Barry N. Preston

The extracellular processing and catabolism of hyaluronan in cultured adult articular cartilage explants

Hyaluronan was shown to have the same turnover time as aggrecan in explant cultures of adult bovine articular cartilage. Inclusion of fetal calf serum in the culture medium resulted in a similar decrease in the rate of catabolism of both hyaluronan and proteoglycan. Less than 9% of the hyaluronan lost from the explants in the course of the experiment was recovered from the culture medium as hyaluronan, suggesting that the catabolism of hyaluronan involves the uptake of this glycosaminoglycan by the chondrocytes. Analysis of the molecular size of the newly synthesized hyaluronan in these cultures showed that the hyaluronan was initially synthesized as large macromolecules that were gradually depolymerized with time within the extracellular matrix. The resulting size distribution of newly synthesized hyaluronan molecules after 12 days in culture was similar to that determined for the endogenous hyaluronan. The kinetics of depolymerization of the newly synthesized hyaluronan was consistent with a random fragmentation of the macromolecule. The rate constants for the depolymerization of hyaluronan indicate that oxygen-derived radicals may be involved in the fragmentation of this macromolecule. Inclusion of either cycloheximide or proteinase inhibitors in the medium of the explant cultures resulted in a marked decrease in the rate of loss of hyaluronan from the tissue and in the inhibition of the depolymerization of the newly synthesized macromolecule. This suggests that both the catabolism and the depolymerization of hyaluronan are cell mediated and depend on metabolically active cells.

Some physical properties of bovine adrenal medullary dopamine -hydroxylase.

(1) Dopamine, β‐hydroxylase (EC 1.14.2.1) was purified from bovine adrenal medullae according to the method of Foldes, Jeffrey, Preston and Austin (1972).

Experimental measurements of polymer unidirectional fluxes in polymer + solvent systems with non-zero chemical-potential gradients

The diffusion properties of two polymers in saline, namely dextran and albumin, have been followed by measurements of the unidirectional fluxes of their tracer (labelled) counterparts. The relationships between the diffusion coefficients obtained by measurement of the forward and back unidirectional fluxes to the polymer mutual-diffusion and intradiffusion coefficients have been analysed for semi-dilute polymer concentrations.

Diffusion of dextran at intermediate concentrations

The diffusion properties of dextran molecules in water have been followed by use of boundary relaxation techniques (refractive index and tracer measurements) and by photon correlation spectroscopy. Measurements have been carried out up to concentrations of ca. 250 kg m–3. The diffusion coefficients and their inter-relationships have been interpreted in terms of the non-ideal behaviour of flexible polymers.

The measurement of negative charge content in cartilage using a colloid titration technique

A colloid titration technique has been used to determine the sulfate and carboxylate content of various glycosaminoglycans and has been validated by comparing the results with data obtained using well-established techniques. The method has been applied to the measurement of the negative charge content of cartilage slices at various depths from the articular surface and to the determination of sulfate and carboxylate contents in bovine nasal septa. Titrations of nasal septa were performed on milled cartilage, on cartilage digested with papain and on proteoglycans purified by cesium chloride gradient centrifugation of guanidinium chloride extracts. The sulfate content was similar for all three preparations (0.5 mu eq per milligram dry cartilage). However, the carboxylate content determined on milled cartilage was 40% higher than that obtained for cartilage digested with papain or for purified proteoglycans; this implies the possible contribution of carboxyl groups from structural glycoproteins present in the extracellular matrix. The carboxylate content determined on purified proteoglycans was in excellent agreement with values calculated from chemical analyses.

Evaluation of nonideality from gel chromatographic partition coefficients: A technique with greater versatility than equilibrium dialysis

Frontal gel chromatography has been used to measure partition coefficients which enable a quantitative evaluation of the thermodynamic nonideality of small solutes generated by the presence of high concentrations of macromolecular solutes. Equivalence of results obtained by the present method and by equilibrium dialysis is demonstrated in a comparison of results for dextran sulfate-NaCl and dextran-sorbitol systems. Interaction coefficients obtained for dextran-sorbitol and protein-polyethylene glycol 4000 systems yields results which are in reasonable agreement with those predicted on the statistical-mechanical basis of excluded volume. Because of its greater versatility in regard to the range of systems that may be studied, the frontal gel chromatographic procedure is likely to be of particular value for the quantitative characterization of thermodynamic nonideality arising from excluded volume effects in concentrated mixtures of macromolecular solutes.

Model connective tissue systems: Measurement of ion flux across gel membranes containing proteoglycan

Abstract The undirectional fluxes of 22 Na, 45 Ca, and 36 C1 across gelatin-agarose membranes containing sulfated proteoglycan under conditions of finite salt gradients, have been measured. The results can be described by use of the integrated Nernst-Plank equations for a membrane carrying a net negative charge. The data are discussed in terms of molecular interactions between the mobile ion and the proteoglycan immobilized within the membrane. A quantitative interpretation of the interactions is made based upon polyelectrolyte theory and expressed in terms of a nonideality parameter of the mobile ions, γ p . The agreement observed between the values of γ p obtained from either influx or outflux measurements demonstrates the internal consistency of the theoretical approach. The application of our gel system as a model for connective tissues is discussed.

Temporal changes in charge content of cultured chondrocytes from bovine cartilaginous tissues.

The effective charge content of the pericellular matrix of chondrocytes has been determined while the matrix is being synthesized by cells grown in culture for several weeks. The data were compared with estimates determined by chemical analysis. When measurements were performed after digestion of the matrix with papain, there was close agreement between results obtained from both techniques for proteoglycans synthesized by chondrocytes from nasal septum (a non-articular cartilage). By contrast, no such agreement was observed for proteoglycans synthesized by chondrocytes from articular cartilage, even after solubilization of the matrix with papain. While the charge calculated from chemical analysis showed a constant increase with time in culture, that measured by colloid titration showed a cyclical pattern, with maximal values occurring on days 7 and 24 of culture and a minimal value on day 14. This inability to detect all negative groups present in the matrix synthesized by articular chondrocytes would suggest the involvement of these groups in electrostatic interactions. Partial characterization of proteins synthesized by the pericellular matrix indicates that the decrease in charge content observed on day 14 could not be attributed to proteins of a particular molecular mass but possibly to an increase in the total amount of protein present. It is concluded that the marked difference in the availability of negative groups between chondrocytes cultured from articular and non-articular cartilages may reflect differences in the interaction of these negative groups with matrix components; these differences would lead to the distinct structural organization of these two cartilaginous tissues which possess different mechanical functions.

CHARACTERISATION OF THE TUMOUR-ASSOCIATED CARBOHYDRATE EPITOPE RECOGNISED BY MONOCLONAL ANTIBODY 4D3

The tumour‐associated epitope recognised by monoclonal antibody (MAb) 4D3 is expressed on a high m.w. mucin glycoprotein preparation known as small intestinal mucin antigen (SIMA). This epitope is detected in tissue from a high proportion of patients with colorectal cancer, and elevated levels occur in serum from a significant number of such patients, highlighting the potential clinical utility of MAb 4D3. In the present study, insight into the composition and structure of the carbohydrate epitope recognised by MAb 4D3 was gained following characterisation of 2 glycopeptides that co‐purified with SIMA. Sequence analysis of 1 of these glycopeptides revealed that it was identical to the glycoprotein α‐1‐anti‐chymotrypsin. This glycoprotein was subsequently deglycosylated to yield 5 forms corresponding to α‐1‐anti‐chymotrypsin substituted with 4, 3, 2, 1 or no branched glycans. MAb 4D3 was reactive with each of the glycosylated forms, including the form carrying only 1 branched glycan, but did not react with fully deglycosylated α‐1‐anti‐chymotrypsin. MAb 4D3 also reacted to different extents with ovine, bovine or porcine submaxillary mucins, each of which has a different amount of the O‐linked sialylated disaccharide known as sialosyl Tn. Of these mucins, MAb 4D3 was most reactive with ovine submaxillary mucin, in which almost all of the carbohydrate chains are sialosyl Tn. Reactivity of MAb 4D3 towards isolated glycans, sialosyl Tn and related structures led to the conclusion that the preferred MAb 4D3 epitope involves the sialylated N‐acetyl galactosamine disaccharide as well as an additional monosaccharide present on a neighbouring carbohydrate chain. Although the preferred epitope recognised by MAb 4D3 involves this sialylated disaccharide, the specificity of MAb 4D3 was different from that of other MAbs with a reported specificity for sialosyl Tn.

A versatile shear cell for diffusion measurements on small sample volumes allowing analytical recording of multicomponent transport: II. Applications

Abstract A diffusion cell described in the preceding paper has been used to determine mutual and intradiffusion coefficients for various compounds in the range 2–2000 × 10−12 m2 s−1. The results are in good agreement with previously reported values. The standard deviation in a series of 13 runs on serum albumin performed in four different instruments was 6%.