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Inhibition of mucopolysaccharide sulfation using rabbit liver sulfotransferases.

A method of measuring the inhibition of sulfation of mucopolysaccharides employed the coupled-enzyme system of Wortman. The procedure utilized the phenol and mucopolysaccharide sulfotransferases of rabbit liver extract. Compounds tested as inhibitors included substituted salicylic acids and related compounds, some hydroxamic acids, other agents capable of metal-ion complexation, and miscellaneous compounds, mostly containing sulfur. The most effective inhibitors were vanillin oxime, salicylhydroxamic acid, and other substituted salicylic acid derivatives of weaker acid strength than salicylic acid.

Ammonium Sulfate↗

Maturation of the extracellular material of the semilunar heart values in the mouse. A histochemical analysis of collagen and mucopolysaccharides.

The developmental changes of collagen and mucopolysaccharides in the semilunar values of the mouse were studied during the embryonic, fetal and postnatal period. The valvular collagen was investigated using Van Gieson and Sirius red-polarization microscopy methods. These procedures showed that the establishment of the fibrosa layer of the cusps does not occur until the second week of the postnatal period. The nature and distribution of the valvular mucopolysaccharides were investigated by staining with Alcian blue at specific pH values and at various critical electrolyte concentrations, with the appropriate enzymatic controls using Streptomyces and testicular hyaluronidase. The results show that hyaluronate and chondroitin 4- and 6-sulphate are the major components during the embryonic and fetal period. In the older fetal stages and during the postnatal period the relative amount of hyaluronate decreases, while chondroitin sulphate increases. It is concluded from this study that the maturation of the valves occurs over a long period of the postnatal life.

Animals↗

Sanfilippo B syndrome (MPS III B): case report with analysis of CSF mucopolysaccharides and conjunctival biopsy.

A case of a child with Sanfilippo B syndrome (MPS III B), born of a consanguineous marriage, is reported. Urinary mucopolysaccharide analysis showed an abnormal excretion mainly of heparan sulphate. N-acetyl-a-glucosaminidase activity was absent in the patient but was present in the heterozygous range in parents and siblings. CSF mucopolysaccharides were also abnormally high. In fibrocytes from conjunctival biopsy and CSF cells numerous vacuoles containing storage material were found. The presence of vacuoles in fibrocytes from conjunctival biopsy and/or in CSF cells can be useful in the diagnosis of many suspected lysosomal storage disorders.

Acetylglucosaminidase↗

[Histochemical investigation of acid mucopolysaccharides in corneal wound healing of lamellar corneal grafts].

Histochemical investigations were carried out in a series of toluidine blue preparations of pH 3.8-5.2 of unfixed frozen corneal sections of lamellar keratoplasties in rabbit eyes to show alterations in the pattern of acid mucopolysaccharides during wound healing. The metachromasia in lamellar auto- and homokeratoplasties is mainly reduced in the region of the vertical host-graft junction. When using virgin silk the zone of reduced metachromasia in the vertical junction is broader compared with a 9/0-Nylon suture. The metachromasia of the lamellar transplant becomes normal in autokeratoplasties (Table 1) after a fortnight and in homokeratoplasties after 3 weeks; otherwise the metachromasia during the wound healing shows no significant difference in auto- and homokeratoplasties. The normal pattern of mucopolysaccharides in the corneal stroma does not return in the vertical host-graft junction, but this scar becomes smaller over a period of time (Fig. 3, 4, 5). There are no significant alterations of metachromasia found in the horizontal host-graft junction during wound healing between 1 day and 6 months, but starting from the 7th day after the lamellar keratoplsty using PAS-stain a small more PAS-intensive coloured line in the horizontal host-graft junction which is still there 6 months after the operation (Fig. 1, 2). When the 9/0 Nylon suture remains for 3 months, corneal vascularisation develops predominantly in the horizontal junction between host and lamellar graft. The graft becomes cloudy and the metachromasia remains reduced as long as 9 months after the suture has been removed (Table 2).

Animals↗

Acid mucopolysaccharides in hair papillae of the stump-tailed macaque (Macaca speciosa).

Acid mucopolysaccharides in dermal papillae of hair follicles from both bald and on-bald regions of the scalp of stump-tailed macaques were studies histochemically. Alcian Blue, Azure A and Periodic acid Schiff methods were used for staining mucopolysaccharides, and Bromphenol Blue for staining basic proteins. In an attempt to identify various polyanions, staining was carried out with Alcian Blue containing different concentrations of electrolytes. Methylation, saponification, mild acid hydrolysis and digestion with streptomyces or testicular hyaluronidase, chondroitinase ABC, or sialidase, were also used. The results indicate that chondroitin sulphate B is present in the papillae of terminal hair follicles in early and intermediate anagen, and degraded chondroitin sulphates are present in the papillae of vellus and terminal hair follicles in late anagen.

Animals↗

Autoradiographic detection of mucopolysaccharide accumulation in single fibroblasts.

A method is described for localizing acid mucopolysaccharides autoradiographically in cultured cells. Normal fibroblasts and fibroblasts, from patients suffering from Mucopolysaccharidosis II disease (MPS II), were cultured for six days in the presence of 35SO4 and one day in unlabelled medium. The cultured cells were transferred to plastic film dish and, after settling, they were rapidly quenched, freeze-dried, fixed in osmium tetroxide vapour and embedded in Epon. Grain counting after autoradiography in 2 mum sections revealed a significant difference (P greater than 0.001) in 35SO4 incorporation in the perinuclear cytoplasm of MPS II cells and control cells grown under the same conditions. Autoradiography was also performed after mixing MPS II cells and control fibroblasts in a ratio 1:1-8 prior to freezing and the same ratio was found between labelled and unlabelled fibroblasts. These results demonstrate the feasibility of the present autoradiographic technique for the detection of the acid mucopolysaccharide storage at the single cell level.

Autoradiography↗

[Acid mucopolysaccharides in human blood and bone marrow cells shown by fluorescence microscopy (author's transl)].

In lymphocytes, monocytes and basophil granulocytes of patients with mucopolysaccharidosis specific fluorescence of acid mucopolysaccharides exists after fixation of blood smears with 9-aminoacridin-hydrochloride and subsequent staining with pseudoisocyanine at pH 4.2. A total of eleven children with various types of mucopolysaccharidosis were investigated. Specificaly stained granula were demonstrated in 5 to 49 percent of all lymphocytes and monocytes. Acid mucopolysaccharides were for the first time also shown in red-cell precursors of the bone marrow of children with mucopolysaccharidosis.

Acridines↗

35S-sulphate incorporation into myelin sulphated mucopolysaccharides during rat brain development.

Rat brain myelin acid mucopolysaccharides (AMPS) incorporate 15%, 8%, 5.5% and 4% of total associated 35S-sulphate, 14, 21, 30 and 75 days after birth, respectively. The course of 35S-sulphate incorporation into total rat brain mucopolysaccharides, as well in those from myelin, had a similar feature with peak on the 2nd week and a significant decrease on the 3rd and 4th week postnatally.

Aging↗

Lipoprotein-acid mucopolysaccharide complexes of human atherosclerotic lesions.

Lipoprotein-acid mucopolysaccharide complexes occurring in different types of human atherosclerotic lesions were isolated and partially characterized. Complexes from fatty streaks and fibrous plaques were extracted from pooled tissues with 0.15 M NaCl, purified by gel filtration, and fractionated by ultracentrifugation. Both low and very low density lipoproteins were present; low density lipoprotein was predominant. The complexes were analyzed for uronic acid, cholesterol, phospholipid, and Ca-2+ contents; there was no significant difference in the relative molar ratios between complexes from fatty streaks and fibrous plaques. Acid mucopolysaccharides were isolated from the complexes and identified by electrophoresis and enzymatic studies. Chondroitin sulfate C and hyaluronic acid were found in complexes from fatty streaks and fibrous plaques. Heparin was detected only in fibrous plaques.

Aorta↗

Enzymatic degradation of heparin-related mucopolysaccharides from the surface of endothelial cell cultures.

When cultures of endothelial cells prelabeled with H2 -35-SO4 are exposed to a purified preparation from induced Flavobacterium heparinum containing heparinase and heparitinase activities, radioactivity accumulates in the supernatant medium. After further treatment in vitro with crude enzyme this material migrates, in part, as glucosamine (N,O-disulfated glucosamine), a break-down product characteristic of heparin and heparin-related mucopolysaccharides. After exposure of the cultures to the purified enzyme, the amount of acid-insoluble -3 5-S radioactivity that can be removed with EDTA is decreased compared to that that can be removed from control cultures. Since the amount of radioactivity that is released as break-down products is much higher than the amount of radioactivity that is secreted into the supernatant medium as intact (non-dialysable) mucopolysaccharide chains in control plates, the action of the enzyme appears to be on the cell itself. The data presented support previous studies suggesting that chains of heparitin sulfate that are accessible to the action of the enzyme are present at the surface of endothelial cells.

Cells, Cultured↗

Dietary carotenoid pigment supplementation influences hepatic lipid and mucopolysaccharide levels in rainbow trout (Oncorhynchus mykiss).

We assessed the effects of dietary carotenoid pigment supplementation on liver histochemistry in the rainbow trout. One hundred and eight rainbow trout (mean mass 266+/-10 g) were assigned to each of three replicate tanks for each of three dietary treatments; astaxanthin, canthaxanthin, or control at a target dietary inclusion of 100 mg/kg, by top-coating a pigment-free commercially extruded basal diet (Trouw Aquaculture, U.K.). Fish were fed for 3 weeks at a ration of 1.2% body mass/day, in a recirculating freshwater system maintained at 16 degrees C. Frozen liver sections were stained for total lipids, unsaturated lipids, glycogen, mucopolysaccharides, glycogen phosphorylase and aspartate aminotransferase. Relative amounts were measured quantitatively by image analysis. Carotenoid treatment significantly (P<0.05) altered the total lipid profile and hepatic mucopolysaccharide contents of livers of rainbow trout. Results are discussed in relation to the catabolic potential of the liver in carotenoid pigment metabolism.

Animals↗

Analysis of urinary mucopolysaccharides using small ion exchange columns.

Methods are presented for the small scale column chromatography of urinary mucopolysaccharides. After isolation by cety pyridinium chloride mucopolysaccharides are applied to a small column of ion exchange resin and eluted by 2 M-sodium chloride. Colorimetric assays on this elutate allow clinically significant disease states to be detected.

Chemical Precipitation↗

Micromethod for fractionation of acid mucopolysaccharides.

A new micromethod for fractionation of acid mucopolysaccharides based upon the use of different concentrations of HC1 to separate the complex of CPC with non-sulphated, monosulphated and polysulphated acid mucopolysaccharides (glycosaminoglycans) is presented. The method utilizes the different binding of the anionic macromolecules to the cationic compound cetyl pyridinium chloride. The method is simple and reproducible. The use of HC1 as eluent allows the exclusion of some steps required when salts are used for elution. Hexuronic acids are determined on the eluents.

Chondroitinases and Chondroitin Lyases↗

The interaction of collagen and acid mucopolysaccharides. A model for connective tissue.

1. The interaction of acid mucopolysaccharides of connective tissue with solubilized collagen of native, or near-native, structure was investigated by free solution electrophoresis at pH7.0. 2. Complex-formation was detected by the appearance of a third peak in the ascending limb only, indicating reversible association. 3. Complex-formation was destroyed by prior heating of solubilized collagen, indicating a probable requirement for high molecular weight or internal structure of the protein. 4. Hyaluronate and chondroitin sulphate of mol.wt. 50000 gave complexes with soluble collagen at I 0.4, whereas heparin and chondroitin sulphate of mol.wt. 15000-18000 did not. All mucopolysaccharides yielded complexes at I 0.1. The stability of the complex appears mainly dependent on electrostatic forces and is increased with increase in chain length of the polysaccharide. 5. Solubilized collagen interacted to yield gels with the ;native' chondroitin sulphate-protein macro-molecule from cartilage. 6. A schematic model for the interaction of collagen and chondroitin sulphate-protein macromolecules shows parallel-ordered interaction of collagen fibrils with chondroitin sulphate side chains of the chondroitin sulphate-protein macromolecule. The biological implications of this model are discussed, particularly in relation to the ordered structures and the ionic-network properties of the intercellular components of connective tissue.

Animals↗

The use of gas-liquid chromatography in the analysis of neutral monosaccharides in hydrolysates of gastric mucopolysaccharides.

1. Conditions are described for the separation and estimation of the neutral monosaccharides obtained on acidic hydrolysis of human gastric mucopolysaccharides. 2. The technique involves the formation of the trimethylsilyl derivatives of the sugars and the analysis of these by gas-liquid chromatography. 3. The monosaccharides estimated in gastric mucopolysaccharides by this technique were l-fucose, d-mannose, d-galactose and d-glucose. 4. The analytical values for glucose and fucose obtained by this method agreed well with values obtained by the glucose oxidase and thioglycollic acid methods respectively. 5. Evidence is presented which clearly indicates that gas-liquid chromatography is a faster, more sensitive and more convenient technique for the measurement of these compounds than any other in use at present.

Chromatography, Gas↗

The 'Lorenzan sulphates'. A new group of vertebrate mucopolysaccharides.

1. The mucopolysaccharide from the glands of Lorenzini of Squalus acanthias has been isolated and purified. 2. The material is a sulphated polysaccharide containing glucosamine, galactosamine, galactose and traces of an uronic acid. 3. This vertebrate polysaccharide is unusual in appearing to have both amino sugars present in the molecule. The amino sugars are N-acetylated. 4. This molecule is one of a group of related mucopolysaccharides which vary in composition with the animal species. The generic name ;lorenzan sulphates' is suggested to describe these substances.

Animals↗

The defect in Hurler and Hunter syndromes. II. Deficiency of specific factors involved in mucopolysaccharide degradation.

Cultured fibroblasts, derived from patients with the Hurler and Hunter syndromes, show defective degradation of sulfated mucopolysaccharide. The aberrant metabolism of Hurler cells can be corrected by secretions of fibroblasts of genotype other than Hurler, and similarly, the defect of Hunter cells can be corrected by secretions of fibroblasts of genotype other than Hunter. The active factors in these secretions, which are heat labile and associated with macromolecules, accelerate the degradation of mucopolysaccharide.

Culture Techniques↗