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J D Esko

Publications and source records attributed to J D Esko.

86 records · Page 5Linked to original sources
PubMed1987

Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose.

Degradation of connective-tissue polysaccharides with bacterial or fungal eliminases and subsequent characterization of the reaction products are now part of standard methodology for the analysis of these compounds. However, the scope of preparative and analytical work based on the use of eliminases has been limited by the lack of procedures for specific removal of the unsaturated uronic acid residues generated in the eliminase reactions. In the present investigation, we have shown that these residues are cleaved by mercuric salts under mild conditions that are not likely to affect other structures in an oligo- or poly-saccharide molecule. Thus the disaccharide generated from hyaluronic acid by digestion with chondroitinase AC or ABC was cleaved into a keto acid and free N-acetylglucosamine within 10 min at room temperature upon exposure to 14 mM-mercuric acetate at pH 5. The reaction of the disaccharide with mercuric salts was used for ready determination of the distribution of radioactivity between the glucuronic acid and N-acetylglucosamine moieties in radioactive hyaluronic acid that had been synthesized by IMR-90 fibroblasts from 3H-labelled monosaccharides. When the precursor was [3H]galactose, over 95% of the incorporated radioactivity was found in the glucuronic acid moiety. In contrast, cells grown in the presence of [3H]glucosamine synthesized a polysaccharide in which almost all of the label was located in the N-acetylglucosamine units. It is apparent from these experiments that the reaction of unsaturated uronic acid residues with mercuric salts provides a new tool with potential for many applications in the study of the structure and metabolism of connective-tissue polysaccharides.

Cell Line↗
PubMed1986

Sulfate transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine.

We isolated 59 Chinese hamster ovary cell mutants defective in 35SO4 incorporation into glycosaminoglycans. Thirty-five mutants incorporated [6-3H]glucosamine into glycosaminoglycans normally, suggesting that they were specifically impaired in sulfate incorporation. Cell hybridization studies revealed that the 35 mutants defined a unique complementation group. Pulse-labeling one of the mutants with 35SO4 showed that it possessed a defect in a saturable, 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid-sensitive transport system required for sulfate uptake. Despite the dramatic reduction in 35SO4 incorporation, the mutant synthesized sulfated heparan and chondroitin chains. Incubation of the mutant with [35S]cysteine resulted in the formation of 35SO4, which was subsequently incorporated into the glycosaminoglycans. Similar results were obtained when wild-type cells were incubated in sulfate-free growth medium containing [35S]cysteine, and isotope dilution analysis indicated that about 15 microM of sulfate was derived from cysteine catabolism. We also found that the sulfate transport deficiency rendered the mutant resistant to 5 microM sodium chromate, whereas wild-type cells did not divide under these conditions. However, the mutant also did not proliferate in medium containing 5 microM chromate when grown in the presence of wild-type cells, suggesting that chromate was transported through cell-cell contacts. Since co-cultivating sulfate transport-deficient mutants with mutants defective in xylosyltransferase or galactosyltransferase I partially restored 35SO4 incorporation into glycosaminoglycans, intercellular sulfate transport occurred as well. Therefore, the availability of sulfate for glycosaminoglycan synthesis depends on sulfate uptake, turnover of sulfur-containing amino acids, and sulfate transport between cells.

Animals↗
PubMed1985

Animal cell mutants defective in glycosaminoglycan biosynthesis.

We have obtained Chinese hamster ovary cell mutants defective in the biosynthesis of glycosaminoglycans by screening replicate colonies immobilized on polyester cloth. Depending upon the strain, the mutants accumulated less 35S-labeled glycosaminoglycans per microgram of cell protein by a factor of 6-60 compared to the wild type. Some of the mutants incorporated [6-3H]glucosamine into glycosaminoglycans to the same extent as the wild type, suggesting that sulfate addition was specifically altered. In contrast, five strains failed to generate 3H-labeled glycosaminoglycans normally. In four of these, the initiation of glycosaminoglycan assembly was specifically altered, since the addition of p-nitrophenyl-beta-xyloside restored sulfation to normal. Enzymatic assay of the xylosyltransferase in extracts prepared from these mutants revealed that one of the strains, S745, contained less enzyme activity by a factor of 15 than the wild type. This mutant provides genetic evidence that the xylosyltransferase assayed in vitro is responsible for the initiation of chondroitin sulfate and heparan sulfate biosynthesis in vivo.

Animals↗
PubMed1983

Biosynthesis of phosphatidylcholine from serum phospholipids in Chinese hamster ovary cells deprived of choline.

We report that Chinese hamster ovary cells assemble membrane phospholipids from choline-linked lipid present in fetal calf serum. This was examined by testing the ability of various serum preparations to satisfy the choline requirement of the cells. Chinese hamster ovary cells divided in growth medium containing lipoprotein-deficient serum and approximately 8 microM lysolecithin. Identical results were obtained in growth medium supplemented with solvent-extracted (delipidated) serum reconstituted with purified egg lysolecithin and the uptake of lipid was inhibited by the addition of bovine serum albumin. Analysis of the phospholipid composition of cells incubated with 32Pi and egg lysolecithin in place of choline revealed that approximately 30% of the phosphorus moieties of the cellular phospholipids were derived from the added lipid, while in the presence of choline less than 10% arose in this fashion. Choline starvation enhanced the formation of lecithin from [32P]lysolecithin without affecting phospholipid turnover and labeled lecithin was converted to other phospholipids, especially sphingomyelin and phosphatidylserine. Unlike endogenous serum lysolecithin, lipoproteins obtained from human and fetal calf sera failed to satisfy the choline requirement of Chinese hamster ovary cells, even though 95% of the lipoprotein phospholipid was phosphatidylcholine and sphingomyelin. Together, these results demonstrate that animal cells can derive all of the choline required for membrane phospholipid synthesis from serum lysolecithin and that its conversion to lecithin within the cell is regulated by the availability of choline. In contrast, serum lipoproteins do not normally serve as a major source of choline moieties.

Animals↗
PubMed1982

Somatic cell cloning in polyester stacks.

Single somatic cells, including fibroblasts, myelomas, and hybridomas, proliferate normally when trapped between a plastic dish and a disc of polyester cloth. Contact between the overlay and the plastic for 8-16 days results in identical colony patterns on the cloth and the plate. When several cloth discs are simultaneously stacked over Chinese hamster ovary cells, three or four-high resolution colony copies can be generated from a single master dish. The colonies on the cloth can be analyzed by radiochemical methods [Esko, J. D. & Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 1190-1193] or by "replica plating" to a new disc. The use of polyester cloth, singly or in stacks, has several major advantages over previous techniques for somatic cell replica plating, including: (i) broad applicability to diverse cell lines such as fragile membrane mutants of Chinese hamster ovary cells and relatively nonadherent myelomas or hybridomas; (ii) the possibility of generating multiple copies of the same colony population, allowing simultaneous analysis for several enzymes or cellular components; and (iii) superior resolution and transfer efficiency in copying colony patterns from one surface to another. The remarkable capacity of animal cell colonies to proliferate upward through "polyester stacks" may reflect chemotropic movement of individual cells and opens new approaches to somatic cell genetics.

Animals↗
PubMed1982

Animal cells dependent on exogenous phosphatidylcholine for membrane biogenesis.

A Chinese hamster ovary cell (CHO) mutant (strain 58), defective in CDP-choline synthetase (cholinephosphate cytidylyltransferase; CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15), is temperature sensitive for growth and contains less than half of the normal amount of phosphatidylcholine under nonpermissive conditions [Esko, J. D. & Raetz, C. R. H. (1980) Proc. Natl. Acad. Sci. USA 77, 5192-5196]. We now report that the addition of 40 microM egg phosphatidylcholine or lysophosphatidylcholine to the medium suppresses the temperature sensitivity of mutant 58 and permits the growth of colonies at the restrictive temperature. Phospholipids with different polar headgroups, lipoprotein-bound phospholipids, sphingomyelin, and glycerophosphocholine do not support prolonged growth at 40 degrees C, whereas phosphatidylcholine analogs such as phosphatidyldimethylethanolamine, D-phosphatidylcholine, and beta-phosphatidylcholine are quite effective. A broad range of saturated phosphatidylcholines, especially those with fatty acids 12-18 carbons in length, suppresses the phenotype. Phospholipids containing ether-linked hydrocarbons are ineffective, whereas polyunsaturated phosphatidylcholines are toxic. Residual endogenous synthesis of phosphatidylcholine by the mutant is not stimulated under conditions of phenotypic bypass, but the uptake of exogenous lipid is enhanced considerably compared to the wild type. Our findings demonstrate that exogenous phospholipid can provide at least 50% of the phosphatidylcholine required for membrane biogenesis in animal cells and that uptake of exogenous phospholipids may be regulated.

Animals↗
PubMed1980

Autoradiographic detection of animal cell membrane mutants altered in phosphatidylcholine synthesis.

We have screened approximately 20,000 colonies of Chinese hamster ovary cells immobilized on filter paper [Esko, J.D. & Raetz, C.R.H. (1978) Proc Natl. Acad. Sci. USA 75, 1190-1193] for strains unable to incorporate [methyl-14C]-choline into trichloroacetic acid-precipitable phospholipid at 40 degrees C. Mutant 58, identified in this way, was specifically defective in choline incorporation, and other isolates were also blocked in thymidine and leucine incorporation into DNA and protein, respectively. Further analysis of mutant 58 revealed that the strain grew almost normally at 33 degrees C, the permissive temperature, but divided only once at 40 degrees C, the restrictive temperature. After a 20-hr incubation at 40 degrees C, the phosphatidyl-choline level dropped from 41% to 20% in the mutant whereas other phospholipids, including sphingomyelin, continued to accumulate. Wild-type cells contained approximately 50% phosphatidylcholine at both temperatures. Anion-exchange chromatography of the water-soluble choline metabolites extracted from mutant 58 revealed that phosphorylcholine accumulation increased from 6 nmol/mg of protein at 33 degrees C to 42 nmol/mg of protein at 40 degrees C whereas CDP-choline decreased from 0.42 nmol to less than 0.07 nmol per mg of protein. Phosphorylcholine also increased in wild-type cells shifted from 33 degrees C to 40 degrees C (from 1.8 nmol to 16 nmol per mg of protein), but the level of CDP-choline was not altered (from 0.52 nmol to 0.58 nmol per mg of protein). Enzymatic assays of extracts prepared from mutant and wild-type cells revealed a reduction of CTP: phosphorylcholine cytidylyltransferase (EC 2.7.7.15) activity (CDP-choline synthetase) in the mutant to 1/40th that in the wild type, and mixing experiments excluded the production of antagonists to CDP-choline synthesis in the mutant. Thus, the inability of the mutant to generate normal amounts of phosphatidylcholine in vivo was correlated with an enzymatic lesion in the biosynthesis of CDP-choline in vitro.

Animals↗
PubMed1978

Replica plating and in situ enzymatic assay of animal cell colonies established on filter paper.

We have developed a simple technique for the replica plating of Chinese hamster ovary (CHO) cells. In this procedure cells are allowed to divide for 8-16 days between the plastic surface of a petri dish and a disc of Whatman no. 50 filter paper, weighted down with glass beads. The culture medium can be replaced when necessary without disturbing the growing colonies. Cells from each developing colony grow into the fibers of the paper, while others remain attached to the plate. The cell colonies transferred to the paper are viable and can be replica plated to a new petri dish with high resolution. In this way several inositol auxotrophs have been identified in a stock of mutagen-treated cells without prior enrichment. Alternatively, the cells on the paper can be rendered permeable in situ, which permits autoradiographic screening for specific biochemical defects, as reported previously for Escherichia coli [Raetz, C. R.H. (1975 Proc. Natl. Acad. Sci. USA 72, 2274-2278]. This technique is applicable to other common cell lines and is especially useful for the identification of single colonies defective in the synthesis of DNA, RNA, protein, and membrane lipids.

Cell Line↗
PubMed1977

Use of a fluorescent probe to determine the viscosity of LM cell membranes with altered phospholipid compositions.

The phospholipid compostition of LM cells grown in tissue culture was altered by substituting ethanolamine for choline in the growth medium. The plasma membrane isolated from cells grown in medium conatining ethanolamine for 83 h had a sixfold increase in the ratio of phosphatidylethanolamine to phosphatidylcholine, the two major phospholipid classes. This was accompanied by small changes in other lipid components of the membrane. There was also a sixfold increase in the amount of triacylglycerols and alkyldiacylglycerols which were not associated with the membrane fraction of the cell. No significant changes occurred in the lipid composition of cells during growth in choline containing medium. The viscosity of plasma membranes was studied in whole cells and isolated membranes using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Plasma membranes isolated from ethanolamine-supplemented cells had greater viscosities than membranes isolated from choline-supplemented cells. When whole cells were labeled with the fluorescent probe, the opposite trend in the apparent membrane viscosity was observed. This was due primarily to the probe penetrating into nonmembranous neutral lipids rather than remaining localized in the surface membrane of the cells. Since the enthanolamine-supplemented cells contained more low viscosity neutral lipids, the whole cells gave an apparently lower viscosity as compared with choline-supplemented cells, thus, measurements carried out on whole cells gave an inaccurate determination of the viscosity of the surface membrane.

Adenosine Triphosphatases↗
PubMed1976

Modification of adenylate cyclase activity in LM cells by manipulation of the membrane phospholipid composition in vivo.

Adenylate cyclase [atp pyrophosphate-lyase (cyclizing): EC 4.6.4.4] activities were examined in mouse LM cell (fibroblast) membranes that were supplemented with ethanolamine and/or fatty acids. The supplements were incorporated into the plasma membrane phospholipids in significant amounts. Fatty acid supplementations had distinct effects as compared to polar head group supplementations. All lipid supplementations increased basal adenylate cyclase activity relative to control cells grown in choline-containing medium. Double supplementation with ethanolamine and linoleate increased the specific activity of adenylate cyclase up to 4-fold. Activity in the presence of fluoride was unaffected by ethanolamine supplementation, but was increased by fatty acid supplementation. In contrast, prostaglandin E1 stimulation was 4.2-fold in controls and ethanolamine and/or elaidate supplements, 6-fold in choline plus linoleate supplements, and 3.1-fold in ethanolamine plus linoleate supplements. Differences in activity could not be ascribed to changes in membrane protein composition in supplemented cells, and could be abolished by detergent solublization. Fluidity of the supplemented membranes was monitored by fluorescence polarization, and no correlation was observed between membrane viscosity and adenylate cyclase activity or hormone stimulation. These results emphasize the importance of the membrane lipid phase for this enzyme.

Adenylyl Cyclases↗
PubMedDate not supplied

Cell surface glycosaminoglycans are not obligatory for Plasmodium berghei sporozoite invasion in vitro.

The malaria circumsporozoite (CS) protein binds to glycosaminoglycan chains from heparan sulfate proteoglycans present on the basolateral surface of hepatocytes and hepatoma cells in vitro. When injected into mice, CS protein is rapidly cleared from the blood circulation by hepatocytes. The binding region for the HSPGs is the evolutionarily conserved region II-plus of the CS protein. Here we have asked whether the presence of glycosaminoglycans on the plasma membrane of target cells is required for sporozoite invasion in vitro. Two types of target cells were used: HepG2 cells, which are permissive for Plasmodium berghei sporozoite development into mature exoerythrocytic forms, and CHO cells, in which the intracellular development of the parasites is arrested early after penetration. The invasion of mutant CHO cells expressing undersulfated glycosaminoglycans or no glycosaminoglycans was only inhibited 41-49% or 24-32%, respectively, in comparison to invasion of CHO-K1 cells. Previous cleavage of HepG2 surface membrane glycosaminoglycans with heparinase or heparitinase had no significant inhibitory effect on subsequent P. berghei sporozoite invasion and EEF development in these cells, although the glycosaminoglycan lyase treatments removed over 80% of CS binding sites from the cell surface. These results suggest that although the presence of glycosaminoglycans on the target cell surface enhances sporozoite invasion, glycosaminoglycans are not required for sporozoite penetration or the development of exoerythrocytic forms in vitro.

Animals↗