chondroitin-sulfates and sodium-sulfate

Light-chain amyloidosis (AL) is characterized by immunoglobulin light-chain fragments aggregating into amyloid fibrils that deposit extracellularly in vital organs such as the kidney, the heart, and the liver, resulting in tissue degeneration and organ failure, leading to death. Cardiac involvement is found in 50% of AL patients and presents the most severe cases with a life expectancy of less than a year after diagnosis. In this study, we have characterized the variable domain of a cardiac AL patient light chain called AL-09. AL-09 folds as a beta-sheet and is capable of forming amyloid fibrils both in the presence of sodium sulfate and in self-seeded reactions under physiological conditions. Glycosaminoglycans such as dermatan sulfate and heparin promote amyloid formation of self-seeded AL-09 reactions, while the glycosaminoglycan chondroitin sulfate A stabilized oligomeric intermediates and did not elongate the preformed fibrils (nucleus) present in the reaction. Finally, the histological dye Congo red, known to bind to the cross beta-sheet structure of amyloid fibrils, inhibits AL-09 amyloid fibril formation in the presence of sodium sulfate and in self-seeded reactions. This paper provides insight into the impact of different reagents on light-chain stability, structure, amyloid fibril formation, and inhibition.

Topics: Amyloid; Amyloidosis; Chondroitin Sulfates; Congo Red; Dermatan Sulfate; Glycosaminoglycans; Heart Diseases; Heparin; Humans; Immunoglobulin Light Chains; Molecular Structure; Sulfates

Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of alpha3, beta3, and gamma2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the alpha3 chain. In this study, we recombinantly expressed the human alpha3LG4/5 fragment in mammalian cells, and we show that this fragment induces adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan- and chondroitin sulfate-dependent manner. Immunoprecipitation experiments with Na2 35SO4-labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the alpha3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to alpha3LG4/5. Furthermore we could show for the first time that unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule.

Topics: Animals; Binding Sites; Cell Adhesion; Cell Adhesion Molecules; Cell Line; CHO Cells; Chondroitin ABC Lyase; Chondroitin Sulfates; Cricetinae; Embryo, Mammalian; Fibrosarcoma; Gene Expression; Heparitin Sulfate; Humans; Immunoblotting; Immunosorbent Techniques; Kalinin; Keratinocytes; Kidney; Laminin; Membrane Glycoproteins; Polysaccharide-Lyases; Proteoglycans; Recombinant Proteins; Sulfates; Sulfur Radioisotopes; Syndecan-1; Syndecans; Transfection; Tumor Cells, Cultured

The concentration of glycosaminoglycans, in an aqueous solution was determined by observing the fluorescent intensity of the ion of 2-hexadecyl-9H-pyrido[4,3b]indole at 450 nm by irradiating 253-nm light after dissolution of the insoluble salt in ethanol, which was formed by the reaction between a solution of a sample of glycosaminoglycan and an aqueous solution of the binary mixture of fluorescent 2-hexadecyl-9H-pyrido[4,3b]indolium bromide and hexadecyl pyridinium chloride. The fluorescent quaternary ammonium salt, which was slightly soluble in water, was solubilized in water by forming mixed micelles with a nonfluorescent soluble quaternary ammonium salt. The present method showed good linearity for the calibration curves between 5 and 1000 micrograms/ml in all samples of glycosaminoglycan except keratan sulfate. The relative standard deviation in determination was less than 3% for the whole calibration range.

Topics: Chondroitin Sulfates; Fluorescent Dyes; Fluorometry; Glycosaminoglycans; Molecular Weight; Quaternary Ammonium Compounds; Reproducibility of Results; Solutions; Sulfates

Cell surface proteoglycans of aortic smooth muscle cells of atherosclerosis-susceptible White Carneau (WC) and atherosclerosis-resistant Show Racer (SR) pigeons were compared to determine differences that may be involved in the greater proliferative properties of cultured WC cells. Using [35S]-sodium sulfate and [3H]-glucosamine as labeling precursors, chondroitin sulfate-proteoglycan (CS-PG) and heparin sulfate-proteoglycan (HS-PG) were identified as distinct molecules associated with the plasma membrane. Heparan sulfate-proteoglycan was reduced up to 50% in WC compared with SR cells, and, based on interaction with ion-exchange resin, had a lower charge density. These differences were not observed for the CS-PG from the two cell types. The mode of association of the cell surface PG with the plasma membrane was examined. Dissociation with 1 mol/l (molar) sodium chloride indicated that less than 10% of total cell surface PG were ironically associated with the cells. The remainder required detergent extraction, suggesting hydrophobic interactions with the plasma membrane. Both CS-PG and HS-PG displayed affinity for octyl sepharose and both were identified in isolated plasma membranes. These data present the first description of a hydrophobic CS-PG that is a significant and distinct cell-associated PG in arterial smooth muscle cells. The observation of decreased and structurally altered HS-PG in WC compared with SR cells is consistent with a potential growth regulatory function for this molecule.

Topics: Animals; Aorta; Cell Membrane; Cells, Cultured; Chondroitin Sulfates; Chromatography, Ion Exchange; Columbidae; Glucosamine; Heparitin Sulfate; Muscle, Smooth, Vascular; Sodium Radioisotopes; Sulfates; Tritium

Binding of calcium to the glycosaminoglycans (GAGs) heparin, chondroitin sulfate (CS), keratan sulfate (KS), and hyaluronic acid (HA) has been studied by equilibrium dialysis using exclusion of sulfate to correct for Gibbs-Donnan effects. Calcium binding occurs to all of these GAG species, suggesting that both sulfate and carboxylate groups are involved in cation binding. For all GAGs, the binding stoichiometry is consistent with a calcium-binding "site" consisting of two anionic groups. The order of calcium binding affinities is heparin greater than CS greater than KS greater than HA, and is critically dependent upon charge density; heparin binds calcium with 10-fold higher affinity than CS. The mode of calcium binding to GAGs is consistent with a recently proposed mechanism of growth plate calcification which states that cartilage proteoglycan functions as a reservoir of calcium for calcification of epiphyseal cartilage.

Topics: Adult; Calcium; Calcium Chloride; Chondroitin Sulfates; Dialysis; Glycosaminoglycans; Humans; Hyaluronic Acid; Keratan Sulfate; Protein Binding; Sulfates

Proteoglycan (PG) metabolism by aortic smooth muscle cell cultures derived from atherosclerosis-susceptible White Carneau (WC) and -resistant Show Racer (SR) pigeons was compared using [35S]sodium sulfate and [3H]serine or [3H]glucosamine as labeling precursors. Chondroitin sulfate (CS) PG and dermatan sulfate (DS) PG were the major PG secreted into the medium by both cell types. Total PG production, whether measured by incorporation of radiolabel into either core protein or glycosaminoglycan chains, was consistently lower in WC compared to SR cultures at several time points. This difference was due in part to lower (30-37%) PG synthesis in WC cells, but degradation of newly synthesized PG was an important contributor. A pulse-chase study indicated that of the total radiolabeled PG present at time O, only 47% was present at 24 h in WC cultures compared to 88% in SR cultures. The large CS-PG appeared to be the primary target for degradation in WC cells, and this selective processing resulted in a higher DS-PG:CS-PG ratio in these cultures. Structural studies indicated similar core protein and glycosaminoglycan chain sizes within a PG type for both cell types. PG monomer composition differed, however, by a higher sulfation of WC CS-PG compared to SR CS-PG and by a disaccharide sulfation position favoring 6-sulfation in WC PG and 4-sulfation in SR PG.

Topics: Animals; Aorta; Arteriosclerosis; Cells, Cultured; Chemical Precipitation; Chondroitin Sulfates; Chromatography; Columbidae; Culture Media; Dermatan Sulfate; Disaccharides; Glucosamine; Glycosaminoglycans; Kinetics; Muscle, Smooth, Vascular; Proteoglycans; Serine; Sulfates

Haemopoietically active mouse bone marrow cultures, incubated for 48 h with [3H]glucosamine and Na2(35)SO4, synthesized radiolabelled hyaluronic acid, heparan sulphate and chondroitin sulphate. Heparan sulphate was enriched in a trypsin extract of the adherent cells whereas hyaluronic acid and chondroitin sulphate were distributed mainly to the culture medium. Analysis of nitrous acid scission products of heparan sulphate by gel chromatography demonstrated the close association of N- and O-sulphate groups along the polysaccharide chain. Chondroitinase AC degradation established the copolymeric nature of chondroitin sulphate in which about 38% of the hexuronic acid residues were in the form of GlcUA. Studies on non-haemopoietic cultures, derived from W/Wv mice or from normal marrow cells maintained in foetal calf serum instead of horse serum, indicated that adherent stromal cells were the major source of glycosaminoglycans.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Chondroitin Sulfates; Chromatography, DEAE-Cellulose; Culture Media; Glucosamine; Glycosaminoglycans; Hematopoiesis; Heparitin Sulfate; Hyaluronic Acid; Mice; Molecular Weight; Sulfates

Morpho-functional changes and synthesis of carbohydrate compounds in the mucous membrane of the rat and cat trachea and bronchi have been studied 2, 4, 7, 14 and 24 days after a single and twice administration of gamma-globulin into the respiratory tract and after repeated intrapleural and intramuscular administration following sensibilization with a complete Freund's adjuvant. Dynamic of the changes observed depends, to a great extent, on the mode and multiplicity of administration of the adjuvant. When gamma-globulin is administered into the respiratory tract or intrapleurally, a more active incorporation of 3H-glucose and 35S-sodium sulfate into chondroitinsulphate A, C, sialic acids and glycogen, which are synthesized by cells of the tracheobronchial system glands, occurs. In 14-24 days after gamma-globulin has been administered by any mode, sulfated glycosamineoglycans (of chondroitinsulfate B and heparin type), as well as proteoglycans and sialic acids resistive to treatment with sialidase are accumulating in discharge from the glands. The accumulation of the sulfated glycosamin sulfates is accompanied with an increased level of serum antibodies specific for the administered antigen.

Topics: Animals; Bronchi; Carbohydrates; Cats; Chondroitin Sulfates; gamma-Globulins; Glucose; Proteoglycans; Rats; Rats, Inbred Strains; Sialic Acids; Sodium; Sulfates; Trachea

A gradual decline in the synthesis of glycosaminoglycans, as evidenced by reduced rates of incorporation of [35S] sulfate and [14C] glucosamine into cellular and medium glycosaminoglycans, was observed during the last (about 5) population doublings before phase-out. The decline was accompanied by a change in the distribution pattern of individual glycosaminoglycans with a relative decrease in the incorporation rate of [14C] glucosamine into cellular and medium hyaluronic acid. The incorporation rate of [14D] glucosamine and [35S] sulfate into cellular and medium heparan sulfate continually increased during the last population doublings ("senescence"). The possibility of a coupling between cell growth and hyaluronic acid synthesis or an involvement of hyaluronic acid in the adhesion of cells (among one another or/and to the substratum), and the functional significance of heparan sulfate as a growth inhibitor were discussed.

Topics: Aging; Cell Survival; Chondroitin Sulfates; Dermatan Sulfate; Fibroblasts; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Sodium; Sulfates

The concentration of inorganic sulfate-sulfur in the serum of vitamin D-deficient rats, 2.6 to 3.5 mg. per cent, was found to be higher than that in the serum of normal rats of the same age, 2.0 mg. per cent. No change was observed following the administration of 25 gamma of vitamin D(2). In accord with the results of others, it was found that a definitely increased deposition of phosphorus in femurs and tibiae had occurred 36 to 48 hours after the administration of vitamin D(2) to vitamin D-deficient rats. An immediate increase in the uptake of sulfate by the skeleton was found using sodium sulfate-S(35). As measured by the specific activity of sulfate-sulfur in samples of chondroitin sulfate isolated from the skeletons of the vitamin D-deficient animals and from normal controls receiving equal doses of sulfur-35, the rate of synthesis of chondroitin sulfate in rachitic rats is similar to the rate in normal rats of the same age. Likewise, the incorporation of labelled sulfate into the sulfomuco-polysaccharides of the pelts was found to be equal at 12 hours to that in normal rats. Following the administration of vitamin D(2) to deficient animals an increase in the rate of synthesis of the chondroitin sulfate of the skeletons was noted. The radiochemical and radioautographic evidence suggest that there is in vitamin D-deficient rats an impaired utilization of chondroitin sulfate and that vitamin D(2) is able to accelerate this process.

Topics: Animals; Cartilage; Chondroitin Sulfates; Ossification, Heterotopic; Osteogenesis; Phosphorus; Phosphorus Radioisotopes; Rats; Sulfates; Sulfur; Sulfur Radioisotopes; Vitamin A; Vitamin D; Vitamin D Deficiency; Vitamins