The Experts below are selected from a list of 174 Experts worldwide ranked by ideXlab platform
David J. Thornton - One of the best experts on this subject based on the ideXlab platform.
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Reorganisation of the Salivary Mucin Network by Dietary Components: Insights from Green Tea Polyphenols
2016Co-Authors: Heather S. Davies, Paul D. A. Pudney, Pantelis Georgiades, Thomas A. Waigh, Nigel W. Hodson, Caroline E. Ridley, Ewan W. Blanch, David J. ThorntonAbstract:The salivary mucins that include MUC5B (gel-forming) and MUC7 (non-gel-forming) are major contributors to the protective mucus barrier in the oral cavity, and it is possible that dietary components may influence barrier properties. We show how one dietary compound, the green tea polyphenol epigallocatechin gallate (EGCG), can substantially alter the properties of both the polymeric MUC5B network and monomeric MUC7. Using Rate-Zonal Centrifugation, MUC5B in human whole saliva and MUC5B purified from saliva sedimented faster in the presence of EGCG. The faster sedimentation by EGCG was shown to be greater with increasing MUC5B concentration. Particle tracking microrheology was employed to determine the viscosity of purified MUC5B solutions and showed that for MUC5B solutions of 200–1600 mg/mL, EGCG caused a significant increase in mucin viscosity, which was greater at higher MUC5B concentrations. Visualisation of the changes to the MUC5B network by EGCG was performed using atomic force microscopy, which demonstRated increased aggregation of MUC5B in a heterogeneous manner by EGCG. Using trypsin-resistant, high-molecular weight oligosaccharide-rich regions of MUC5B and recombinant N-terminal and C-terminal MUC5B proteins, we showed that EGCG causes aggregation at the protein domains of MUC5B, but not at the oligosaccharide-rich regions of the mucin. We also demonstRated that EGCG caused the majority of MUC7 in human whole saliva to aggregate. Furthermore, purified MUC7 also underwent a large increase in sedimentatio
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Aggregation of MUC7 in human whole saliva by EGCG.
2014Co-Authors: Heather S. Davies, Paul D. A. Pudney, Pantelis Georgiades, Thomas A. Waigh, Nigel W. Hodson, Caroline E. Ridley, Ewan W. Blanch, David J. ThorntonAbstract:Agarose gel electrophoresis of Rate-Zonal Centrifugation fractions of human whole saliva with (A) PBS, (B) 100 µM EGCG, (C) 500 µM EGCG and (D) 1 mM EGCG in 10–50% (w/v) sucrose gradients, probed with EUMUC7a and developed using the Odyssey Imaging system.
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MUC5B in human whole saliva sediments faster in the presence of EGCG.
2014Co-Authors: Heather S. Davies, Paul D. A. Pudney, Pantelis Georgiades, Thomas A. Waigh, Nigel W. Hodson, Caroline E. Ridley, Ewan W. Blanch, David J. ThorntonAbstract:Human whole saliva was incubated with (A) PBS, (B) 0.5 mM EGCG, (C) 1 mM EGCG or (D) 4 mM EGCG and subjected to Rate-Zonal Centrifugation in 10–50% (w/v) sucrose gradients for 1 hour. MUC5B was detected by agarose gel electrophoresis and western blotting with the MAN-5BIII antiserum (insets). Bands were quantified using the Odyssey Imaging system.
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Increased sedimentation Rate of purified MUC5B in the presence of 1 mM EGCG.
2014Co-Authors: Heather S. Davies, Paul D. A. Pudney, Pantelis Georgiades, Thomas A. Waigh, Nigel W. Hodson, Caroline E. Ridley, Ewan W. Blanch, David J. ThorntonAbstract:Rate-Zonal Centrifugation of MUC5B (A: 200 µg/mL, B: 400 µg/mL, C: 800 µg/mL, D: 1.6 mg/mL) with 1 mM EGCG (grey triangles) or PBS (black circles), performed in 10–35% (w/v) sucrose gradients in PBS. MUC5B was detected by agarose gel electrophoresis and western blotting with the MAN-5BIII antiserum (insets; MUC5B (top panel) and MUC5B+EGCG (bottom panel)). Bands were quantified using the Odyssey Imaging system.
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MUC5B oligosaccharide-rich regions do not aggregate in the presence of excess EGCG.
2014Co-Authors: Heather S. Davies, Paul D. A. Pudney, Pantelis Georgiades, Thomas A. Waigh, Nigel W. Hodson, Caroline E. Ridley, Ewan W. Blanch, David J. ThorntonAbstract:MUC5B T-domains were geneRated by trypsin-digestion and made to 200 µg/mL (A) and 600 µg/mL (B) before Rate-Zonal Centrifugation with PBS (black circles) or 4 mM EGCG (grey triangles), in 5–20% (w/v) sucrose gradients. EGCG was removed from fractions using a HiTrap desalting column, fractions were slot blotted and PAS stained to detect glycoprotein (inset). Blots were scanned using Biorad ChemiDoc MP Imaging System and intensities measured using ImageLab software.
Reinhard Jahn - One of the best experts on this subject based on the ideXlab platform.
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Small-scale isolation of synaptic vesicles from mammalian brain
Nature Protocols, 2013Co-Authors: Saheeb Ahmed, Matthew Holt, Dietmar Riedel, Reinhard JahnAbstract:Synaptic vesicles (SVs) are essential organelles that participate in the release of neurotransmitters from a neuron. Biochemical analysis of purified SVs was instrumental in the identification of proteins involved in exocytotic membrane fusion and neurotransmitter uptake. Although numerous protocols have been published detailing the isolation of SVs from the brain, those that give the highest-purity vesicles often have low yields. Here we describe a protocol for the small-scale isolation of SVs from mouse and rat brain. The procedure relies on standard fractionation techniques, including differential Centrifugation, Rate-Zonal Centrifugation and size-exclusion chromatography, but it has been optimized for minimal vesicle loss while maintaining a high degree of purity. The protocol can be completed in less than 1 d and allows the recovery of ∼150 μg of vesicle protein from a single mouse brain, thus allowing vesicle isolation from transgenic mice.
Ingemar Carlstedt - One of the best experts on this subject based on the ideXlab platform.
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muc5b is a major gel forming oligomeric mucin from human salivary gland respiratory tract and endocervix identification of glycoforms and c terminal cleavage
Biochemical Journal, 1998Co-Authors: Claes Wickström, Julia R. Davies, Enno C. I. Veerman, Gitte V Eriksen, Ingemar CarlstedtAbstract:Mucins from human whole saliva, as well as from respiratory- and cervical-tract secretions, were subjected to density-gradient Centrifugation in CsCl/0.5 M guanidinium chloride. A polydisperse population of MUC5B mucins was demonstRated in all samples using anti-peptide antisera (LUM5B-2, LUM5B-3 and LUM5B-4) raised against sequences within the MUC5B mucin. The sequences recognized by the LUM5B-2 and LUM5B-3 antisera are located within the domains flanking the highly glycosylated regions of MUC5B, and reduction increased the reactivity with these antibodies, suggesting that the epitopes are partially shielded and that these regions are folded and stabilized by disulphide bonds. Rate-Zonal Centrifugation before and after reduction showed MUC5B to be a large oligomeric mucin composed of disulphide-linked subunits. In saliva and respiratory-tract secretions, populations of MUC5B mucins with different charge densities were identified by ion-exchange HPLC, suggesting the presence of MUC5B ‘glycoforms ’. In trachea, the F2 monoclonal antibody against the sulpho-Lewis C structure reacted preferentially with the later-to-be-eluted populations. An antibody (LUM5B-4) recognizing a sequence in the C-terminal domain of MUC5B identified, after reduction, the mucin subunits as well as smaller fragments, suggesting that some of the MUC5B mucins are cleaved within the C-terminal domain. Immunohistochemistry revealed that MUC5B is produced by cells dispersed throughout the human submandibular and sublingual glands, in the airway submucosal glands as well as the goblet cells, and in the epithelium and glands of the endocervix. The F2 antibody stained a subpopulation of the MUC5B-producing cells in the airway submucosal glands, suggesting that different cells may produce different glycoforms of MUC5B in this tissue.
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different mucins are produced by the surface epithelium and the submucosa in human trachea identification of muc5ac as a major mucin from the goblet cells
Biochemical Journal, 1996Co-Authors: Hans W Hovenberg, Julia R. Davies, Ingemar CarlstedtAbstract:Mucins were extracted from the epithelial surface and the submucosal tissue of human trachea in order to enrich glycoproteins from the goblet cells and the submucosal glands respectively. The macromolecules were purified using density-gradient Centrifugation, and the presence of the MUC5AC mucin was investigated using an antiserum raised against a synthetic peptide based on the sequence of the MUC5AC apoprotein. Mucins from the surface epithelium showed a higher reactivity with the antiserum relative to carbohydRate than those from the submucosa, and ion-exchange HPLC of reduced subunits revealed the presence of two distinct mucin populations in the samples. The predominant species from the surface epithelium was more acidic than the major population from the submucosa and showed a strong reactivity with the anti-MUC5AC antiserum. In contrast, the major portion of the submucosal mucins were less acidic and showed no MUC5AC reactivity, although a more acidic population did react with the antibody. Rate-Zonal Centrifugation showed that the MUC5AC mucin from the surface epithelium is smaller than the major submucosal mucin, and that both are composed of subunits. Immunolocalization confirmed that the MUC5AC mucin from human trachea originates from the goblet cells and that this glycoprotein is not a major product of the submucosal glands.
Roy E. Gondo - One of the best experts on this subject based on the ideXlab platform.
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Organellar clusters formed by mitochondrial-rough endoplasmic reticulum associations: An ordered arrangement of mitochondria in hepatocytes
Hepatology, 1995Co-Authors: Joseph Cascarano, Patricia A. Chambers, Eileen Schwartz, Parvoneh Poorkaj, Roy E. GondoAbstract:Abstract Our objective was to determine if mitochondrialrough endoplasmic reticulum (mt-RER) associations provide for an ordered arrangement of mitochondria in the cell. If such an ordered arrangement exists, it might be manifested by grouping of mitochondria according to size and biochemical properties. Liver homogenate was subjected to Rate Zonal Centrifugation for fractionating mitochondrial clusters. These clusters were then examined for morphological and biochemical characteristics. Scanning electron microscopy (SEM) showed that (1) mitochondria were held together in clusters by rough endoplasmic reticulum, (2) clusters consisted of mitochondria of comparable size, and (3) a 45-fold difference in average mitochondrial volume existed between the organelles of the fastest and slowest sedimenting clusters. Transmission electron microscopy (TEM) affirmed that all of the organellar clusters examined were mitochondria associated with rough endoplasmic reticulum. Cytochrome oxidase and mitochondrial DNA were found to be proportional to mitochondrial volume, indicating that these components were synthesized in proportion to increases in volume. Conversely, succinic dehydrogenase and ornithine carbamoyl transferase were increased disproportionately (2.9-fold and six-fold, respectively) with increase in mitochondrial volume. It is evident from this biochemical heterogeneity that clusters composed of larger mitochondria differ functionally from clusters of smaller mitochondria. The size-ordered arrangement suggests that this organization is in some way related to the biogenesis of hepatocyte mitochondria. It is also conjectured that the biochemical heterogeneity is a consequence of addition of selected proteins (e.g., succinic dehydrogenase and carbamoyl transferase) to mitochondria in a developmental process as they mature into larger organelles.
William M Shih - One of the best experts on this subject based on the ideXlab platform.
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WM: Purification of DNAorigami nanostructures by Rate-Zonal Centrifugation. Nucleic Acids Res 2013, 41:e40. Page 4 of 4 (page number not for citation purposes
2016Co-Authors: Chenxiang Lin, Steven D Perrault, Minseok Kwak, Franziska Graf, William M ShihAbstract:Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1–1mg purified DNA nanostructures, obtaining>100mg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing Rate-Zonal Centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40–80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1–100 mg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures
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Purification of DNA-origami nanostructures by Rate-Zonal Centrifugation
Nucleic Acids Research, 2013Co-Authors: Chenxiang Lin, Steven D Perrault, Minseok Kwak, Franziska Graf, William M ShihAbstract:Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1-1 µg purified DNA nanostructures, obtaining >100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing Rate-Zonal Centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40-80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1-100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.
