Degree Of Sulphation Research Articles

Modulation of neurite promoting proteoglycans by neuronal differentiation

A human cell line committed to neuronal lineage was used to examine the influence of differentiation on proteoglycan synthesis and function. Where the LA-N-2 cells were stimulated to differentiate towards a phenotype of cholinergic neurons, proteoglycans of the heparan sulphate class increased relative to chondroitin sulphate proteoglycans and displayed more homogenously shorter glycosaminoglycan chains with increasing degrees of sulphation. The changes were accompanied by increasing potency of the heparan sulphate proteoglycans in neurite growth-promoting activity when immobilized on a laminin substrate. These studies begin to address the role of activity-independent growth and differentiation on the synthesis and release by neurons of neurite growth-promoting proteoglycans. The observations have implications for understanding the role of proteoglycan overexpression and the production of dystrophic neurites in Alzheimer disease.

Blood coagulation is inhibited by sulphated copolymers of vinyl alcohol and acrylic acid under in vitro as well as in vivo conditions

Biological effects of the modification of the sulphate ester and carboxyl group content of poly(vinyl alcohol-acrylic acid) copolymers (PAVAS) and sulphated polyvinyl alcohol copolymers (PVAS) with mol.weight of 5,000 to 20,000 D were studied. The in vitro anticoagulant potency of PAVAS assessed by activated partial thromboplastin time (APTT) increased with increasing the overall anionic charge, while differences in mol.weight yielded few obvious effect. The degree of sulphation played an essential part, but the carboxyl group content also contributed to the in vitro anticoagulant activity of PAVAS. On i.v. administration to rats (40 mg/kg), the anticoagulant potency of PAVAS was found to be comparable to that observed in vitro. The ability of PAVAS to induce a state of leukocytosis and decrease serum triglyceride level in rats was also dependent on charge density, and both these effects were increased with elevation of charged groups content irrespectively of mol.weight. Sulphated polyvinyl alcohol copolymers (PVAS) showed similarity to PAVAS charge-dependent biological activities.

Preliminary chemical, biochemical, and pharmacological characterization of a low molecular weight dermatan sulphate

The aim of this study was to set up a depolymerization process which resulted in the formation of a low molecular weight dermatan sulphate (LMWDS), retaining the chemical properties possessed by native dermatan sulphate (DS), fundamental for the expression of its specific biological activity. The depolymerization of DS by a β elimination process led to the production of oligosaccharide chains having a 4,5 unsaturated uronic acid at the nonreducing end. The chemical evaluation has shown that the most important parameters (degree of sulphation, sulphate to carboxyl ratio, and specific rotation) have not undergone any particular modification compared to native DS. The biochemical results demonstrate that the LMWDS obtained retains most, if not all, of the specific biological activity. The reduction in molecular weight significantly enhanced the bioavailability of the product after subcutaneous administration.

Low tissue gastrin content in the porcine distal duodenum is associated with increased percentage of G34

In the gastric antrum of pigs aged between 2 and 171 days, G17 accounted for most of the gastrin-like immunoreactivity and G34 for up to 2%. G34 increased progressively from a mean of 7% in the duodenal bulb to 39% in the most distal segment of the duodenum. In both the proximal duodenum and for the whole duodenum, the percentage of G34 correlated negatively with the tissue gastrin content. The degree of sulphation was similar in the antrum and proximal duodenum (mean 67% G17 non-sulphated).

Bile salt sulphotransferase activity in the liver of cholestatic infants.

A simple assay of bile salt sulphotransferase activity in human liver was developed. The system used glycolithocholate and PAPS as substrates. Km values for glycolithocholate and PAPs were 2.8 microM and 11.5 microM, respectively. Furthermore bile salt sulphation capacity in infants with cholestasis was investigated by measuring the activity of the bile salt sulphotransferase in the liver. No significant difference was found between the sulphotransferase activity in cholestatic infants and non-cholestatic adults. In addition the magnitude of the bile salt sulphotransferase activity in as neonatal liver did not differ from the enzymatic activity in adult liver. It is thus considered unlikely that low degree of sulphation of bile salts in infants is due to reduced capacity of this enzyme system.

Occurrence of 2- O-sulphated d-glucuronic acid in rat liver heparan sulphate

Heparan sulphates (HS) are a complex and heterogeneous family of glycosaminoglycans with a polysaccharide chain composed of alternating units of o-glucosamine and uranic acid (D-glucuronic or L-iduronic) with a (1 + 4) linkage. Sulphate substituents occur as N-sulphate (at C-2 of the o-glucosamine residues) or as O-sulphate groups (at C-6 of the o-glucosamine or at C-2 of the L-iduronic acid units). In our previous studiesle3 we have isolated and characterized a highly sulphated HS from rat liver tissues, Ll. In spite of its high degree of sulphation (2.0 sulphate groups/disaccharide), only 50% of the hexosamine residues bear Nsulphate groups, and r_-iduronic acid accounts for 25% of the total uranic acids. The high sulphate content of Ll could not be explained on the basis of the sulphation pattern normally observed in heparin-like polysaccharides. As the polymer is only partially N-sulphated, the high degree of sulphation is presumably due to the O-sulphate groups. The low proportion of L-iduronic acid suggests that positions other than C-2 or C-6 of o-glucosamine are substituted. In order to determine the location of the remaining O-sulphate groups, we continued our structural studies on Ll through 13C NMR and methylation analysis, which allowed us to detect the presence of 2-O-sulphated o-glucuronic acid residues in Ll.

Structural differences between heparan sulphates of proteoglycan involved in the formation of basement membranes in vivo by Lewis-lung-carcinoma-derived cloned cells with different metastatic potentials

This study addresses the characterization of heparan sulphates of the basement-membrane proteoglycans in tumour formed after the subcutaneous implantation of Lewis-lung-carcinoma-derived different metastatic clones (P29, LM12-3 and LM60-D6 clones with low, medium and high metastatic potentials respectively). Heparan sulphate proteoglycans (125-158 micrograms of hexuronate/g dry weight of tissue) were isolated from chondroitin ABC lyase digests of a proteoglycan fraction obtained after DEAE-Sephacel chromatography of tissue extracts. The proteoglycans were separated into three molecular species by Sepharose CL-4B chromatography followed by CsCl-density-gradient centrifugation: large proteoglycans with an estimated M(r) of 820,000-130,000, which consisted of two components with low (< 1.34 g/ml; PGII-M) and high (> 1.37 g/ml; PGII-B) density, and a small proteoglycan with an M(r) of less than 80,000 (PGIII). Of these, only the PGII-M proteoglycan (34-37 micrograms of hexuronate/g dry weight) reacted with the antiserum against proteoglycan of Engelbreth-Holm-Swarm-tumour basement membrane, and represented, therefore, a basement-membrane proteoglycan. Digestion with heparan sulphate lyases I and II of the heparan sulphates (M(r) 36,000) from the PGII-M proteoglycan of the three tumours resulted in almost complete depolymerization to give six unsaturated disaccharides identified as 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-D-glucose, 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-D-glucose and 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-6-O-sulpho-D-glucose. Comparison of the relative amounts of these disaccharides produced from the three tumour-derived heparan sulphates demonstrated that the degree of sulphation of the heparan sulphates correlated with the degree of morphological organization of the tumour basement membranes; the heparan sulphate from the more highly metastatic tumour with more highly organized basement membrane exhibited a higher degree of overall sulphation along the glycosaminoglycan chains, which was due to an increased content of the three repeating disaccharides having 6-O-sulphated glucosamine residues.

Human melanoma cell-derived factor(s) stimulate fibroblast glycosaminoglycan synthesis.

Conditioned media from cultures of human metastatic Hs294T melanoma cells contain a factor/factors that promote(s) fibroblast-mediated contraction of collagen lattices, and stimulate(s) fibroblast glycosaminoglycan (GAG) synthesis. Complete medium from melanoma cell cultures stimulated fibroblast hyaluronate synthesis 9.3-fold, and sulphated GAG synthesis 2.6-fold, as measured by 3H-glucosamine incorporation. 35SO4 incorporation into sulphated GAGS was essentially unaltered, the net result being a decrease in the degree of sulphation. Fibroblasts synthesized hyaluronate with an increased molecular weight when grown in the presence of the melanoma-cell culture medium, while the molecular weights of heparan and chondroitin sulphates remained essentially unaltered. Our results indicate that the tumour-cell-derived factor(s) stimulate(s) changes in fibroblast glycosaminoglycan synthesis, and that these changes may facilitate tumour cell invasion in vivo.

Effects of detergent on the sulphation of chondroitin by cell-free preparations from chick-embryo epiphyseal cartilage.

The effects of the non-ionic detergent Triton X-100 on 6-sulphation of two species of endogenous nascent proteochondroitin by a chick-embryo cartilage microsomal system was examined. Sulphation of the larger (Type I) species with adenosine 3'-phosphate 5'-phosphosulphate was slightly diminished when Triton X-100 was present, whereas sulphation of the smaller (Type II) species was slightly enhanced. An ordered rather than random pattern of sulphation was obtained for the smaller proteoglycan, but with a considerably lower degree of sulphation than that of the larger proteochondroitin. These differences were consistent with other differences between these two species as described previously. Sulphation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system with Triton X-100 present produced ordered rather than random sulphation patterns. When a 100,000 g supernatant fraction was utilized for sulphation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and ordered sulphation was still obtained. When hexasaccharide was used, sulphation of multiple N-acetylgalactosamine residues of the individual hexasaccharides resulted. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulphation of multiple N-acetylgalactosamine residues on the individual hexasaccharide molecules was even greater, so that tri-sulphated products were found. This suggests that ordered rather than random sulphation of chondroitin with these enzyme preparations is due to enzyme-substrate interaction rather than to membrane organization.

Conserved charge of glomerular and mesangial cell proteoglycans: possible role of amino acid-derived sulphate.

Sulphation of proteoglycans was studied in isolated glomeruli and cultured rat mesangial cells. Both preparations produced heparan, dermatan, and chondroitin sulphates, recoverable both from the tissue layers and the conditioned media. The proportion of heparan sulphate made by mesangial cells was independent of the age of the culture, but declined in later passage. These preparations differed from several other nontransformed cell types studied to date in that the degree of proteoglycan sulphation was independent of the concentration of inorganic sulphate provided. Even when no exogenous sulphate was added, sulphation-dependent charge density of newly synthesized proteoglycans was conserved. Both isolated glomeruli and cultured mesangial cells produced proteoglycans with 35S-labelled sulphate esters when [35S]methionine was provided as the sole source of labelled sulphate. Conversion of methionine to cysteine and subsequent oxidation of organic sulphate via the sulphinyl pyruvate pathway is the only mechanism known in eukaryotic cells that can account for this observation. We conclude that facile oxidation of sulphur-containing amino acids can contribute to sulphation of glomerular proteoglycans and may serve to sustain the charge density of these multifunctional molecules when the supply of inorganic sulphate is otherwise compromised.

Glycosaminoglycan patterns in gingival proteoglycans of rat with age

Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-,6-,8-,12- and 18-week-old rats were incubated for 4 h in medium containing [ 3H]-glucosamine and [ 35S]-Na 2SO 4, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61–76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development.

Performance of silica-supported copper oxide sorbents for SO x/NO x-removal from flue gas : II. Selective catalytic reduction of nitric oxide by ammonia

The selective catalytic reduction (SCR) of nitric oxide by ammonia was studied for silica-supported copper oxide particles to be used as a sorbent/catalyst in a continuous process for the simultaneous removal of SO x and NO x from flue gases. The SCR-behaviour was determined as a function of the sulphation degree, i.e. the fraction of copper oxide converted to copper sulphate, at temperatures ranging from 20 to 450°C. Up to 350°C, the fresh catalyst with 0% CuSO 4 showed a high selectivity towards production of nitrogen and water by the reaction of nitric oxide with ammonia and oxygen. At higher temperature, nitric oxide removal efficiencies decreased due to the oxidation of ammonia by oxygen. With an increase of the sulphation degree, the maximum temperature for selective catalytic reduction of nitric oxide gradually increased up to 420°C for a sulphation degree of 80%. In addition, the maximum nitric oxide removal efficiency increased as well. After regeneration of catalyst particles with a sulphation degree of 80%, realised by reduction with hydrogen and subsequent re-oxidation, the catalytic behaviour was similar to that of fresh catalyst particles with a sulphation degree of 5%. This is ascribed to the formation of some Cu 2S during the reduction, which is oxidised to CuSO 4 in the subsequent oxidation step. Since the selectivity towards the reduction of nitric oxide with ammonia is maintained up to about 375°C, a temperature which is very suitable for SO x removal as well, the silica-supported CuO investigated can be applied as a sorbent/catalyst for the simultaneous removal of SO x and NO x from flue gases. The reaction rate constants for SO x and NO x removal appeared to be of the same order of magnitude provided that the reduced sorbent/catalyst enters the absorber directly, i.e. without a separate pre-oxidation.

Antiangiogenic effect of sulphated and nonsulphated glycosaminoglycans and polysaccharides in the chick embryo chorioallantoic membrane

The inhibiting effect of sulphated and nonsulphated glycosaminoglycans and polysaccharides on the normal outgrowth of capillaries was tested in the chick embryo chorioallantoic membrane (CAM) with and without the presence of hydrocortisone. An antiangiogenic response to 50 micrograms of heparin and heparan sulphate (without hydrocortisone present) was observed in 38.8% and 23.1% of the CAMs, respectively, while the antiangiogenic response rate for dermatan sulphate, chondroitin sulphate A or C, hyaluronic acid and keratan sulphate was 15.9-0%. All sulphated homopolysaccharides tested were more effective than the naturally occurring glycosaminoglycans. Nonsulphated dextran and (methyl) cellulose had no antiangiogenic effect, while largely desulphated heparin retained such an effect. Hydrocortisone generally improved the antiangiogenic effect, a 100% response was obtained when it was combined with cellulose sulphate or fucoidan (polyfucose sulphate derived from marine algae), but the antiangiogenic effect of the largely desulphated heparin was unaffected by the presence of hydrocortisone. The results show that different polysulphated polysaccharides also have an antiangiogenic effect, without the addition of corticosteroids. The effect was apparently independent of their degree of sulphation, but the glycosidic structure may be of critical importance.

High-temperature reaction between sulphur dioxide and limestone—III. A grain-micrograin model and its verification

A modified grain model of the reaction between calcined chalk, sulphur dioxide and oxygen has been developed, and the model has been verified by comparison with a large volume of experimental data. A chalk particle is constituted of grains that are non-porous in the uncalcined state. The pore volume of calcined chalk is distributed between macropores corresponding to the interstices between grains and micropores formed in the grains during calcination. The porous grains are assumed to be constituted of non-porous micrograins. Mass transfer in the pores takes place by molecular diffusion and Knudsen diffusion, and micrograins react with sulphur dioxide and oxygen according to a shrinking, unreacted-core mechanism. Since calcium sulphate formed by the reaction has a significantly higher molar volume than calcium oxide, micrograins will grow in volume with increasing degree of sulphation, eventually filling the micropores at a degree of sulphation of approximately 50%. Further reaction in the grains takes place according to a shrinking, partially-reacted-core mechanism, accompanied by an increase in grain volume. The only unknown parameters in the model are the tortuosity factor and the diffusion coefficient in the solid product layer encasing micrograins and grains.

Physiological Activity of New Heparinoids Derived from Plant Polysaccharides

Derivatisation and anticoagulant effects of three heparinoid model substances are described which are derived from three different types of plantpolysaccharides. Degree of sulphatation and structural prerequisites for inhibiting coagulation are discussed.

Sulphated Polymers are Potent and Selective Inhibitors of Various Enveloped Viruses, Including Herpes Simplex Virus, Cytomegalovirus, Vesicular Stomatitis Virus, Respiratory Syncytial Virus, and Toga-, Arena- and Retroviruses

The sulphated polymers, such as polyvinylalcohol sulphate (PVAS) and its co-polymer with acrylic acid (PAVAS), have proved to be potent inhibitors for herpes simplex virus, human cytomegalovirus, vesicular stomatitis virus, respiratory syncytial virus, Sindbis virus, Semliki Forest virus, Junin virus, Tacaribe virus, murine sarcoma virus and human immunodeficiency virus. They are not inhibitory to non-enveloped viruses, such as poliovirus and reovirus. The broad-spectrum antiviral effects of these compounds depend on their molecular weight and degree of sulphation. Pharmacokinetic studies in rabbits have indicated that after intravenous bolus injection the serum concentrations of these compounds decay biphasically, with an initial half-life of approximately 90–120 min.

Hydrodynamic properties of connective-tissue polysaccharides

The major hydrodynamic properties of the connective-tissue polysaccharides are those that describe polysaccharide-water interaction as embodied in their osmotic-pressure and hydraulic-conductivity properties. This study shows that, for polysaccharides such as chondroitin sulphate, hyaluronate and the heparin-like polysaccharides, their hydrodynamic properties depend primarily on the presence of the uronic residue and the nature of the glycosidic linkage. Other parameters such as the degree of N-acetylation and sulphation were found not to influence these properties to any great extent. These studies particularly delineate structural-functional aspects of the connective-tissue polysaccharides in terms of their primary structure.

Two sulphonated dye compounds which compete for inositol 1,4,5-trisphosphate binding to rat liver microsomes: Effects on 5′-phosphatase activity

The ability of heparin to interact with the Ins 1,4,5-P3 receptor is dependent on its chain length and degree of sulphation. Here we report results obtained with two sulphonated dye compounds of known structures and molecular weights below 1000, Cibacron blue and Patent blue. Both compounds compete for Ins 1,4,5-P3 binding to rat liver microsomes and also inhibit Ins 1,4,5-P3 5′-phosphatase activity in the same preparation. Comparison with the effects of heparin show these to be two separate actions of the compounds.

Release of diamine oxidase into plasma by glycosaminoglycans in rats

Plasma diamine oxidase (DAO) values are enhanced by intravenous injection of heparin which releases the enzyme, synthesized in small bowel enterocytes, from binding sites located on endothelial cells of the intestinal microvasculature. Intestinal DAO, in analogy with lipoprotein lipase (another heparin-released enzyme), is believed to be electrostatically linked to endothelial binding sites composed of a glycosaminoglycan (GAG) which is presumably heparan sulphate, but the complete mechanism of enzyme release is not known. In this study we assayed in rats the DAO-releasing capability of heparan sulphate, dermatan sulphate, chondroitin sulphate A and hyaluronic acid, all heparin related compounds. Heparan sulphate, a compound with the same hexosamine as heparin but with a lower concentration of sulphated iduronic acid, induced a very high release of DAO (3-fold less than heparin), while the other tested GAGs, composed of higher proportions of non sulphated uronic acid and with galactosamine instead of glucosamine, induced a significantly lower release. In rats treated with 60 mg heparan sulphate the significant decrease in ileal mucosal DAO activity indicates that, in analogy with heparin, the high plasma enzymatic activity induced is of enterocytic origin. It is suggested that the high charge density of the compounds tested, due to the degree of sulphatation, is the decisive factor in promoting the release of intestinal DAO.

Release of lipoprotein lipase and hepatic triglyceride lipase in rats by heparin and other sulphated polysaccharides

The ability of parenteral heparin to release the capillary-bound enzymes lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) into the circulation is shared by several other sulphated polysaccharides. The lipase-releasing activity in rats of two unfractionated heparins has been compared with that of characterised preparations of other glycosaminoglycans and of two partially synthetic polysaccharides. The results suggest that whilst both molecular weight and degree of sulphation are important in determining the potency, there is also some specific structural element associated with the heparin class. The clearance of lipases was also investigated. The half-lives of LPL (29 mins) and of HTGL (36 mins) did not appear to be affected by the nature of the releasing agent.