Studies on the synthesis, secretion and assembly of proteoglycan aggregates by chondrocytes.
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Animals↗
Biomedical subjects
A Ratcliffe
Publications and source records attributed to A Ratcliffe.
Cartilage proteoglycan binding region and link protein. Radioimmunoassays and the detection of masked determinants in aggregates.
Antibodies have been raised in rabbits to the hyaluronate-binding region and link-protein components of aggregated proteoglycans from pig laryngeal cartilage. The anti-(binding region) antibodies did not bind 125I-labelled link protein, nor was 125I-labelled binding region bound by the anti-(link protein) antibodies. The antisera were applied in sensitive inhibition radioimmunoassays to determine binding region and link protein in purified proteoglycan preparations. With intact proteoglycan aggregates, the antigenic sites of link protein, and to a lesser extent binding region, were masked. Heat treatment in the presence of sodium dodecyl sulphate (0.025%, w/v) was found to overcome this masking, thereby allowing the determination of link protein and binding region in aggregated proteoglycan preparations in pure and impure samples.
Animals↗
Technical difficulties overcome in the use of Lowicryl 4KM EM embedding resin.
Several technical difficulties have been overcome in the use of Lowicryl 4KM resin. In order to embed and section tissue satisfactorily in the resin, it has been found necessary to thoroughly degass the resin before infiltration and polymerisation. After irradiation with UV light, the blocks are further polymerised by exposure to daylight for 2-3 weeks and then stored under partial vacuum over dessicant.
Acrylic Resins↗
Measurement of the binding activity of defined IgG aggregates to macrophage Fc receptors.
Small soluble IgG aggregates of defined size were prepared from pooled human IgG by gel filtration chromatography, and examined by analytical ultracentrifugation. Three such fractions, dimer-rich, trimer-rich and 25S aggregate were used to inhibit IgG monomer binding in a study of the influence of aggregation in the binding of human IgG1 to mouse macrophage Fc receptors. Of the polymers tested, IgG in the trimeric form was found to bind with the greatest avidity, being 158 times more active than monomeric IgG, whereas IgG as a larger 25S aggregate had an increased binding activity of 80 times; the avidity of IgG as dimer was increased by a factor of 2 over monomeric IgG. The possible mechanisms involved in achieving enhanced binding are discussed.
Animals↗
The localization of the binding site(s) on human IgG1 for the Fc receptors on homologous monocytes and heterologous mouse macrophages.
Two different methods, a rosette assay and a direct binding assay, have been employed in an examination of the binding of human IgG1 to mouse macrophages. In both cases, inhibition of IgG binding was demonstrated by Fc (CH2 + CH3 domains) and pFc' (CH3 domains) fragments of human IgG. In a homologous system, the binding of 125I-human IgG to human peripheral-blood monocytes was inhibited by the Fc fragment whereas the pFc' fragment was inactive. Scatchard plot analysis of the binding data from both the heterologous and homologous systems allowed association constants and numbers of receptors per cell to be calculated. A more thorough examination of the possible location of IgG Fc-receptor binding sites was made using less orthodox proteolytic cleavage fragments of IgG. The site on human IgG1 responsible for binding to mouse macrophage Fc receptors was confirmed to be within the CH3 domains. Human IgG1 binding to homologous monocytes was shown, using a dimeric C gamma 2 domain fragment, to be via the CH2 domains, and was dependent on the integrity of the covalent interaction between the C gamma 2 domains at the hinge region.
Animals↗
The use of synthetic gamma-chain peptides in the localization of the binding site(s) on human IgG1 for the Fc receptors of homologous monocytes and heterologous mouse macrophages.
In an attempt to locate precisely the sites on human IgG responsible for binding to monocytes and macrophages, synthetic peptides representative of sequences of the human gamma-chain have been used as potential inhibitors of human [125I]IgG1 binding to human monocytes and mouse macrophages. One peptide, comprising the sequence of Tyr407--Arg416 of the C gamma 3 domain, showed the same maximum inhibition of [125I]IgG binding to mouse macrophages as unlabelled IgG. Two peptides derived from sequences in the C gamma 2 domain were shown to exhibit limited inhibition of Igg binding to homologous human monocytes.
Animals↗
The sterols and carbohydrates in fungi: Boletus edulis.
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Journal Article↗
Formulation of PEG-based hydrogels affects tissue-engineered cartilage construct characteristics.
The limited supply of cartilage tissue with appropriate sizes and shapes needed for reconstruction and repair has stimulated research in the area of hydrogels as scaffolds for cartilage tissue engineering. In this study we demonstrate that poly(ethylene glycol) (PEG)-based semi-interpenetrating (sIPN) network hydrogels, made with a crosslinkable poly(ethylene glycol)-dimethacrylate (PEGDM) component and a non-crosslinkable interpenetration poly(ethylene oxide) (PEO) component, and seeded with chondrocytes support cartilage construct growth having nominal thicknesses of 6 mm and relatively uniform safranin-O stained matrix when cultured statically, unlike constructs grown with prefabricated macroporous scaffolds. Even though changing the molecular weight of the PEO from 100 to 20 kDa reduces the viscosity of the precursor polymer solution, we have demonstrated that it does not appear to affect the histological or biochemical characteristics of cartilaginous constructs. Extracellular matrix (ECM) accumulation and the spatial uniformity of the ECM deposited by the embedded chondrocytes decreased, and hydrogel compressive properties increased, as the ratio of the PEGDM:PEO in the hydrogel formulation increased (from 30:70 to 100:0 PEGDM:PEO). Total collagen and glycosaminoglycan contents per dry weight were highest using the 30:70 PEGDM:PEO formulation (24.4+/-3.5% and 7.1+/-0.9%, respectively). The highest equilibrium compressive modulus was obtained using the 100:0 PEGDM:PEO formulation (0.32+/-0.07 MPa), which is similar to the compressive modulus of native articular cartilage. These results suggest that the versatility of PEG-based sIPN hydrogels makes them an attractive scaffold for tissue engineering of cartilage.
Journal Article↗
Knee joint immobilization decreases aggrecan gene expression in the meniscus.
Aggrecan is the major proteoglycan of the meniscus, and its primary function is to give the meniscus its viscoelastic compressive properties. The objective of this study was to determine the effect of joint immobilization on aggrecan gene expression in the meniscus. The right hindlimbs of six mature beagles were knee cast-immobilized in 90 degrees of flexion and supported by a sling to prevent weightbearing, while the contralateral limb was left free to bear weight. The animals were sacrificed at 4 weeks, and the anterior and posterior halves of the medial and lateral menisci were analyzed separately. Analysis of aggrecan gene expression by quantitative polymerase chain reaction showed decreased aggrecan gene expression in menisci from immobilized knees (P < 0.01, two-way analysis of variance). Aggrecan gene expression decreased by a factor of 2 to 5.5 in the different regions examined. Analysis of the composition of the meniscus also showed decreased proteoglycan content and increased water content with immobilization (P < 0.05, two-way analysis of variance). These results show that joint immobilization can significantly affect meniscal cellular activity and composition and can therefore potentially affect meniscal function.
Aggrecans↗