Effect of magnesium deficiency on the metabolism of glycosamino-glycans in rats

Magnesium deficiency in rats has significant effect on the concentration of different glycosaminoglycans in the tissues, the nature of the change being different in different tissues. Total glycosaminoglycans, chondroitin-4-sulphate + chondroitin-6-sulphate and dermatan sulphate increased in the aorta while hyaluronic acid, heparan sulphate and heparin decreased. In the liver, total glycosaminoglycans, hyaluronic acid, chondroitin-4-sulphate + 6-sulphate and heparin decreased while total glycosamino-glycans and all the glycosaminoglycan fractions increased in the heart. In the kidney, total glycosaminoglycans showed no significant alteration, hyaluronic acid and heparin decreased while chondroitin-4-sulphate + 6-sulphate increased. Activity of biosynthetic enzymesviz. glucosamine-o-phosphate isomerase and UDPG-dehydrogenase showed decrease in the liver. The concentration of 3’-phosphoadenosine 5’-phosphosulphate, activity of sulphate activating system and sulphotransferase were also similarly altered in the liver in magnesium deficiency.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS IN HUMAN BRAIN OF DIFFERENT AGE GROUPS

—Five distinct glycosaminoglycan fractions have been isolated from human brain of various age groups, by employing an improved fractionation procedure. Analysis of these fractions showed that human brain contains hyaluronic acid, chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan sulphate, heparan sulphate and two unidentified low sulphated fractions. The pattern of variation of these compounds with age, indicates that they may be playing an important role in the process of myelination and brain maturation.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS IN BRAIN OF DIFFERENT SPECIES

Abstract— The uronic acid-containing glycosaminoglycans present in the brains of rat, monkey, chicken, sheep and rabbit were isolated into various fractions by combining the cetyl pyridinium procedure and DEAE-Sephadex column chromatography. The analyses of the fractions show that hyaluronic acid, chondroitin-4-sulphate, chondroitin-6-sulphate, heparan sulphate and a testicular hyaluronidase-resistant galactosamine-containing GAG are present in the brain of all the species studied. Hyaluronic acid is the major GAG (33–41 per cent). Chondroitin-4-sulphate (19–35 per cent), and heparan sulphate (11–19 per cent), are the next prominent GAGs, in all the species except chicken. The results indicate the similarity in the pattern of GAGs in the brain of all the species.     

THE NATURE OF SULPHATION OF URONIC ACID-CONTAINING GLYCOSAMINOGLYCANS CATALYSED BY BRAIN SULPHOTRANSFERASE

—A sulphotransferase system of rat brain catalyses the transfer of sulphate from 3′-phosphoadenosine 5′-phosphosulphate to the low-sulphated glycosaminoglycans isolated from normal adult human brain. These were shown to be precursors of higher-sulphated glycosaminoglycans by DEAE-Sephadex column chromatography and paper electrophoresis. Nitrous acid degradation and mild acid hydrolysis of enzymically-sulphated fractions further confirmed the presence of heparan sulphate in human brain. A partially purified sulphotransferase preparation was obtained from neonatal human brain using chondroitin-4-sulphate as sulphate acceptor. This sulphotransferase catalyses the transfer of sulphate to the various uronic acid containing glycosaminoglycans. Heparan sulphate was the best sulphate acceptor followed by dermatan sulphate, N-desulphoheparin, chondroitin-4-sulphate and chondroitin-6-sulphate in decreasing order. Sulphotransferase obtained from 1-day-old rat, rabbit and guinea pig brain also had the same pattern of specificity towards various sulphate acceptors. This sulphotransferase catalyses both N-sulphation and O-sulphation. Studies on the sulphotransferase obtained from both rat and human brain of various age groups indicate that the ratio of N-sulphation: O-sulphation decreases as the brain matures.     

The synthesis of glycosaminoglycans by cultures of rabbit corneal endothelial and stromal cells.

Confluent monolayer cultures of rabbit corneal endothelial and stromal cells were incubated independently with [35S]sulphate and [3H]glucosamine for 3 days. AFter incubation, labelled glycosaminoglycans were isolated from the growth medium and from a cellular fraction. These glycosaminoglycans were further characterized by DEAE-cellulose column chromatography and by sequential treatment with various glycosamino-glycan-degrading enzymes. Both endothelial and stromal cultures synthesized hyaluronic acid as the principal product. The cell fraction from the stromal cultures, however, had significantly less hyaluronic acid than that from the endothelial cultures. In addition, both types of cells synthesized a variety of sulphated glycosaminoglycans. The relative amounts of each sulphated glycosaminoglycan in the two cell lines were similar, with chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate as the major components. Heparan sulphate was present in smaller amounts. Keratan sulphate was also identified, but only in very small amounts (1-3%). The presence of dermatan sulphate and the high content of hyaluronic acid are similar to the pattern of glycosaminoglycans seen in regenerating or developing tissues, including cornea.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS IN PERIPHERAL NERVE AND SPINAL CORD OF MONKEY

—The isolation of uronic acid-containing glycosaminoglycans from peripheral nerve and spinal cord of monkey was done by combining the cetyl pyridinium procedure and DEAE-Sephadex column chromatography. The constituent analyses of the isolated GAG-fractions indicated that hyaluronic acid, chondroitin-4-sulphate, chondroitin-6-sulphate, heparan sulphate and a testicular hyaluronidase-resistant galactosamine-containing GAG were present in both tissues. Hyaluronic acid was the predominant GAG (63 per cent) in both tissues and its level was much higher than in brain. Chondroitin-4-sulphate constituted 16 per cent in both tissues. The levels of heparan sulphate and hyaluronidase-resistant galactosamine-containing GAG in these tissues were much lower than in brain. The results indicate that the patterns of GAGs in peripheral nerve and spinal cord of monkey are similar but differ from that of brain.     

Distribution of proteoglycans and hyaluronic acid in transverse sections of bovine thoracic aorta

Summary The distribution of hyaluronic acid and proteoglycans in bovine thoracic aorta was studied by Alcian Blue staining of frozen tissue sections under controlled electrolyte conditions with and without prior enzymic digestion. Some sections were digested with chondroitinase ABC, testicular hyaluronidase or bacterial collagenase and subsequent staining permitted conclusions to be drawn about the distribution of specific glycosaminoglycans within the tissue. The total glycosaminoglycan content was maximal in the intima and decreased across the arterial wall to the outermost adventitial layer. The content of proteoglycan containing chondroitin sulphate and/or dermatan sulphate chains paralleled this distribution. However, other glycosaminoglycans also contributed significantly to staining, although there was no evidence for any appreciable concentration of heparin or highly sulphated heparan sulphate.Several experiments indicated that proteoglycan containing chondroitin sulphate and/or dermatan sulphate was associated with elastic laminae which were often seen stained along their periphery. Hyaluronic acid was present at significant concentrations in all locations of the aorta and there was evidence for a similar distribution of heparan sulphate which was possibly also present at a high concentration in the endothelium. Staining of sections after treatment with 4m guanidinium chloride confirmed that this extractant removed most of the proteoglycan from the tissue section.     

Tissue structure-specific distribution of glycosaminoglycans in the human penis

The aim of this work was to isolate and characterise the glycosaminoglycans present in the different tissue structures of the human penis in view of their potentially significant role in the physiology of erection. Penile tissue samples were obtained from patients who underwent penectomy and were subsequently dissected into individual tissue structures. Total glycosaminoglycans were isolated and purified from tunica albuginea, corpora cavernosa and corpus spongiosum, following tissue mincing, ultrasonication, lipid extraction, extensive digestion with pronase and DNase, treatment with alkali-borohydride and ethanol precipitation. Isolated glycosaminoglycans were separated by cellulose acetate electrophoresis and fractionated by anion exchange chromatography on DEAE Sephacel columns. Different glycosaminoglycan fractions were identified using glycosaminoglycan-degrading enzymes of known specificity. Gradient polyacrylamide gel electrophoresis was used to determine the average molecular mass of the glycosaminoglycans. The corpus cavernosum and the corpus spongiosum extracts contained almost twice the amount of glycosaminoglycan-associated uronic acids as compared to the tunical extracts (1.47+/-0.09, and 1.49+/-0.15 as opposed to 0.75+/-0.15 microg/mg dry defatted tissue, respectively; S.E.M., n=5). With the exception of hyaluronic acid, the relative amount of individual glycosaminoglycan types varied significantly among extracts of different origin. Heparan sulphate was more abundant in cavernosal, dermatan sulphate in tunical, and chondroitin-6-sulphate in corpus spongiosum extracts. No structure-specific differences were detected with respect to the molecular mass distribution of each glycosaminoglycan type. Our study shows that the different structures of the human penis produce distinct profiles of glycosaminoglycans, which are well suited to the individual functional characteristics of these structures.     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.     

Action pattern of polysaccharide lyases on glycosaminoglycans

The action pattern of polysaccharide lyases on glycosaminoglycansubstrates was examined using viscosimetric measurements andgradient polyacrylamide gel electrophoresis (PAGE). Heparinlyase I (heparinase, EC 4.2.2.7 [EC] ) and heparin lyase II (no ECnumber) both acted on heparin in a random endolytic fashion.Heparin lyase II showed an ideal endolytic action pattern onheparan sulphate, while heparin lyase I decreased the molecularweight of heparan sulphate more slowly. Heparin lyase III (heparitinase,EC 4.2.2.8 [EC] ) acted endolytically only on heparan sulphate anddid not cleave heparin. Chondroitin ABC lyase (chondroitinaseABC, EC 4.2.2.4 [EC] ) from Proteus vulgaris acted endolytically onchondroitin-6-sulphate (chondroitin sulphate C) and dermatansulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate(chondroitin sulphate A) at a reduced rate, decreasing its molecularweight much more slowly. Two chondroitin AC lyases (chondroitinaseAC, both EC 4.2.2.5 [EC] ) were examined towards chondroitin-4- and-6-sulphates. The exolytic action of chondroitin AC lyase Afrom Arthrobacter aurescens on both chondroitin-4- and -6-sulphateswas demonstrated viscosimetrically and confirmed using bothgradient PAGE and gel permeation chromatography. ChondroitinAC lyase F from Flavobacterium heparinum (Cytophagia heparinia)acted endolytically on the same substrates. Chondroitin B lyase(chondroitinase B, no EC number) from F.heparinum acted endolyticallyon dermatan sulphate giving a nearly identical action patternas observed for chondroitin ABC lyase acting on dermatan sulphate. action pattern chondroitin lyase glycosaminoglycan heparin lyase.     

Histochemical analysis of urea-unmasked glycosaminoglycans in the skin of the rat and mouse

Summary Histochemical analysis of urea-unmasked glycosaminoglycans has been performed in connective tissues of the rat and mouse skin by means of combined staining and enzyme digestion procedures. The staining procedures used were Alcian Blue pH 1.0, Alcian Blue pH 2.5, Aldehyde Fuchsin, periodic acid-Schiff (PAS), Alcian Blue pH 2.5-PAS, high iron diamine and low iron diamine methods. The digestive enzymes employed wereStreptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained indicated that the major components of the glycosaminoglycans in the connective tissues of the skin were hyaluronic acid, dermatan sulphate and chondroitin sulphate A and/or C, whereas the tissues were devoid of keratan sulphate.     

Observations on the glycosaminoglycans of aging bronchial cartilage studied with Alcian Blue

Synopsis The changes in the distribution of acidic glycosaminoglycans in the extracellular matrix of aging human bronchial cartilage have been studied with Alcian Blue using the critical electrolyte technique described by Scott & Dorling (1965). Keratan sulphate was detected in the interterritorial matrix early in the first decade. The factors initiating the synthesis of keratan sulphate by chondrocytes are discussed and a hypothesis is proposed to explain the subsequent localization of this glycosaminoglycan in the extracellular matrix.     

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry

Summary Selected commonly used cationic dyes, viz. Thionin, Safranin O, Toluidine Blue O, Dimethylmethylene Blue, Cuprolinic Blue, Cupromeronic Blue,N, N-Diethylpseudoisocyanine, and a modified PAS-method, and staining method, with a variety of alternative procedures, e.g., variation of pH, use of the critical electrolyte concentration method, and blocking reactions (methylation-saponification, carboxymethylation), were tested to select optimal staining procedures for the semiquantitative histochemical estimation of glycosaminoglycans by microspectrophotometry in sections of articular cartilage. The methods were carried out on 3 m-thick paraffin and 1 m-thick glycolmethacrylate sections of bovine articular cartilage. The staining intensity of the sections was measured from spots 25 m apart using a leitz MPV 3 microspectrophotometer, starting at the surface of the cartilage and ending up at the tidemark. The result was compared with the fixed-charge density graph determined from the adjacent articular cartilage.Of the dyes tested, Thionin and Safranin O proved to be excellent cationic dyes for the histochemical quantification of cartilage matrix proteoglycans, since the staining intensity curves showed a linear correlation (r=0.900–0.995) with the fixed charge density curves from the adjacent cartilage. Also, the stain distribution was consistently uniform across the sections. In 1 m-thick glycolmethacrylate sections, the Safranin O staining gradient showed almost perfect identity with the fixed-charge density curve. Cuprolinic Blue and Cupromeronic Blue combined with the critical electrolyte concentration technique were also useful for the microspectrophotometric assays of glycosaminoglycans, but the presence of metachromasia should be checked prior to the measurements. The reliability of blocking procedures for quantitative histochemical work was not convincing.     

The influence of the type of sulphate bond and degree of sulphation of glycosaminoglycans on their interaction with lysosomal enzymes.

Significant differences occur between the interaction of several sulphated glycosaminoglycans with a particular lysosomal protein, leading to inhibition in the case of lysosomal enzymes. The order of strength of inhibition at pH4 was: heparin greater than chondroitin 4-sulphate = chondroitin 6-sulphate greater than dermatan sulphate.     

Investigation of the ability of several naturally occurring and synthetic polyanions to bind to and potentiate the biological activity of acidic fibroblast growth factor

The ability of several animal, plant, and bacterial derived polyanions (PAs) as well as synthetic PAs to compete with heparin for the binding of acidic fibroblast growth factor (aFGF) was correlated with their ability to potentiate the mitogenic and neurotrophic actions of this factor. Dextran sulphate, K-carrageenan, pentosan sulphate, polyanethole sulfonate, heparin, and fucoidin competed for the heparin binding site on aFGF at relatively low concentrations (≤50 μg/ml). λ-carrageenan, ι-carrageenan, and polyvinyl sulphate exhibited lower affinity for aFGF, whereas hyaluronic acid, dermatan sulphate, chondroitin-6-sulphate, chondroitin-4-sulphate, and uncharged dextran displayed very low or no demonstrable affinity. Potentiation of the mitogenic action of aFGF for Balb/c 3T3 fibroblasts tended to be in general agreement with the aFGF binding affinity of the PAs. However, polyanethole sulfonate, the carrageenans, polyvinyl sulphate, fucoidin, and pentosan sulphate exerted a mitogenic action on the 3T3 cells that was independent of, and in addition to, the ability of these GAGs to potentiate the action of aFGF. The ability to potentiate the neurotrophic action of aFGF for E8 chick ciliary neurons was a general property of those PA with low or no activity in the mitogen assay. Thus hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, and even uncharged dextran all potentiated aFGF induced neuronal survival. The differential effects of these PA in potentiating the biological activities of aFGF are discussed in relation to their ability to compete for the heparin-binding site of aFGF. © 1993 Wiley-Liss, Inc.     

The proteoglycans and glycosaminoglycan chains of rabbit synovium

The synovial lining of joint capsules is important because it controls the flow of fluid into and out of the joint cavity. Physiological studies have shown that the glycosaminoglycans, particularly hyaluronan, have an important role in the control of fluid flow. The distribution of the glycosaminoglycans and proteoglycans in the synovium and subsynovium of rabbits (approximately 12 weeks old) was, therefore, determined immunohistochemically. Hyaluronan, chondroitin-4- and chondroitin-6-sulphates and keratan sulphate are present in the synovium and subsynovium; chondroitin-4-sulphate is at higher concentrations than chondroitin-6-sulphate. The core proteins of the chondroitin sulphate proteoglycans, biglycan and decorin, and of the keratan sulphate proteoglycan, fibromodulin, are also present. To date, fibromodulin has not been located in other synovial linings, and its presence corroborates that of keratan sulphate.     

Quantitative measurements of the proton-motive force and its relation to steady state lactose accumulation in Escherichia coli.

1. Developing tail tendons from rats (19-day foetal to 126 days post partum) were examined by electron microscopy after staining for proteoglycan with a cationic copper phthalocyanin dye. Cuprolinic Blue, in a "critical electrolyte concentration" method. Hydroxyproline was measured on papain digests of tendons, from which glycosaminoglycuronans were isolated, characterized and quantified. 2. Mean collagen fibril diameters increased more than 10-fold with age according to a sigmoid curve, the rapid growth phase 2 being during 30-90 days after conception. Fibril periodicities were considerably smaller (50-55 nm) in phases 1 and 2 than in phase 3 (greater than 62 nm). 3. Dermatan sulphate is the main glycosaminoglycuronan in mature tendon. Chondroitin sulphate and hyaluronate preponderate in foetal tissue. 4. Proteoglycan was seen around but not inside collagen fibrils. Proteoglycan and collagen were quantified from electron micrographs. Their ratios behaved similarly to uronic acid/hydroxyproline and hyaluronate/hydroxyproline ratios, which decreased rapidly around birth, and then levelled off to a low plateau coincident with the onset of rapid growth in collagen fibril diameter. 5. Dermatan sulphate/hydroxyproline ratios suggest that the proteoglycan orthogonal array around the fibril is largely dermatan sulphate. In the foetus hyaluronate and chondroitin sulphate exceed that expected to be bound to collagen. 6. An inhibiting action of chondroitin sulphate-rich proteoglycan on fibril diameter growth is suggested. 7. The distributions of hyaluronate, chondroitin sulphate and dermatan sulphate are discussed in the light of secondary structures suggested to be present in hyaluronate and chondroitin sulphate, but not in dermatan sulphate.     

Electrophoretic and enzymatic analysis of glycosaminoglycans in the blood serum of patients with hyperthyroidism

An effect of hyperthyroidism on the composition and levels of glycosaminoglycans in the blood serum was studied. Glycosaminoglycans isolated from 1-ml blood samples were assayed with the following techniques: carbazole, electrophoretic and enzymatic. Separation and assay of particular GAG were made with bidirectional electrophoresis. Isomers of the remaining chondroitin sulphates were assayed enzymatically. Electrophoretograms of GAG in blood serum of healthy women have shown two fractions: low sulphate chondroitin sulphate and chondroitin-4-sulphate. The same fractions of GAG were found in blood serum of the female patients with hyperthyroidism. Mean concentration of GAG in the blood serum of hyperthyroid patients increased by 51%: low sulphate chondroitin sulphate and chondroitin-4-sulphate concentrations increased by 22% and 190% respectively. Chondroitin sulphates in the blood serum of both groups were degraded to unsaturated disaccharides not containing sulphur and unsaturated 4-sulphate disaccharides. Concentrations of unsaturated 4-sulphate and unsaturated sulphur-free disaccharides increased by 71% and 17% in hyperthyroidism. Observed changes in the blood serum GAG concentrations reflect changes in the connective tissue metabolism in hyperthyroidism.     

Copper retention,calcium release and ultrastructural evidence indicate specific Cuprolinic Blue uptake and peculiar modifications in mineralizing aortic valves

Previously, reactions with copper phthalocyanines at 0.05M critical electrolyte concentration were found to cause demineralization in calcifying porcine aortic valves after subdermal implantation in rat, as well as simultaneous visualization of peculiar phthalocyanine-positive layers around cells and cell-derived matrix vesicles. In the present investigation, an appraisal was made of the mechanism and specificity of reactions with Cuprolinic Blue by comparing quantitatively calcium release and copper retention by calcified aortic valves reacted with this phthalocyanine under different critical electrolyte concentration conditions, and the corresponding ultrastructural patterns. It was found that (i) decalcifying properties are inversely proportional to salt molarity; (ii) reactivity to Cuprolinic Blue is critical electrolyte concentration-dependent, since the greatest copper retention occurred in 0.05M critical electrolyte concentration Cuprolinic Blue-reacted samples, the only ones that also exhibited phthalocyanine-positive layers; (iii) the appearance of phthalocyanine-positive layers depends on Cuprolinic Blue uptake, revealing pericellular clustering of calcium-binding, anionic molecules; and (iv) minor Cuprolinic Blue uptake occurs by residual proteoglycans which still remain in the extracellular matrix after 6-week-long subdermal implantation. The present results indicate that this method is appropriate for the study of mineralized tissues and illustrate peculiar tissue modifications occurring at least in the experimental conditions used here.     

Glycosaminoglycan (GAG) level and activities of certain enzymes of GAG metabolism in Culex quinquefasciatus and Setaria digitata

Significant differences were observed in GAG metabolism of S. digitata and one of its intermediate vectors, C. quinquefasciatus. Distribution of different components such as hyaluronic acid, heparin-sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan sulphate and heparin was comparable in both. However, there were quantitative differences; the difference was marked in the activity of enzymes of GAG metabolism in presence and absence of diethylcarbamazine (DEC) a known antifilarial drug. While the activities of beta-galactosidase and beta-N-acetyl glucosaminidase of S. digitata systems showed an inhibition of 96.5 and 92.6% respectively, in the Culex systems they showed an inhibition of 93.3% and an activation of 18% respectively. The differences clearly indicate the existence of basic differences in GAG metabolism of vector and parasite.