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Biomedical subjects

E A Davidson

Publications and source records attributed to E A Davidson.

At least 91 records · Page 5Linked to original sources
PubMed1979

Glycoproteins and human cancer. 1. Circulating levels in cancer serum.

Total protein and sialic acid levels were determined in the supernatant of serum treated with perchloric acid. Patients with either localized or advanced metastatic malignancy have significantly elevated mean serum values. The highest levels occur in patients with lung, GI, GYN cancer, lymphoma and malignant melanoma. Patients with leukemia and multiple myeloma have slightly elevated values, but they were not significantly different from normal. Patients following curative surgery have normal values while patients in clinical remission following chemotherapy have elevated mean serum protein and NANA levels. Elevated values also occur in patients with benign tumors and 12% of patients with nonmalignant disease. Tumor cells appear to shed macromolecules which contribute to the observed elevation of serum protein and sialic acid levels.

Adult↗
PubMed1979

Glycosaminoglycans of normal and malignant cultured human mammary cells.

Glycosaminoglycans have been characterized from a normal human breast cell line (HBL-100) and two different cell lines from human breast carcinoma (MDA-MB-231 and MCF-7). The glycosaminoglycans were labeled by exposure of cell cultures to [3H]glucosamine and [35S]sulfate and then isolated from both spent media and cells by pronase digestion and cetylpyridinium chloride fractionation. They were further characterized by (a) hexosamine composition, (b) controlled-pore glass exclusion chromatography, (c) reactivity with specific enzymes (hyaluronidase chondroitinase, heparitinase, and heparinase), (d) nitrous acid degradation, and (e) DEAD-Sephadex chromatography. The results indicate that the HBL-100 line synthesizes mainly hyaluronic acid, most of which is secreted into the medium. Chondroitin sulfate and heparan sulfate are the predominant glycosaminoglycans synthesized by the cancer lines; both are found mainly in the spent medium, but the hyaluronic acid synthesized by the MDA-MB-231 line remains cell associated. The cell-associated heparan sulfate had a molecular weight in excess of 13,000 and may contain linkages susceptible to testicular hyaluronidase. The MCF-7 cells produce significantly lower amounts of glycosaminoglycans than do the other two lines.

Breast↗
PubMed1978

Isolation and characterization of glycoconjugates from B16 mouse melanoma tumors.

The major glycosaminoglycans isolated from B16 mouse melanoma tumors after Pronase digestion were shown to be a family of chondroitin 4-sulfates with different degrees of sulfation and a wide molecular-weight range. Ultracentrifugation data gave molecular weight values as high as 88 000, in contrast to that of costal cartilage chondroitin 4-sulfate which is about 14 000. A mucin-type sialoglycopeptide, isolated from the tumors by cetylpyridinium chloride precipitation of the Pronase digest, was shown to contain O-glycosylically linked tetra- and tri-saccharides consisting of sialic acid, galactose, and N-acetylgalactosamine. The sialoglycoprotein, which on Pronase digestion gave rise to the glycopeptide, was isolated from the tumor by extraction with lithium diiodosalicylate and affinity chromatography on a wheat-germ agglutinin-Sepharose 4B column. It was homogeneous on the basis of gel filtration on Sepharose 4B and Sephadex G-200 columns, lectin affinity, and ion-exchange chromatography. The compounds isolated from the B16 mouse melanoma tumors are similar to those produced by the cultured melanoma cells, which suggests that the latter compounds are not artifacts of the culture system.

Amino Acids↗
PubMed1977

Purification and properties of an endo-alpha-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae.

An enzyme that hydrolyzes the O-glycosidic linkage between alpha-N-acetyl-D-galactosamine and serine or threonine in mucins and mucin-type glycoproteins was purified by chromatography on an Affi-Gel 202 column or isoelectric focusing from filtrates of Diplococcus pneumoniae cultures. The final preparations were free of protease and a wide range of other glycosidase activities. The preparation obtained by isoelectric focusing was shown to consist of a single protein by gel filtration and sodium dodecyl sulfate-gel electrophoresis. This preparation had an apparent molecular weight of about 160,000, determined by gel filtration, an optimum pH of 7.6, and an isoelectric point in the range pH 8 to 9. The enzyme releases the disaccharide Gal-GalNAc from a variety of glycopeptide and glycoprotein substrates and appears to have a specific requirement for an unsubstituted galactose in the nonreducing terminus and an alpha linkage between N-acetylgalactosamine and the aglycone. This is the only endoenzyme known capable of cleaving the linkage between a carbohydrate and serine or threonine residues in glycoproteins. The ability of this enzyme to act on macromolecular substrates and its pH optimum makes it ideally suited to explore the distribution and function of mucin-type glycoproteins on normal and cancer cell surfaces.

Acetylglucosaminidase↗
PubMed1977

Release of O-sulfate groups under mild acid hydrolysis conditions used for estimation of N-sulfate content.

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100 degrees C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing 35S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution is necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.

Animals↗
PubMed1977

Characterization of the chondroitin sulfate produced by B16 mouse melanoma cells.

The mucopolysaccharides produced by B16 mouse melanoma cells have been isolated in milligram quantities from the spent media in which the cells were grown in the presence of 2-amino-2-deoxy-D-glucose-t and [35S]-sulfate. The mucopolysaccharides obtained by precipitation with cetylpyridinium chloride from the Pronase digest of the media were further purified by gel filtration, ion-exchange chromatography, and treatment with nucleases. The major components were identified as chondroitin-4-sulfates by identification of the hexosamine as 2-amino-2-deoxy-D-galactose, and by digestibility with hyaluronidases, chondroitinase AC, and chondro-4-sulfatase. The o.r.d. curve and i.r. spectra of these components also confirmed their similarity to chondroitin-4-sulfate from cartilage. The molecular weight of the polysaccharide chains was estimated to be in the range 90,000-120,000 by sedimentation equilibrium analysis.

Carbohydrates↗
PubMed1977

Chemical and biological properties of B16 murine melanoma cells grown in defined medium containing bovine serum albumin.

The addition of 1 percent (w/v) bovine serum albumin (BSA) to the F12 medium utilized for the growth of the B16 melanoma cells significantly stimulated the growth of this cell line. The synthesis of mucopolysaccharides and sialoglycopeptides in this medium is identical with that in Eagle's minimal essential medium with Earle's balanced salt solution supplemented with 2 mM L-glutamine, twice the recommended concentration of vitamins, nonessential amino acids, sodium pyruvate, and 10 percent (v/v) fetal calf serum. Cell volume and morphology did not change significantly, under the different growth conditions and tumorigenicity, as assayed by injection of cultured cells into syngeneic animals, was not decreased. Analysis of the BSA used indicated the presence of a sialoglycoprotein contaminant. This sialoglycoprotein contaminant was present in all lots examined and contains N-acetyl-and N-glycolylneuraminic acid, mannose, galactose, and glucosamine. The sialoglycoprotein can be removed by chromatography on acetate form anion-exchange resin at pH 4.3. F12 media containing the purified BSA plus selenite and the sodium salts of palmitic, oleic, and linoleic acids supported growth of the melanoma cells to the same extent as did the media containing unpurified BSA, indicating that the sialoglycoprotein has no role in sustaining the growth of the cells.

Cell Division↗
PubMed1976

Anionic polysaccharide production and tyrosinase activation in cultured human melanoma cells.

A human melanoma cell line established in our laboratory was characterized in terms of tyrosinase activity and anionic polysaccharide production. Tyrosinase levels were diluted during the growth phase and increased after the cell culture became confluent. The anionic polysaccharides produced included hyaluronic acid, heparitin sulfate, and a high-molecular-weight condroitin 4-sulfate. In contrast, a primary culture of human melanocytes derived from embryonic iris produced much greater amounts of hyaluronic acid, about 30-fold less heparitin sulfate, and a mixture of chondroitin 4-sulfate and dermatan sulfate. Saccharides secreted into the culture medium were generally identical to those remaining cell associated except for the melanoma heparitin sulfate, wherein the latter fraction appeared to be of lower molecular weight.

Animals↗
PubMed1975

The location of proteins labeled by the 125I-lactoperoxidase system in the NIL 8 hamster fibroblast.

NIL 8 hamster fibroblast cells were labeled by lactoperoxidase-catalyzed iodination. Their membranes were fractionated by sedimentation-rate and isopycnic zonal centrifugation. All the iodinated proteins except the very prominently labeled high molecular weight protein (greater than 200,000 daltons) were located in a fraction identified enzymically and compositionally as plasma membrane. The high molecular weight protein that was previously shown to be sensitive to virus transformation (Hynes, 1973) is concentrated in a very high density particle (rho equals 1.253-1.259) which contains mainly carbohydrate and protein and very low levels of lipid. 5'-nucleotidase was the only enzyme reproducibly demonstrated in this fraction, and electron micrographs revealed a predominantly amorphous morphology together with a few membraneous structures. The iodine label in this fraction was very sensitive to trypsinization prior to homogenization. All the available evidence indicates that this fraction is derived from the surface coat. Mitochondria, nuclei, and soluble protein were labeled to an insignificant extent. The presence of the iodinated surface proteins associated with the endoplasmic reticulum fraction is discussed in the light of these results.

Acid Phosphatase↗
PubMed1975

Mucopolysaccharides associated with nuclei of cultured mammalian cells.

Mucopolysaccharides have been isolated, fractionated, and characterized from the nuclei of cultured B16 mouse melanoma cells grown in the presence of (3-H)-glucosamine and (35-S)sulfate. Digestion of the nuclei with DNase followed by Pronase gave a mixture of complex carbohydrates from which the mucopolysaccharides were isolated by precipitation with cetylpyridinium chloride. After fractionation by differential salt extraction and chromatography on controlled pore glass bead columns, the components were identified by chemical and enzymatic methods. The major polysaccharide components were a family of high-molecular-weight chondroitin sulfates with different degrees of sulfation; a minor component has been characterized as heparan sulfate.

Animals↗
PubMed1975

The linkage region in the polypeptide of pig costal cartilage proteoglycan.

Proteoglycan from pig costal cartilage and fragments obtained by proteolytic digestion were characterized by equilibrium ultracentrifugation and amino acid analysis. The proteoglycan extractable in 4 M guanidinium chloride yielded, after proteolytic digestion with trypsin and chymotrypsin, a chondroitin sulfate peptide containing four chains of polysaccharide. The unextractable residue yielded chondroitin sulfate peptide containing only two chains. The amino acid composition indicated a fairly uniform spacing between all four chains with an average of eight amino acid residues between the serine residues involved in linkage. Following the alkaline sulfite elimination-addition reaction, free peptide was isolated and found to contain one unsubstituted serine residue for every two linked glycosidically. Glycine and glutamic acid were the only two amino acids sufficiently abundant to be part of an invariant sequence near to serine residues destined to be glycosylated. The linkage region of the polypeptide also contains some substituted serine residues which do not carry a full chondroitin sulfate chain.

Amino Acid Sequence↗
PubMed1975

The production of acidic polysaccharides by 5-bromodeoxyuridine-treated B16 mouse melanoma cells.

B16 melanoma cells were treated in culture with 5-bromo-2-deoxyuridine. The cell-associated and released proteoglycans and sialoglycopeptides were compared to those of control cultures treated with thymidine. The 5-bromo-2-deoxyuridine-treated cultures showed a marked reduction in the proportion of cell-associated proteoglycans and sialoglycopeptides, an increase in the synthesis of hyaluronic acid, the absence of high-molecular-weight chondroitin sulfate, and the presence of increased amounts of heparan sulfate in the media. In addition, the 5-bromo-2-deoxyuridine-treated cells had a higher DNA content and were larger than controls.

Animals↗