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H Muir

Publications and source records attributed to H Muir.

At least 19 recordsLinked to original sources
PubMed1992

Protein kinase C modulates parathyroid hormone- but not prostaglandin E2-mediated stimulation of cyclic AMP production via the inhibitory guanine nucleotide binding protein in UMR-106 osteosarcoma cells.

In UMR-106 osteosarcoma cells we found that PTH activated both the cAMP/protein kinase A and the Ca(2+)-dependent phosphoinositide/protein kinase C (PKC) pathways, but prostaglandin E2 (PGE2) activated only the cAMP pathway. Activation of PKC by the phorbol ester PMA had no effect on cAMP production but enhanced PTH-stimulated cAMP production by 50% or more; the effect on PGE2-induced cAMP was negligible. Inhibition of the alpha-subunit of the inhibitory guanine nucleotide binding protein (Gi) by pertussis toxin pretreatment also enhanced PTH-mediated cAMP production but had no effect on PGE2-induced cAMP production. These results suggest that although PTH-mediated adenylate cyclase activity is regulated via both the stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, only Gs regulates PGE2-mediated adenylate cyclase activity in UMR-106 cells. Costimulation with pertussis toxin and PMA did not increase PTH-stimulated cAMP production above that obtained with PMA alone. This implies a similar target of action for pertussis toxin and PMA, that is, the alpha-subunit of Gi. The alpha-subunit of Gi was found to be a substrate for in vitro PKC phosphorylation of membrane fractions from UMR-106 cells, seen as a +/- 40 kD band on SDS-PAGE. Stimulation of in situ 32P-labeled cells with either PMA or PTH also enhanced incorporation of 32P into the 40 kD band. Using the peptide antisera AS/7 and EC/2, we showed that pertussis toxin-labeled subunits of both Gi1 alpha/Gi2 alpha and Gi3 alpha could be immunoprecipitated, respectively, but immunoprecipitation of membrane proteins after in situ phosphorylation and stimulation with PMA precipitated only Gi2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗
PubMed1992

Increased release of matrix components from articular cartilage in experimental canine osteoarthritis.

The release rates of specific components of the proteoglycan aggregates (G1 domain, the chondroitin sulfate and keratan sulfate containing portion of the protein core, and link protein) of the articular cartilage of mature beagles were studied at early stages of canine experimental osteoarthritis (OA), generated by transection of the anterior cruciate ligament. Analysis of cartilage explants and synovial fluids indicates that at early stages of experimental OA, there is increased release of the proteoglycan aggregates of the articular cartilage. This involves a release from the tissue of the components of the proteoglycan that are specifically involved with aggregation together with the glycosaminoglycans of the proteoglycan. These components were detected at elevated levels in the media of explants of cartilage from the operated joint, and in the synovial fluids of the operated joints.

Animals↗
PubMed1992

Changes in proteoglycan turnover in experimental canine osteoarthritic cartilage.

The metabolism of newly-synthesised and total ("resident") proteoglycans was examined in control and osteoarthritic cartilage explants obtained from an experimental model (Pond and Nuki, 1973) of canine osteoarthritis. The following findings were obtained: (i) Non-labelled proteoglycans extracted from normal cartilage with 4 M guanidine HCl showed two bands visualised by staining with toluidine blue. The electrophoretic mobilities of proteoglycans from osteoarthritic cartilage were unchanged but the relative abundance of the slower migrating band increased with time after surgery. (ii) There were qualitative differences in the proteoglycan breakdown products released into the medium of explant cultures of osteoarthritic compared with control cartilage. This was apparent for both labelled and total unlabelled proteoglycans. (iii) There were similarities in the electrophoretic mobilities of the major labelled and non-labelled proteoglycan breakdown products suggesting that total ("resident") proteoglycans and newly-formed proteoglycans were degraded by similar mechanisms. There were however some differences in the labelled and non-labelled proteoglycans, suggesting that the mechanisms of breakdown were not identical. (iv) Immunoblotting techniques showed differences in the distribution of various glycosaminoglycans in proteoglycan breakdown products from control compared with osteoarthritic cartilage explant cultures. (v) Monoclonal antibodies 7-D-4 and 3-B-3 (which recognise unusual native chondroitin sulphate epitopes) showed greatly increased expression on proteoglycans from osteoarthritic cartilage compared with controls.

Animals↗
PubMed1990

Modulation of native chondroitin sulphate structure in tissue development and in disease.

Chondroitin sulphate proteoglycans are synthesised by different tissues and cell types, and the chondroitin sulphate chains are variably sulphated. Three monoclonal antibodies 3B3, 7D4 and 6C3 that recognise different native chondroitin sulphate epitopes have been used to investigate changes in structure during embryonic tissue development in the chick and in the response of mature canine articular cartilage during experimental osteoarthritis. Strong focal expression of the epitopes was seen during development of chick bursa, which was different for the three epitopes and which changed during 5 days of development. In embryonic chick limb, although chondroitin sulphate is present throughout the cartilage, the 3B3 epitope, which is at the non-reducing terminus of chains, was only expressed on chondroitin sulphate within one region of the sub-articular cartilage. In mature canine articular cartilage the expression of this epitope on proteoglycans was very low, but when determined 3 or 6 months after induction of experimental osteoarthritis the level was greatly increased in all joints tested (23/23). The abundance of the other two native chondroitin sulphate epitopes was also increased in this experimental disease. The results show that expression of the chondroitin sulphate epitopes detected by the monoclonal antibodies changes during cellular differentiation and development and suggests that it is closely controlled by the cells synthesising chondroitin sulphate chains.

Animals↗
PubMed1989

The influence of link protein stabilization on the viscometric properties of proteoglycan aggregate solutions.

The dynamic, steady-shear and transient shear flow properties of precisely prepared link-stable (s0 136, 66% aggregate) and link-free (s0 93, 59% aggregate) proteoglycan aggregate solutions at concentrations ranging from 10 to 50 mg/ml were determined using a cone-on-plate viscometer in a mechanical spectrometer. All proteoglycan solutions tested possessed: (1) linear viscoelastic properties - as measured by the dynamic complex modulus under small amplitude steady oscillatory conditions (1 less than or equal to omega less than or equal to 100 rad/s) - and (2) nonlinear shear-rate dependent apparent viscosities and primary normal stress difference under steady shearing conditions (0.25 less than or equal to gamma less than or equal to 250 s-1). Our transient flow data show that all proteoglycan aggregate solutions exhibited transient stress overshoot effects in shear stress and normal stress. From these steady and transient flow data, we conclude that link protein stabilized aggregates have significant effects on their dynamic and steady-shear properties as well as transient flow properties. The transient stress overshoot data provide a measure of the energy per unit volume of fluid required to overcome the proteoglycan networks in solution from a resting state. Thus we found that link-stable aggregates form much stronger networks than link-free aggregates. This is corroborated by the fact that link-stable aggregates form more elastic (lower than delta) and stiffer (higher [G*]) networks than link-free aggregates. The complete spectrum of viscometric flow data is entirely compatible with the proposed role of link protein in adding structural stability to the proteoglycan-hyaluronate bond. In cartilage, the enhanced strength of the networks formed by link-stable aggregates may play an important role in determining the material properties of the tissue and thereby contribute to the functional capacity of cartilage in diarthrodial joints.

Animals↗
PubMed1989

Adhesion of lymphoid cells to fibroblasts in tissue culture.

In this study we have examined the cellular and molecular specificity of lymphocyte interaction with fibroblasts. Using mitogen-activated T-cells, we found that attachment to fibroblasts was highly sensitive to protease treatment, and to an antibody raised against the purified lymphocyte plasma membrane, but it was not mediated by the MEL-14 surface antigen or phosphomannosyl receptors. Lymphocyte interaction with fibroblasts was also unaffected by monoclonal antibodies against the LFA-1, Mac-1, and Class II MHC antigen complexes. In contrast, adhesion of both T- and B-lymphocytes was strongly inhibited by fucoidan, a polymer of sulphated fucose, whereas fucose, mannan, and mannose 6-phosphate had no effect. Both B- and T-lymphoid cell lines were able to recognise and adhere to fibroblasts, although the marked differences between the attachment of the different types of cell did not appear to be related to their immunological function. The attachment of most of the cell lines was prevented by the presence of fucoidan, whereas the inhibition of binding of each of the lymphoid lines in the presence of the anti-T-lymphocyte plasma membrane antibody varied widely. These findings suggest that lymphocyte attachment to fibroblasts involves multiple cell surface receptors, and that these are expressed at different levels on specific T- and B-cells.

Animals↗
PubMed1989

Fibroblast matrix and surface components that mediate cell-to-cell interaction with lymphocytes.

The interaction between lymphocytes and fibroblasts in vitro has been examined using a quantitative ELISA assay to measure the binding of T and B cells to monolayer cultures of human dermal fibroblasts. This was carried out on microtiter culture plates, using an anti-Thy-1 monoclonal antibody, to determine the attachment of murine T lymphocytes and an affinity-purified polyclonal anti-IgM antibody to measure B cell binding. Both types of lymphocyte were found to adhere strongly to intact human fibroblasts, and also had high levels of attachment to purified fibroblast plasma membranes and extracts of the fibroblast extracellular matrix. Attachment, particularly of B lymphocytes, also took place onto plastic surfaces coated with fibronectin, but not to collagens or to intact fibroblasts that had been fixed with a low concentration of paraformaldehyde. Lymphocyte binding to fibroblasts was partially prevented by a monoclonal antibody against fibroblast MHC class II antigens, but not against the class I membrane complex, or by polyclonal antiserum to the cell surface mannose 6-phosphate receptor. In addition, although both lymphocyte types were able to adhere to fibro-nectin, the presence of antibody against fibronectin or the synthetic peptide Arg-Gly-Asp-Ser, had no effect on their attachment to fibroblasts. Thus, lymphocyte adhesion may occur by fibronectin, but other types of interactions with fibroblasts also appear to take place.

Animals↗
PubMed1988

Dermatan sulphate proteoglycan from human articular cartilage. Variation in its content with age and its structural comparison with a small chondroitin sulphate proteoglycan from pig laryngeal cartilage.

Low molecular mass proteoglycans (PG) were isolated from human articular cartilage and from pig laryngeal cartilage, which contained protein cores of similar size (Mr 40-44 kDa). However, the PG from human articular cartilage contained dermatan sulphate (DS) chains (50% chondroitinase AC resistant), whereas chains from pig laryngeal PG were longer and contained only chondroitin sulphate (CS). Disaccharide analysis after chondroitinase ABC digestion showed that the human DS-PG contained more 6-sulphated residues (34%) than the pig CS-PG (6%) and both contained fewer 6-sulphated residues than the corresponding high Mr aggregating CS-PGs from these tissues (86% and 20% from human and pig respectively). Cross-reaction of both proteoglycans with antibodies to bovine bone and skin DS-PG-II and human fibroblasts DS-PG suggested that the isolated proteoglycans were the humans DS-PG-II and pigs CS-PG-II homologues of the cloned and sequenced bovine proteoglycan. Polyclonal antibodies raised against the pig CS-PG-II were shown to cross-react with human DS-PG-II. SDS/polyacrylamide-gel analysis and immunoblotting of pig and human cartilage extracts showed that some free core protein was present in the tissues in addition to the intact proteoglycan. The antibodies were used in a competitive radioimmunoassay to determine the content of this low Mr proteoglycan in human cartilage extracts. Analysis of samples from 5-80 year-old humans showed highest content (approximately 4 mg/g wet wt.) in those from 15-25 year-olds and lower content (approximately 1 mg/g wet wt.) in older tissue (greater than 55 years). These changes in content may be related to the deposition and maintenance of the collagen fibre network with which this class of small proteoglycan has been shown to interact.

Age Factors↗
PubMed1988

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts.

The activity of a lysosomal enzyme, alpha-D-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of alpha-D-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.

Animals↗
PubMed1988

Hyaluronic acid in human articular cartilage. Age-related changes in content and size.

Total tissue content and molecular mass of hyaluronic acid was determined in papain digests of human articular cartilage using a sensitive radiosorbent assay [Laurent & Tengblad (1980) Anal. Biochem. 109, 386-394]. 1) Hyaluronic acid content increased from 0.5 microgram/mg wet wt. to 2.5 micrograms/mg wet wt. between the ages of 2.5 years and 86 years. 2) Hyaluronic acid chain size decreased from Mr 2.0 x 10(6) to 3.0 x 10(5) over the same age range. 3) There was no age-related change in the size of newly-synthesized hyaluronic acid, which was of very high molecular mass, in both immature and mature cartilage. The results are consistent with an age-related decrease in proteoglycan aggregate size and suggest that modification of the hyaluronic acid chain may take place in the extracellular matrix.

Adolescent↗
PubMed1988

Lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact.

We have examined the changes in the activities of the different types of alpha-D-mannosidase when fibroblasts from patients deficient in the lysosomal form of the enzyme are cultured together with normal lymphocytes. Our results show that whereas the mannosidosis cells acquired high levels of this enzyme, the activities of both the Golgi and the endoplasmic reticulum forms of alpha-D-mannosidase remained the same as in the fibroblasts cultured alone in the absence of lymphocytes. The increase in the activity of the lysosomal enzyme in the cocultured fibroblasts was not affected by the presence of mannose 6-phosphate or alpha-methyl mannoside, inhibitors of receptor- and lectin-mediated uptake of lysosomal enzymes, respectively, but it did require cell-to-cell contact. Ion-exchange HPLC and electrophoresis in polyacrylamide gradient gels showed that the acquired enzyme had the same elution profile and molecular size as the lysosomal form of the enzyme present in the lymphocytes. Immunoprecipitation studies using antibody specific for the lymphocyte type of lysosomal alpha-D-mannosidase confirmed that the increased activity in the cocultured mannosidosis cells resulted from the acquisition of the lymphocyte enzyme. Cytochemical examination revealed, however, that the transferred lymphocyte enzyme was localized in cytoplasmic organelles in the peripheral regions of the recipient fibroblasts. These results show that lymphocytes transfer only the lysosomal form of alpha-D-mannosidase during cell-to-cell contact with mannosidosis cells.

Animals↗
PubMed1988

Effects of hypervitaminosis A on the bone and mineral metabolism of the rat.

Vitamin A toxicity has been associated with alterations in mineral metabolism and may result in osteopenia, fractures, deformities, and growth arrest. The pathogenesis of the bone lesions that occur in vitamin A toxicity is, however, ill defined and was examined in the present study. The administration of pharmacological doses of vitamin A to growing male rats resulted in weakness and spontaneous fractures. Undecalcified bone histology of vitamin A toxic animals was characterized by increased bone resorption, osteoclastosis, a paucity of trabecular surfaces covered with osteoid, and lesions which appear to be pathognomonic of hypervitaminosis A. The serum calcium and magnesium levels of vitamin A-toxic animals were unremarkable, but serum phosphate levels were significantly higher than control values. Urinary hydroxyproline excretion reflected bone histology and was significantly increased in experimental rats. Circulating levels of the potent bone resorbers, PTH, 1,25-dihydroxyvitamin D, and 25-hydroxyvitamin D, were, however, comparable in vitamin A-toxic and control animals, suggesting a possible direct effect of vitamin A on bone. Subsequently, the effects of vitamin A (retinol) on in vitro collagen synthesis (incorporation of [3H]proline into collagen) and bone resorption (45Ca release from bone) were examined using a fetal rat calvarial culture. Retinol added to the culture medium for 20-24 h in concentrations ranging from 0.5-10 micrograms/ml selectively inhibited collagen synthesis in a dose-dependent fashion. Higher concentrations of retinol were toxic and resulted in a general inhibition of protein synthesis. Bone resorption was stimulated by 0.5 and 2.5 micrograms/ml retinol. We conclude that vitamin A toxicity in rats causes bone lesions, the genesis of which can be explained, at least in part, by a direct effect of the vitamin on skeletal tissue.

Alkaline Phosphatase↗
PubMed1988

Effects of tiaprofenic acid and other NSAIDs on proteoglycan metabolism in articular cartilage explants.

Using in vitro methods, we have determined the effects of four NSAIDs on the biosynthesis and turnover of proteoglycans in articular cartilage explants. Over the concentration ranges examined (5 X 10(-7) to 10(-5) mol/L for tiaprofenic acid, naproxen and indomethacin; 5 X 10(-5) to 10(-3) mol/L for aspirin), all the drugs examined, except tiaprofenic acid, reduced the rate of proteoglycan synthesis compared with control tissue. All of the compounds examined reduced the turnover rate of newly synthesised proteoglycans. The products of turnover of explants treated with the various NSAIDs did not appear to be significantly different from those produced by control tissue, as assessed by gel chromatography and electrophoresis. There were some indications, however, of quantitative differences in distribution on both these analytical systems. These results indicate that with the exception of tiaprofenic acid all the NSAIDs examined produced a general reduction in chondrocyte metabolic activity. Thus it seems unlikely that tiaprofenic acid would have a direct detrimental effect on articular cartilage during long term administration.

Animals↗
PubMed1988

Adhesion of T and B lymphocytes to fibroblasts in tissue culture.

We have examined the direct interaction of lymphocytes with human fibroblasts in vitro. The results of our study, using radiolabelled lymphocytes, show that activated T cells rapidly adhered to the fibroblasts when the two types of cell were cultured together at 37 degrees, and that the residency time of lymphocyte-fibroblast contact was approximately 30 min. Lymphoblast adhesion did not require the presence of divalent cations, but it was temperature-dependent. Although the binding of the lymphocytes was not prevented by the presence of monosaccharides or by treatment with exoglycosidases, it was highly sensitive to low concentrations of trypsin. Attachment was also inhibited partially in the presence of the sulphated fucose polysaccharide, fucoidan. These results suggest that at least two distinct receptors are involved in lymphocyte adhesion to fibroblasts. The binding of B lymphoblasts to fibroblasts was much greater than that of the activated T cells. Resting, non-stimulated, B and T lymphocytes both bound only very poorly, showing that only mature lymphocytes express high levels of functionally effective adhesion proteins. Moreover, the results of competition experiments suggest that different cell surface components mediate the interaction of T and B cells with fibroblasts.

B-Lymphocytes↗
PubMed1987

Viscoelastic properties of proteoglycan solutions with varying proportions present as aggregates.

Monomer and aggregated proteoglycans were prepared from pig laryngeal cartilage. Vascoelastic flow properties, comprising linear complex dynamic shear modulus, nonlinear steady-state shear-rate dependent viscosity, and primary normal stress difference, were measured in proteoglycan solutions containing varying proportions of aggregate (0-80%) and at different concentrations (10-50 mg/ml). Results were analyzed using the simple Oldroyd four-parameter nonlinear rate-type rheological equation. All solution properties were strongly dependent on proteoglycan concentration and on the proportion of aggregates present. Aggregation was found to have a great effect on the zero shear-rate viscosity at 50 mg/ml, which increased fivefold from 0-100% aggregate. The results showed that network formation in proteoglycan solutions increased with concentration from 10-50 mg/ml and also increased with aggregation. All proteoglycan solutions showed shear thinning, which was most marked with aggregated proteoglycan at high concentration (50 mg/ml), where the viscosity decreased tenfold from the zero shear-rate limit to the infinite shear-rate limit. The intermolecular interactions in the network were therefore increasingly disrupted by increasing shear rate, but repeated measurements showed that these were reversible changes and that testing did not induce disaggregation or degradation of proteoglycan. These rheological properties show that aggregation is likely to immobilize proteoglycan at high concentration within cartilage and to contribute to the material properties of the porous solid matrix of articular cartilage that are important for its load-bearing function.

Animals↗