Biomedical subjects

W Knudson

Publications and source records attributed to W Knudson.

39 records · Page 3Linked to original sources

PubMed1984

Selective hydrolysis of chondroitin sulfates by hyaluronidase.

Chondroitin 4-sulfate and chondroitin 6-sulfate were incubated with testicular hyaluronidase in the presence of excess beta-glucuronidase. The beta-glucuronidase caused rapid removal of the nonreducing terminal beta-D-glucuronosyl residues from the oligosaccharides formed by the action of the hyaluronidase, destroying the oligosaccharide acceptors required for the transglycosylation activity of hyaluronidase and releasing free D-glucuronic acid at a rate that was equal to the rate of the hyaluronidase-catalyzed hydrolysis. When hyaluronidase was assayed at 37 degrees C in the presence of 0.05 M NaCl, 0.05 M Na2SO4, and 0.1 M sodium acetate at pH 5, chondroitin 4-sulfate was hydrolyzed at 1.5 times the rate found for chondroitin 6-sulfate. When hyaluronidase was assayed at 45 degrees C in 0.06 M sodium acetate at pH 6, chondroitin 4-sulfate was hydrolyzed at 8 times the rate observed for chondroitin 6-sulfate. Under the pH5 conditions, the chondroitin 4-sulfate was converted to a mixture of tri- and pentasaccharides, while the chondroitin 6-sulfate was converted primarily to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide. Under the pH 6 conditions, the chondroitin 4-sulfate was converted to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide, but the products from chondroitin 6-sulfate were a mixture of oligosaccharides ranging in degree of polymerization from 7 to 25 monosaccharides per oligosaccharide. End-group analyses of the products formed at pH 6 showed that both substrates were cleaved preferentially at the glycosidic bonds of the 4-sulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Stimulation of glycosaminoglycan production in murine tumors.

Three types of murine tumors, B-16 melanoma, A-10 carcinoma, and S-180 sarcoma, were shown to contain elevated glycosaminoglycan (GAG) concentrations in vivo as compared to normal muscle or subcutaneous tissue. Hyaluronate was especially concentrated in the A-10 carcinoma, which contained approximately six times more hyaluronate than subcutaneous tissue and 18 times more than muscle. In all three tumors, chondroitin sulfates, especially chondroitin-4-sulfate, were present in higher concentrations than in the normal tissues. In culture, however, all three tumor cell lines produced less than 5% as much GAG as mouse fibroblasts, when measured by incorporation of [3H] acetate or by chemical analysis. Varying the culture passage number or the medium composition, ie, glucose, serum, and insulin concentrations, had little effect on GAG synthesis by the tumor cells. The low GAG levels in the tumor cell cultures were not due to hyaluronidase activity in their media. In an attempt to mimic possible host-tumor cell interactions that could account for the elevated GAG levels in vivo, tumor cells were cocultured with fibroblasts, but no stimulation above the amount made by the tumor cells alone plus that by the fibroblasts alone was observed. Conditioned media from the tumor cells, either dialyzed or not against fresh complete medium, had no effect on fibroblast GAG synthesis. Tumor extracts, however, were found to stimulate synthesis of hyaluronate by fibroblasts. Stimulation by extracts of A-10 carcinoma was greater than and additive to that of serum. The above results strongly suggest that GAG production in these tumors is in part regulated by host-tumor interactions.

Acetates↗

PubMed1984

Interactions between human tumor cells and fibroblasts stimulate hyaluronate synthesis.

Several types of tumors contain high concentrations of hyaluronate, yet isolated tumor cells in culture often produce little glycosaminoglycan. To explore the possibility that interactions between tumor cells and host fibroblasts stimulate hyaluronate synthesis, human tumor cells were grown separately from and in coculture with normal human fibroblasts. Stimulation was observed with each of the three types of tumor cells used: LX-1 lung carcinoma, DAN pancreatic carcinoma, and TRIG melanoma. The interaction between LX-1 cells and fibroblasts was studied in detail. Under serum-free conditions, cocultures of LX-1 and fibroblasts synthesized 3-fold more hyaluronate than the sum of that produced by LX-1 and fibroblast cultures grown separately. This stimulation was linear over 72 hr and hyaluronate represented 80% of the glycosaminoglycan synthesized. Maximum stimulation occurred at a ratio of fibroblasts to LX-1 cells of 1-2:1. Quantitation of unlabeled glycosaminoglycans by HPLC analysis of disaccharides generated by digestion with chondroitin ABC and AC lyases (EC 4.2.2.4 and 4.2.2.5) demonstrated that net accumulation of hyaluronate increased 2-fold and that hyaluronate represented 80% of total chondroitinase-sensitive glycosaminoglycan produced by the cocultures. The disaccharide patterns obtained showed that accumulations of chondroitin-4- and chondroitin-6-sulfates were stimulated proportionately to that of hyaluronate in these cocultures. Similar levels of stimulation due to coculture were obtained in serum-containing and serum-free media. Stimulation was not effected by addition of LX-1-conditioned medium to fibroblast cultures or by culturing LX-1 and fibroblasts under conditions where they shared the same medium but were physically separated. Cell contact between LX-1 and fibroblasts thus appears to be necessary for the stimulation of hyaluronate synthesis.

Blood↗