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Biomedical subjects

R M Mason

Publications and source records attributed to R M Mason.

At least 127 records · Page 7Linked to original sources
PubMed1986

Evidence for rapid metabolic turnover of hyaluronate synthetase in Swarm rat chondrosarcoma chondrocytes.

Synthesis of [3H]hyaluronate from [6-3H]glucosamine was investigated in cultures of Swarm rat chondrosarcoma chondrocytes treated with various concentrations (0.1 microM-0.1 mM) of cycloheximide for various times. Concentrations greater than 1 microM inhibited protein synthesis by greater than 90%. Hyaluronate synthesis was decreased, with a t1/2 for 50% inhibition of 80-120 min, depending on the concentration of cycloheximide present. Similar experiments using [1-3H]glucose as a precursor label gave similar results. Experiments using [6-3H]glucosamine as a precursor label and 0.18 mM-puromycin to inhibit protein synthesis inhibited hyaluronate synthesis (t1/2 = 82 min) with similar kinetics to cycloheximide-induced inhibition. Cultures incubated with 3.6 microM-cycloheximide for up to 9 h and supplemented with p-nitrophenyl beta-D-xyloside during the last 75 min of treatment showed increased synthesis of [3H,35S]chondroitin sulphate, demonstrating that UDP-hexose precursors for glycosaminoglycan synthesis are not rapidly depleted on blockage of protein synthesis. Rapid metabolic turnover of hyaluronate synthetase is the most likely cause for decreased hyaluronate synthesis in chondrocytes in which protein synthesis is inhibited.

Animals↗
PubMed1986

Proteoglycan synthesis in suspension cultures of Swarm rat chondrosarcoma chondrocytes and inhibition by exogenous hyaluronate.

Conditions were established for short-term primary suspension culture of chondrocytes from the Swarm rat chondrosarcoma. Proteoglycan and hyaluronate synthesis on Day 0 to Day 2 in culture was investigated and compared with that for plated cultures. Incorporation of [35S]sulfate into proteoglycans was the same for both suspension and plated cultures. 35S-Proteoglycan synthesis decreased by about 80% between Days 0 and 1 irrespective of culture conditions. Suspension culture chondrocytes synthesized proteoglycans which were very similar to those made in plated cultures, with respect to hydrodynamic size, glycosaminoglycan, chain length, and composition. [3H]Hyaluronate synthesis accounted for 18 and 23% of the total 3H-glycosaminoglycans synthesized from [3H]glucosamine by suspension and plated cultures, respectively. Suspension culture chondrocytes responded to exogenous hyaluronate (1 mg/ml) by reducing their 35S-proteoglycan synthesis by about 50%. [3H]Hyaluronate synthesis was inhibited by 13% under these conditions. The inhibition was dependent on the concentration of exogenous hyaluronate and reached a plateau level within 2 h. Plated chondrocyte cultures showed little or no response to hyaluronate. Suspension cultures of chondrocytes were prelabeled with [3H]lysine and lysed, and a heavy membrane fraction (12,000g) was extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. A Sepharose-hyaluronate affinity gel was used to show that the extract contained hyaluronate binding 3H-labeled proteins and evidence was obtained suggesting that these came from the external face of the plasma membrane.

Animals↗
PubMed1986

Changes in mouse intervertebral-disc proteoglycan synthesis with age. Hereditary kyphoscoliosis is associated with elevated synthesis.

Mice with hereditary kyphoscoliosis (ky/ky) develop intervertebral-disc degeneration at the cervico-thoracic junction. Disc proteoglycans were investigated to determine whether changes in synthesis or structure were associated with this. Elevated 35S-proteoglycan synthesis was found in one or more cervico-thoracic discs in 80-day-old ky/ky mice. The hydrodynamic size and aggregation properties of ky/ky-mouse disc 35S-proteoglycans extracted with 4 M-guanidinium chloride were normal. Increased proportions of small 35S-proteoglycans were extracted with 0.5 M-guanidinium chloride from discs of normal and ky/ky mice with increasing age.

Age Factors↗
PubMed1985

Absence of keratan sulphate from skeletal tissues of mouse and rat.

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.

Animals↗
PubMed1984

Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen.

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.

Animals↗
PubMed1984

Intervertebral disc degeneration in adult mice with hereditary kyphoscoliosis.

Breeding experiments confirmed that a hereditary form of kyphoscoliosis in the BDL strain mouse was due to an autosomal recessive gene (ky). Sagittal sections of whole vertebral columns from adult homozygous recessive mice (ky/ky) were examined histologically. All mice showed varying degrees of degenerative change in one or more intervertebral discs between the fifth cervical and the second thoracic vertebrae. The changes comprised loss of cells, loss of distinction between nucleus pulposus and annulus fibrosus, loss of characteristic ring-like structure in the annulus, and development of wedge-shaped discs. In most animals, degenerative disc substance protruded from the disc space, usually posteriorly, sometimes anteriorly, and occasionally through the vertebral end plate cartilage. Posterior protrusions impinged on the spinal cord.

Animals↗
PubMed1983

Biosynthesis and metabolism in vivo of intervertebral-disc proteoglycans in the mouse.

The synthesis and turnover in vivo of 35S-labelled proteoglycans in mouse cervical, thoracic and lumbar intervertebral discs, and in costal cartilage, was investigated after intraperitoneal injection of [35S]sulphate. Intervertebral discs and costal cartilage synthesize similar amounts of 35S-labelled proteoglycans per microgram of DNA. Discs and cartilage all synthesize a major proteoglycan species (approx. 85%) of large hydrodynamic size and a minor species (approx. 15%) of small size. Both proteoglycans carry chondroitin sulphate chains. Keratan sulphate was not found associated with either species. The total 35S-labelled proteoglycan pool had a metabolic half-life (t1/2) of 10-12 days in discs, and 17 days in cartilage. The extractable major and minor species turned over at similar rates. Those proteoglycans left in the tissue after 29 days turn over very slowly. Approx. 50% of the major 35S-labelled proteoglycan species formed mixed aggregates with hyaluronic acid and rat chondrosarcoma proteoglycan. The ability to form aggregates did not decrease up to 45 days after synthesis. Of the heterogeneous population of proteoglycans comprising the major species, those remaining in the tissue 9 days after synthesis were of smaller average hydrodynamic size and had shorter chondroitin sulphate side chains than the average size at the time of synthesis. With increasing time after synthesis, proteoglycans were less readily extracted from the tissue by 4.0 M-guanidinium chloride than at the time of synthesis.

Animals↗
PubMed1983

Effects of serum and insulin on hyaluronate synthesis by cultures of chondrocytes from the Swarm rat chondrosarcoma.

Incorporation of [3H]glucosamine into hyaluronate synthesized by chondrocyte cultures was dependent on the concentration of foetal calf serum in the culture medium. [3H]Hyaluronate levels in cultures supplemented with 2% serum, or maintained without serum, were about 60 and 43%, respectively, of that in cultures maintained with 15% serum. Addition of insulin to cultures maintained with 15% serum had no significant effect on [3H]hyaluronate synthesis. Addition of the hormone to cultures maintained with 2% serum increased [3H]hyaluronate synthesis to levels either the same (1 ng insulin/ml), or greater than (100 ng insulin/ml) that in cultures maintained with 15% serum. The [3H]hyaluronate synthesized by the cultures was of very high molecular weight irrespective of the level of synthesis. [3H]Hyaluronate formed about 12% of the total [3H]glycosaminoglycan synthesized under all culture conditions. Synthesis of 35S, 3H-labelled proteoglycan was reduced, or increased, by the same relative amounts as [3H]hyaluronate, under the different culture conditions. Incorporation of [3H]glucosamine into hyaluronate by near confluent cultures of fibroblasts derived from the Swarm rat chondrosarcoma was reduced by 50% in cultures treated with 2% foetal calf serum compared to those maintained with 15% serum. [3H]Hyaluronate synthesis by fibroblast cultures treated with 2% serum was not stimulated by addition of insulin.

Animals↗
PubMed1983

Characterisation of collagen from normal and scoliotic human spinal ligament.

Acid-soluble and pepsin-soluble collagens have been isolated from spinal ligaments of normal and scoliotic individuals. Polyacrylamide gel electrophoresis of native and cyanogen bromide-treated collagens, and amino acid analysis, showed that the ligament collagen is almost all of the Type I variety with only trace amounts of Type III present. There was no evidence for abnormal ratios of collagen alpha-chains, or underhydroxylation of proline and lysine in the scoliotic ligament. These results indicate that collagen biochemistry is normal with respect to type, post-translational modification and cross-linking in spinal ligaments of patients with idiopathic scoliosis. Elastin and proteoglycan were only minor components of the ligaments. The nature of the non-collagenous part of the ligament is unknown, although it contains some proteins with a hydrophobic nature.

Amino Acids↗
PubMed1983

Mineral status of grazing beef cattle in the warm climate region of Florida.

An experiment was conducted to determine the mineral status of purebred Brahman beef cattle grazing Bermuda--Bahia pastures grown on sandy, well-drained soils in Central Florida. Soil, plant, blood serum and liver tissue concentrations of calcium, potassium, sodium, magnesium, manganese and cobalt were generally adequate. Sixty-nine per cent of forages and 40% of serum samples were deficient in phosphorus. Seventy-five per cent of soils, 38% of forages and 18% of liver samples were deficient in copper. Sixty-nine per cent of forage samples were low, less than 30 ppm, in iron. All samples of forage, liver and serum were deficient in selenium. Likewise, all soil and forage samples and 47 and 88% of serum and liver samples, respectively, were deficient in zinc. Mineral elements most likely deficient and needed in supplements for grazing cattle on sandy, well-drained soils in the warm climate region of Central Florida are phosphorus, copper, selenium and zinc.

Animals↗
PubMed1982

Absence of covalently linked core protein from newly synthesized hyaluronate.

1. Primary cultures of chondrocytes from the Swarm rat chondrosarcoma were labelled with either [3H]glucosamine or [14C]glucosamine, and hyaluronate synthesized by the cells was isolated from the cell layer. Parallel cultures were labelled with either [3H]serine or [3H]lysine, and identical fractions were isolated from the cell layer. Some cultures were dual-labelled. 2. In cultures labelled with [3H]serine for between 30 min and 24 h and extracted with 4.0 M-guanidine, a procedure that solubilizes predominantly extracellular macromolecules, small amounts of [3H]serine-labelled molecules were found associated with the hyaluronate fraction purified from the extract by dissociative CsCl-density-gradient centrifugation and dissociative Sepharose CL-2B chromatography. About 75% of the [3H]serine-labelled molecules in the fraction were specifically associated with hyaluronate, since they could be removed by prior treatment with proteinase-free Streptomyces hyaluronidase. The association of the [3H]serine-labelled molecules with hyaluronate was non-covalent, since they could be separated from it by further centrifugation in CsCl density gradients containing 4 M-guanidinium chloride and a zwitterionic detergent. 3. In other experiments the cultures were extracted with a sequential zwitterionic-detergent/guanidinium chloride procedure that completely solubilized the cell layer and enabled fractions containing newly synthesized cell-associated hyaluronate to be isolated. Zwitterionic detergent was present throughout. No [3H]lysine was incorporated into these fractions, irrespective of whether the cultures were pulsed concurrently with [3H]lysine and [14C]glucosamine or sequentially with [3H]lysine to prelabel the protein pool (24 h) followed by [14C]-glucosamine to label hyaluronate (1 h). 4. The results show that newly synthesized hyaluronate is not associated with covalently bound protein, and suggest that chain synthesis is initiated by a mechanism other than on to a core protein. Small amounts of [3H]serine-labelled molecules are, however, non-covalently associated with extracellular hyaluronate. Their identity is at present unknown, but they are probably of low molecular weight.

Animals↗
PubMed1982

Biosynthesis of hyaluronic acid in cultures of chondrocytes from the Swarm rat chondrosarcoma.

The synthesis of hyaluronic acid by cultured chondrocytes from the Swarm rat chondrosarcoma was examined using [3H]glucosamine as a precursor. [3H]Hyaluronate in samples was estimated as the specific unsaturated disaccharide released by incubation with chondroitinase ABC under conditions where all [3H]hyaluronate was converted to disaccharide even in the presence of excess chondroitin sulfate. The products of digestion of chondroitin sulfate and hyaluronate were well separated by cellulose thin layer chromatography. A second method involving quantitation of the 3H-oligosaccharides released by digestion with Streptomyces hyaluronidase gave nearly identical results. [3H]Hyaluronate formed about 12% of the [3H]glycosaminoglycans synthesized by the chondrocytes, although levels as high as 20% were found in one set of cultures. The incorporation of [3H]glucosamine into chondroitin sulfate and hyaluronate was linear and at a constant ratio over a 24-h period. The distributions of [35S]proteoglycans, [3H]chondroitin sulfate, and [3H]hyaluronate between the medium, a 4.0 M guanidine HCl extract of the cell layer, and the cell residue, were investigated over 24 h. Both hyaluronate and proteoglycan accumulated in the medium with little retention in the cell layer. Pulse-chase experiments indicated that a greater proportion of the [3H]hyaluronate than the [3H]chondroitin sulfate was initially retained in the cells, but that subsequently [3H]hyaluronate accumulated more rapidly in the medium, suggesting differences in the transit time through the extracellular matrix for the two molecules once secreted from the cell. In cultures labeled for 6 h, 4.0 M guanidine HCl extracted 66% of the hyaluronate and 58% of the chondroitin sulfate associated with the cell layer, while 1% Zwittergent, a zwitterionic detergent, extracted 59% and 29%, respectively. The selective extraction with detergent suggests that there is a pool of hyaluronate in the cell layer which is not associated with proteoglycan aggregates. Equilibrium density gradient centrifugation of cell layer extracts and culture media, under dissociative conditions, showed that 89% of the [3H]hyaluronate banded at densities between 1.43-1.55 g/ml with a peak at 1.47 g/ml. Disaccharide analyses of the gradient fractions showed that the main proteoglycan component contained primarily chondroitin 4-sulfate but also revealed a minor proteoglycan at lower densities which was considerably enriched in chondroitin 6-sulfate.

Animals↗
PubMed1982

The team approach in orofacial management.

Successful treatment of head and neck deformities by the plastic and maxillofacial surgeon often involves participation of the speech pathologist and orthodontist in planning therapy and helping the patient adapt to the surgery performed. Patients are presented demonstrating the communication in treatment planning and implementation recommended between surgeon, speech pathologist, and orthodontist. We describe the tools of examination used in the Division of Plastic and Maxillofacial Surgery at Duke University Medical Center.

Adolescent↗