X-ray fibre diffraction of cartilage proteoglycan aggregates containing hyaluronic acid.
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ABSTRACT:
SUBMITTER: Atkins ED
PROVIDER: S-EPMC1168199 | biostudies-other | 1974 Sep
REPOSITORIES: biostudies-other
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Project description:1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.
| S-EPMC1166321 | biostudies-other
Project description:Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.
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Project description:Oriented films of the sodium salt of heparin crystallize in a triclinic unit cell. The X-ray diffraction results favour a tetrasaccharide covalent repeating sequence but the measured molecular repeat of 1.59nm is inconsistent with all alpha-(1-->4)(1ax-->4eq)-glycosidic linkages as generally accepted. Three models are proposed for the molecular shape of heparin, which depend on the chair conformation adopted by the hexuronic acid moieties. Model I has both uronic acids in the C1 chair form, model II has one uronic acid in each of the C1 and 1C chairs and model III has both in the 1C chair form. The merits of these models are discussed.
| S-EPMC1165889 | biostudies-other
Project description:Hyaluronic acid (HA) is an extracellular matrix molecule with multiple physical and biological functions found in many tissues, including cartilage. HA has been incorporated in a number of biomaterial and scaffold systems. However, HA in the material may be difficult to control if it is not chemically modified and chemical modification of HA may negatively impact biological function. In this study, we developed a poly(ethylene glycol) hydrogel with noncovalent HA-binding capabilities and evaluated its ability to support cartilage formation in vitro and in an articular defect model. Chondrogenic differentiation of mesenchymal stem cells encapsulated in the HA-interactive scaffolds containing various amounts of exogenous HA was evaluated. The HA-binding hydrogel without exogenous HA produced the best cartilage as determined by biochemical content (glysocaminoglycan and collagen), histology (Safranin O and type II collagen staining), and gene expression analysis for aggrecan, type I collagen, type II collagen, and sox-9. This HA-binding formulation was then translated to an osteochondral defect model in the rat knee. After 6 weeks, histological analysis demonstrated improved cartilage tissue production in defects treated with the HA-interactive hydrogel compared to noninteractive control scaffolds and untreated defects. In addition to the tissue repair in the defect space, the Safranin O staining in cartilage tissue surrounding the defect was greater in treatment groups where the HA-binding scaffold was applied. In sum, incorporation of a noncovalent HA-binding functionality into biomaterials provides an ability to interact with local or exogenous HA, which can then impact tissue remodeling and ultimately new tissue production.
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Project description:The suitability of point focus X-ray beam and area detector techniques for the determination of the uniaxial symmetry axis (fibre texture) of the natural mineral satin spar is demonstrated. Among the various diffraction techniques used in this report, including powder diffraction, 2D pole figures, rocking curves looped on φ and 2D X-ray diffraction, a single simple symmetric 2D scan collecting the reciprocal plane perpendicular to the apparent fibre axis provided sufficient information to determine the crystallographic orientation of the fibre axis. A geometrical explanation of the 'wing' feature formed by diffraction spots from the fibre-textured satin spar in 2D scans is provided. The technique of wide-range reciprocal space mapping restores the 'wing' featured diffraction spots on the 2D detector back to reciprocal space layers, revealing the nature of the fibre-textured samples.
| S-EPMC11001398 | biostudies-literature
Project description:The aim of this study is to identify gene expression profiles associated with hyaluronic acid (HA) treatment of normal and osteoarthritis (OA)-like tissue-engineered cartilage. 3D cartilage micromasses were treated with tumour necrosis factor-α (TNF-α) (OA-inducer) and/or HA for 7 days. Viability was examined by PI/FDA staining. To document extracellular matrix (ECM) formation, glycosaminoglycans (GAG) were stained with Safranin-O and cartilage-specific type II collagen was detected immunohistochemically. Genome-wide gene expression was determined using microarray analysis. Normal and OA-like micromasses remained vital and showed a spherical morphology and homogenous cell distribution regardless of the treatment. There was no distinct difference in immunolabeling for type II collagen. Safranin-O staining demonstrated a typical depletion of GAG in TNF-α-treated micromasses (-73%), although the extent was limited in the presence of HA (-39%). The microarray data showed that HA can influence the cartilage metabolism via upregulation of TIMP3 in OA-like condition. The upregulation of VEGFA and ANKRD37 genes implies a supportive role of HA in cartilage maturation and survival. The results of this study validate the feasibility of the in vitro OA model for the investigation of HA. On the cellular level, no inhibiting or activating effect of HA was shown. Microarray data demonstrated a minor impact of HA on gene expression level.
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Project description:1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.
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Project description:An electric field causes partial alignment of macromolecules in a dilute solution. The accompanying changes in the solution birefringence offer a sensitive and quick means of monitoring the rates of particle orientation and hence the size of the solute molecules. Such measurements are reported for dilute solutions of proteoglycans in the absence and presence of added hyaluronic acid. The proteoglycan molecules are shown to be some 580 nm long. In the presence of hyaluronic acid they form aggregates that appear to be consistent with the model previously proposed in which the proteoglycans attach radially to the extended hyaluronic acid chain. The electric-birefringence relaxation rates indicate aggregates of similar length to that of the extended hyaluronic acid chain, with the proteoglycans spaced on average at 29nm intervals. A proteoglycan sample the cystine residues of which had been reduced and alkylated showed no evidence of aggregation with hyaluronic acid up to the concentrations of the acid corresponding to 1% of the total uronic acid content. The electric-birefringence method is shown to have a large potential in the study of associating polysaccharide solutions.
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Project description:Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.
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Project description:Crystal structure solutions from thin films are often performed by grazing-incidence X-ray diffraction (GIXD) experiments. In particular, on isotropic substrates the thin film crystallites grow in a fibre texture showing a well defined crystallographic plane oriented parallel to the substrate surface with random in-plane order of the microcrystallites forming the film. In the present work, analytical mathematical expressions are derived for indexing experimental diffraction patterns, a highly challenging task which hitherto mainly relied on trial-and-error approaches. The six lattice constants a, b, c, α, β and γ of the crystallographic unit cell are thereby determined, as well as the rotation parameters due to the unknown preferred orientation of the crystals with respect to the substrate surface. The mathematical analysis exploits a combination of GIXD data and information acquired by the specular X-ray diffraction. The presence of a sole specular diffraction peak series reveals fibre-textured growth with a crystallographic plane parallel to the substrate, which allows establishment of the Miller indices u, v and w as the rotation parameters. Mathematical expressions are derived which reduce the system of unknown parameters from the three- to the two-dimensional space. Thus, in the first part of the indexing routine, the integers u and v as well as the Laue indices h and k of the experimentally observed diffraction peaks are assigned by systematically varying the integer variables, and by calculating the three lattice parameters a, b and γ. Because of the symmetry of the derived equations, determining the missing parameters then becomes feasible: (i) w of the surface parallel plane, (ii) the Laue indices l of the diffraction peak and (iii) analogously the lattice constants c, α and ß. In a subsequent step, the reduced unit-cell geometry can be identified. Finally, the methodology is demonstrated by application to an example, indexing the diffraction pattern of a thin film of the organic semiconductor pentacenequinone grown on the (0001) surface of highly oriented pyrolytic graphite. The preferred orientation of the crystallites, the lattice constants of the triclinic unit cell and finally, by molecular modelling, the full crystal structure solution of the as-yet-unknown polymorph of pentacenequinone are determined.
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