Experimental mucopolysaccharidosis: preservation and ultrastructural visualization of intralysosomal glycosaminoglycans by use of the cationic dyes cuprolinic blue and toluidine blue

The cationic dyes Cuprolinic Blue (CB) and Toluidine Blue (TB) were used to preserve the intralysosomal storage material accumulating in tilorone-induced mucopolysaccharidosis. As shown in previous studies, the stored glycosaminoglycans (GAGs) are leached during the conventional fixation procedure, with the result that the lysosomes appear empty. In the present study, the liver, spleen, and cornea-conjunctiva of tilorone-treated rats were examined. The application of CB in the presence of 0.1 M or 0.3 M MgCl2 simultaneously with, or subsequently to the primary fixative yielded electron-dense precipitates within the storage lysosomes. When TB (0.1%) was added to the primary fixative, the storage lysosomes contained filamentous structures arranged in reticular patterns. With increasing TB concentrations (up to 1%) the lysosomes increasingly often showed apparently amorphous storage material which was continuous with the reticular filamentous structures. Similar ultrastructural patterns were obtained with GAG-TB complexes prepared in vitro. The intralysosomal storage material preserved by TB is interpreted as GAG-TB precipitates. In conclusion, the use of CB provides a method which allows direct cytochemical demonstration of the subcellular sites of GAG-storage. The use of TB represents an easy method to obtain electron micrographs pathognomonic of the mucopolysaccharidosis induced by tilorone and congeners. Either method may be helpful to detect this adverse drug effect at the subcellular level.     

Experimental mucopolysaccharidosis: Preservation and ultrastructural visualization of intralysosomal glycosaminoglycans by use of the cationic dyes cuprolinic blue and toluidine blue

Summary The cationic dyes Cuprolinic Blue (CB) and Toluidine Blue (TB) were used to preserve the intralysosomal storage material accumulating in tilorone-induced mucopolysaccharidosis. As shown in previous studies, the stored glycosaminoglycans (GAGs) are leached during the conventional fixation procedure, with the result that the lysosomes appear empty. In the present study, the liver, spleen, and cornea-conjunctiva of tilorone-treated rats were examined. The application of CB in the presence of 0.1 M or 0.3 M MgCl₂ simultaneously with, or subsequently to the primary fixative yielded electron-dense precipitates within the storage lysosomes. When TB (0.1%) was added to the primary fixative, the storage lysosomes contained filamentous structures arranged in reticular patterns. With increasing TB concentrations (up to 1%) the lysosomes increasingly often showed apparently amorphous storage material which was continuous with the reticular filamentous structures. Similar ultrastructural patterns were obtained with GAG-TB complexes prepared in vitro. The intralysosomal storage material preserved by TB is interpreted as GAG-TB precipitates. In conclusion, the use of CB provides a method which allows direct cytochemical demonstration of the subcellular sites of GAG-storage. The use of TB represents an easy method to obtain electron micrographs pathognomonic of the mucopolysaccharidosis induced by tilorone and congeners. Either method may be helpful to detect this adverse drug effect at the subcellular level.     

Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans.

The secretory enzyme extracellular superoxide dismutase (EC-SOD) occurs in at least three forms, which differ with regard to heparin affinity: A lacks affinity, B has intermediate affinity, and C has relatively strong affinity. The affinity of EC-SOD C for various sulphated glycosaminoglycans (GAGs) was assessed (a) by determining the concentration of NaCl required to release the enzyme from GAG-substituted Sepharose 4B and (b) by determining the relative potencies of the GAGs to release EC-SOD C from heparan sulphate-Sepharose 4B. Both methods indicated the same order of affinity. Heparin bound EC-SOD C about 10 times as avidly as the studied heparan sulphate preparation, which in turn was 10 and 150 times as efficient as dermatan sulphate and chondroitin sulphate respectively. Chondroitin sulphate showed weak interaction with EC-SOD C at physiological ionic strength. Heparin subfractions with high or low affinity for antithrombin III were equally efficient. The binding of EC-SOD C to heparin-Sepharose was essentially independent of pH in the range 6.5-9; below pH 6.5 the affinity increased, and beyond pH 9.5 there was a precipitous fall in affinity. The inhibitory effect of NaCl on the binding of EC-SOD C to GAGs indicates that the interaction is of electrostatic nature. EC-SOD C carries a negative net charge at neutral pH, and it is suggested that the binding occurs between the negative charges of the GAG sulphate groups and a structure in the C-terminal end of the enzyme that has a cluster of positive charges. These results are compatible with the notion that heparan sulphate proteoglycans on cell surfaces or in the intercellular matrix may serve to bind EC-SOD C in tissues.     

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 µg 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.     

Improved quantitation of plasma lipids by direct gas-liquid chromatography

A preliminary digestion of total plasma lipid extracts with phospholipase C, which converts the lysolecithins, lecithins, and sphingomyelins into monoglycerides, diglycerides, and ceramides, respectively, has been shown to facilitate subsequent determination of the plasma lipids by gas-liquid chromatography. A further improvement in the chromatographic elution pattern results from acetylation or trimethylsilylation of the liberated alcohol moieties prior to injection into the chromatograph. If tridecanoin is used as internal standard, quantitative estimates can be rapidly obtained for plasma lysolecithins, free cholesterol, lecithins, sphingomyelins, cholesteryl esters, and triglycerides, as well as for free fatty acids. Other plasma lipids do not occur in sufficiently high concentrations to interfere with the analysis. The determination requires 0.1-0.5 ml of plasma and about 6 hr of processing, but many samples can be processed at a time.     

Influence of cetylpyridinium chloride on the ultrastructural appearance of sulphated glycosaminoglycans in human colonic mucosa

Summary The effect of adding cetylpyridinium chloride to the fixative on the preservation of sulphated glycosaminoglycans (SGs) was studied in human normal colonic mucosa. SGs were visualized at the ultrastructural level through the application of Spicer's High Iron Diamine (HID) technique followed by a post-fixation with potassium ferrocyanide-reduced osmium tetroxide. SGs were mainly localized in basement membranes of epithelium and capillary wall and along collagen fibers. The morphology of the reactive sites depended on the presence of cetylpyridinium chloride, SGs being granular in absence of the salt and more or less elongated when cetylpyridinium chloride was added to the fixative.We suggest that the use of cetylpyridinium chloride during fixation may help to preserve SG molecule at the ultrastructural level.     

The quantitative discrimination of corneal type I, but not skeletal type II, keratan sulfate in glycosaminoglycan mixtures by using a combination of dimethylmethylene blue and endo-β- -galactosidase digestion

The quantitation of individual glycosaminoglycans in mixtures of polyanions using the dimethylmethylene blue (DMB) method described by R. W. Farndale, D. J. Buttle, and A. J. Barrett (1986, Biochim. Biophys. Acta 883, 173) is dependent on enzymatic hydrolysis by specific polysaccharidases. While using this method to examine the keratan sulfate (KS) of the intervertebral disc we found that digestion with commercially available keratanase decreased binding to DMB by less than 30%, whereas corneal KS was reduced by 85%. However, by preincubating the KS fractions with endo-beta-D-galactosidase prior to keratanase treatment the corneal KS could be completely digested and disc KS digestion increased to 60%. It is suggested that the resistance of the disc KS to these digestive procedures arises from branching and/or sites of multisulfation on the polysaccharide chain. Agarose gel electrophoresis and compositional analyses of the keratan sulfates supported such an interpretation.     

Influence of cetylpyridinium chloride on the ultrastructural appearance of sulphated glycosaminoglycans in human colonic mucosa

The effect of adding cetylpyridinium chloride to the fixative on the preservation of sulphated glycosaminoglycans (SGs) was studied in human normal colonic mucosa. SGs were visualized at the ultrastructural level through the application of Spicer's High Iron Diamine (HID) technique followed by a post-fixation with potassium ferrocyanide-reduced osmium tetroxide. SGs were mainly localized in basement membranes of epithelium and capillary wall and along collagen fibers. The morphology of the reactive sites depended on the presence of cetylpyridinium chloride, SGs being granular in absence of the salt and more or less elongated when cetylpyridinium chloride was added to the fixative. We suggest that the use of cetylpyridinium chloride during fixation may help to preserve SG molecule at the ultrastructural level.     

Estimation of glycosaminoglycans by atomic absorption determination of copper in their alcian blue complexes

Glycosaminoglycans can be quantitated by determining the copper content of their alcian blue complexes. The use of this method is demonstrated with mixtures containing heparan sulfate, hyaluronic acid, and chondroitin sulfate after they have been resolved by cellulose acetate electrophoresis. Quantitation of alcian blue by atomic absorption is more sensitive than spectrophotometric techniques previously published. The method can be used to estimate the glycosaminoglycan composition in small amounts of tissue. This report demonstrates the use of this methodology in the quantitation of glycosaminoglycans in fetal and postnatal mouse brain and in the determination of the specific activities of glycosaminoglycans of fetal mouse brain labeled in vitro with [1-¹⁴C]glucosamine.     

Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions

Summary The application of the critical electrolyte concentration (CEC) concept to the differentiation of acidic glycosaminglycans (mucopolysaccharides) is described. Alcian Blue 8GX stains with increasing selectivity as increasing amounts of magnesium chloride are incorporated into the dye solution. Model experiments with pure polyanions, or artifically carboxylated, phosphorylated and sulphated liver sections, showed that binding of dye to carboxylate or phosphate groups ceased at low electrolyte concentrations (< 0.3M) whereas dye continued to be held by sulphate ester groups at concentrations five to ten times as high. The similarity to the well established cetylpyridinium system for polyanion fractionation is discussed.Sections of tissues chosen to contain predominantly or characteristically carboxylated mucins, and/or sulphate ester polyanions showed a staining pattern entirely similar to the model sections. Goblet cell mucin in rat ileum stained at < 0.4M MgCl₂, Cartilage at < 0.6M MgCl₂, mast cells at < 0.75M, and corneal stroma at < 1.0M. These results are in agreement with the known contents of sialo-mucin, chondroitin sulphate, heparin and keratansulphate, respectively. The conditions in which this principle can be used in a practical technique are described.The new and more precise terminology of Jeanloz (1960) is used in preference to the older nomenclature.     

A simple method for the quantitation of glycuronic acid-containing glycosaminoglycans with mucopolysaccharidases

A simple method for the quantitative determination of glycuronic acid-containing glycosaminoglycans (UA-GAG) is described. Sample solutions of glycosaminoglycans were digested with chondroitinase AC, chondroitinase C, chondroitinase B, heparitinases, and Streptomyces hyaluronidase, respectively, and the absorbance was read at 232 nm after digestion. The contents of 4-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-4S), 6-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-6S) plus N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-OS), 4-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)L-idosiduronyl units (D-4S) plus N-acetylgalactosaminyl beta (1 leads to 4)L-idosiduronyl units (D-OS), heparan sulfate, and hyaluronic acid in the sample solutions were calculated from the absorbance with reference to that of the digestion products of known amounts of standard UA-GAG. The analytical data obtained with the mixtures of authentic UA-GAG were in close agreement with the theoretical values. Application of this procedure to the urinary GAG fractions from orthopedic patients gave satisfactory results.     

Quantitation and interaction of glycosaminoglycans with Alcian blue in dimethyl sulfoxide solutions.

An improved method is described for the quantitation of glycosaminoglycans separatedon cellulose acetate, stained with Alcian blue, and dissolved in a dimethyl sulfoxide solution. Standard curves are shown for all eight glycosaminoglycans. It is shown that absorption at the Alcian blue orthochromatic E_max is depressed under conditions which favor formation of dye-glycosaminoglycan complexes. The interaction between Alcian blue and the eight glycosaminoglycans was studied in dimethyl sulfoxide solutions of varying composition. It was shown that the extent of complex formation depends both on the glycosaminoglycan and the composition of the dimethyl sulfoxide solution. A dimethyl sulfoxide solution which contains 0.094 m H₂SO₄ is described which maximizes dye-glycosaminoglycan dissociation and thus the absorbance. Also, an improved staining method is described which improves dye uptake by the glycosaminoglycans and consequently increases the sensitivity of glycosaminoglycan quantitation.     

A new continuous, monodimensional electrophoretic system for the separation and quantitation of individual glycosaminoglycans

A rapid and sensitive method for agarose gel electrophoresis is described. By simply miniaturizing a conventional gel electrophoresis apparatus, we have decreased the time necessary for the separation of nucleic acid molecules by a factor of 10. The ability to detect DNA molecules by ethidium bromide fluorescence has simultaneously been increased fivefold. Transfer of DNA from these “minigels” onto nitrocellulose filters followed by hybridization using the procedure of C. M. Southern (1975, J. Mol. Biol.98, 503–517) was found to be efficient and rapid. This technique is sufficiently sensitive to detect radioactive quantities of [³²P]phosphate-labeled DNA or RNA microinjected into 500 chick embryo fibroblasts.     

The use of lectins in the quantitation and analysis of macromolecules by affinoelectrophoresis

The technique of rocket affinoelectrophoresis, initially introduced for the quantitation of a succinylated mannan by concanavalin A [Owen, P., and Salton, M. R. J. (1976) Anal. Biochem. 73, 20–26] has been extended to (a) the quantitation of four other macromolecules: vz. streptococcal lipoteichoic acid, I blood group substance, desialylated bovine submaxillary mucin, and desialylated pig submaxillary mucin; and (b) the use of three other lectins: vz. wheat germ agglutinin, soybean agglutinin and peanut agglutinin. In all cases stable-affinity precipitin rockets were observed the heights of which bore an approximately linear relationships to the amount of sample analyzed. For all lectins, the detection limits were in the range of 15–25 ng. Furthermore, a new technique has been introduced called crossed affinoelectrophoresis which is basically a two-dimensional variant of rocket affinoelectrophoresis. This technique can be used with concanavalin A, wheat germ agglutinin, soybean agglutinin, or peanut agglutinin in the affinity gel and allows the examination of glycoprotein homogeneity. Modification of this technique, involving the use of other lectins or antiserum in intermediate gels, is also described and evaluated.     

Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies

This article describes a simple and selective procedure used for direct measurement of sulfated glycosaminoglycans (GAGs) in biological samples and its application to the determination of GAGs during tissue regeneration and myogenic differentiation. We describe a modified procedure of previous GAG assays that has improved specificity, reproducibility, and sensitivity. The assay is based on the ability of sulfated GAGs to bind the cationic dye 1,9-dimethylmethylene blue. We describe conditions that allow isolation of the GAG-dye complex. This complex was dissociated; the optical density measurement of the dissociated dye permitted quantification of GAGs in biological samples. Applied to the study of myogenic cell differentiation in vitro, muscle repair, and skin ulceration, this method revealed significant modifications in the patterns of expression of different sulfated GAGs in these tissues. In particular, application of the method after nitrous acid treatment revealed that heparan sulfate and chondroitin sulfate ratio changed during muscle regeneration process.     

Improved quantitation of DNA curvature using ligation ladders.

It is often desirable to estimate accurately the local shape of DNA molecules. Such measurements are useful in understanding the intrinsic contribution of DNA sequence to curvature, as well as in assessing the effects of chemical modifications. We have been investigating the effects of asymmetric phosphate neutralization on DNA shape using the well-characterized ligation ladder approach developed by Crothers and co-workers [D.M.Crothers and J.Drak (1992) Meth. Enzymol.,212, 46-71]. This technique is remarkably sensitive to differences in DNA shape. We now report a general quantitative assay of DNA curvature that we have validated using a set of phased A(5)tract standards. This approach allows simultaneous estimation of helix axis deflection magnitude and direction when a test sequence is monitored in at least three phasings relative to a reference A(5-6)tract in short DNA duplexes. Analysis using this improved approach confirms our published data on DNA curvature due to electrostatic effects.