Biomedical subjects

G Huey

Publications and source records attributed to G Huey.

3 recordsLinked to original sources

PubMed1990

Ascorbic acid stimulates production of glycosaminoglycans in cultured fibroblasts.

The effect of ascorbic acid on collagen synthesis is well characterized. Proteoglycans and their attached glycosaminoglycans are components of the extracellular matrix closely associated with collagen fibers. We examined whether ascorbic acid also plays a role in glycosaminoglycan production. Synthesis and deposition of glycosaminoglycans into the extracellular matrix and secretion into the media were followed in human skin fibroblasts cultured in the presence and absence of ascorbic acid. Specific glycosaminoglycans were identified and quantitated by differential enzyme digestion, ion-exchange column chromatography, and cellulose-acetate electrophoresis. No major qualitative changes in glycosaminoglycans were observed. However, quantitatively, synthesis of glycosaminoglycans increased 30 to 90%, and deposition into the extracellular matrix increased 80% in the presence of ascorbic acid. This effect was only in part secondary to decreased levels of collagen, and the diminished capacity of underhydroxylated collagen to bind proteoglycans. The effect of ascorbic acid on extracellular macromolecules is thus more pervasive than previously assumed.

Ascorbic Acid↗

PubMed1990

Hyaluronic acid determinations: optimizing assay parameters.

Assay conditions for determining hyaluronic acid levels in cultured cells have been examined. In cultures labeled with [3H]glucosamine, hyaluronic acid is measured by digestion with a highly specific hyaluronidase from Streptomyces hyaluronlyticus. Products obtained in the presence and absence of preliminary enzyme digestion are precipitated with cetylpyridinium chloride. The precipitation step has been optimized for ion concentration, glycosaminoglycan carrier and for cetylpyridinium chloride levels. Chondroitin sulfate is an effective carrier in the precipitation of radiolabeled product, while unlabeled hyaluronic acid is not. Addition of sulfate to the mixture yields a flocculent precipitate that facilitates subsequent steps of the determination. Optimizing these steps in hyaluronic acid determination can generate two- to three-fold increases in apparent levels of deposition in cultured cells.

Animals↗

PubMed1990

Levels of [3H]glucosamine incorporation into hyaluronic acid by fibroblasts is modulated by culture conditions.

Tissue culture conditions can modulate apparent levels of incorporation of the radiolabeled precursor [3H]glucosamine into hyaluronic acid in cells. A careful study was made on the effects of culture conditions on human skin fibroblasts. A newly described technique to measure hyaluronic acid was utilized based on incorporation of [3H]glucosamine into cetylpyridinium chloride-precipitable hyaluronidase-digestible material. The precipitate was collected on glass fiber filters using a manifold suction apparatus. A six-fold greater level of incorporation occurred in rapidly growing preconfluent than in confluent fibroblasts. Ascorbic acid stimulated incorporation with a maximum at 25 micrograms/ml. The same ascorbic acid optimum was observed for collagen prolylhydroxylation. When beta-hydroxybutyrate was used as an energy source instead of D-glucose, a 3.5-fold increase in levels was observed. All tissue-culture media examined supported comparable levels of incorporation, except for Roswell Park Memorial Institute Media-1640, in which cells had only half the level. Fetal calf serum supported high levels of incorporation in a dose-dependent manner, while newborn calf and calf sera supported much lower levels of incorporation. Under serum-free conditions, lactalbumin hydrolysate was best able to support incorporation of hyaluronic acid. In the search for mechanisms that modulate hyaluronic acid, it is critical to consider the tissue culture conditions under which incorporation of radiolabeled precursors are being examined.

3-Hydroxybutyric Acid↗