of reactivating AChE. We have screened a collection of organic molecules to assess their ability to inhibit the enzymatic activity of AChE, aiming to find lead compounds for further optimization leading to drugs with increased efficacy and/or decreased side effects. 124 inhibitors were discovered, with considerable chemical diversity regarding size, polarity, flexibility and charge distribution. An extensive structure determination campaign resulted in a set of crystal structures of protein-ligand complexes. Overall, the ligands have substantial interactions with the peripheral anionic site of AChE, and the majority form additional interactions with the catalytic site (CAS). Reproduction of the bioactive conformation of six of the ligands using molecular docking simulations required modification of the default parameter settings of the docking software. The results show that docking-assisted structure-based design of AChE inhibitors is challenging and requires crystallographic support to obtain reliable results, at least with currently available software. The complex formed between C5685 and Mus musculus AChE (C5685.mAChE) is a representative structure for the general binding mode of the determined structures. The CAS binding part of C5685 could not be structurally determined due to a disordered electron density map and the developed docking protocol was used to predict the binding modes of this part of the molecule. We believe that chemical modifications of our discovered inhibitors, biochemical and biophysical characterization, crystallography and computational chemistry provide a route to novel AChE inhibitors and reactivators.
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| 8. |
- Gullberg, Elisabet, et al.
(författare)
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Identification of Cell Adhesion Molecules in the Human Follicle-Associated Epithelium That Improve Nanoparticle Uptake into the Peyer's Patches
- 2006
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Ingår i: Journal of Pharmacology and Experimental Therapeutics. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0022-3565 .- 1521-0103. ; 319:2, s. 632-639
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to identify cell adhesion molecules that could serve as targets of the human follicle-associated epithelium (FAE) overlying Peyer's patches and to assess nanoparticle uptake levels across this epithelium. We first studied the expression of the mouse M-cell marker beta(1)-integrin and used a model of human FAE derived from intestinal epithelial Caco-2 cells and Raji B-cells to identify additional potential targets by cDNA array. The protein expression of potential targets in the model FAE and in human ileal FAE tissues was quantified by immunofluorescence. Integrin targeting was studied by investigating the transport of Arg-Gly-Asp (RGD)-coated (integrin- binding), Arg-Gly-Glu (RGE)-coated (nonintegrin-binding), and uncoated nanoparticles across ileal specimens mounted in Ussing chambers. Both beta(1)-integrin and the cell adhesion molecule CD9 were more abundantly expressed in the model and human FAE compared with the Caco-2 control cells or villus epithelium (VE). Uncoated nanoparticles were not taken up across either FAE or VE. General integrin targeting with RGD improved the nanoparticle transport dramatically across the FAE and to a lower extent across the VE. Compared with RGE, RGD improved transport 4-fold across the FAE. There was no difference in the transport of RGD- and RGE-coated nanoparticles across the VE. In conclusion, beta(1)-integrin and CD9 were identified as targets in human FAE. The difference in RGD- and RGE-mediated transport across the FAE, but not the VE, suggests that a specific integrin interaction was the dominating mechanism for improved nanoparticle uptake across the FAE., whereas charge interaction contributed substantially to the improved VE uptake.
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| 9. |
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| 10. |
- Gullberg, Elisabet, 1972-
(författare)
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Particle Transcytosis Across the Human Intestinal Epithelium : Model Development and Target Identification for Improved Drug Delivery
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of nano- and micro-particulate carriers as delivery systems for oral vaccines has been under investigation for several decades. Surprisingly little is known of their uptake in the human intestine, despite the fact that substantial improvement is required to achieve adequate immune responses in man after oral administration. In this thesis, various aspects of particle transcytosis across the human intestinal epithelium were studied, in order to identify strategies for improved uptake of nano- and micro-particulate drug delivery systems. The follicle associated epithelium (FAE) overlying Peyer´s patches contains M-cells, which have an increased capacity for uptake of particulate antigens. Therefore, a model of human FAE was developed to study mechanisms of particle uptake and transport.Receptors that could be used for targeting to the FAE had previously not been identified in humans. By use of the model FAE, two new targets were identified on human intestinal FAE; CD9 and β1-integrin. Furthermore, studies of isolated human intestinal tissue showed that an integrin-adherent peptide motif, RGD, could be utilized to achieve selective and improved transport of nanoparticles into human Peyer´s patches.Studies of factors influencing intestinal particle uptake and transcytosis revealed that two cytokines, TNF-α and LTα1/β2, but also one growth factor, TGF-β1, induced uptake of particles in Caco-2 cells and transcytosis of particles in the model FAE. Furthermore, it was shown that an enteric bacterium, Yersinia Pseudotuberculosis, could trigger uptake and transcytosis of particles across model absorptive epithelial cells.In conclusion, this thesis provides a platform for further investigations of particle transcytosis across the human intestinal epithelium. The identification of two new proteins with increased expression in human FAE and a targeting sequence that improves particle uptake into Peyer’s patches, gives new hope for the development of subunit oral vaccines.
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