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.     

The histochemistry of urea-unmasked glycosaminoglycans in the skin of the eel, Anguilla japonica

In the connective tissues of the dermis and subcutis of the eel skin, the histochemistry of urea-unmasked glycosaminoglycans has been studied by means of combined staining and enzyme digestion procedures. The staining procedures employed were alcian blue (AB) pH 1.0, AB pH 2.5, aldehyde fuchsin (AF), periodic acid-Schiff (PAS), AB pH 2.5-PAS, high iron diamine (HID) and low iron diamine (LID) methods, whereas the enzymes used were Streptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained have shown that a substantial amount of dermatan sulfate and a relatively small amount of hyaluronic acid, chondroitin, chondroitin sulfate A and/or C were the glycosaminoglycans involved in the connective tissues of the eel skin and that the tissues were devoid of keratan sulfate.     

The effect of digestion with keratanase (Pseudomonas sp.) on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues

Summary The effect of digestion with keratanase (Pseudomonas sp.) on the Alcian Blue (AB) pH 1.0, pH 2.5, Aldehyde Fuchsin, high iron diamine, low iron diamine and dialysed iron-ferrocyanide reactions has been tested in the costal and ear cartilage tissues of the rabbit and corneal tissues of the rat and rabbit. The effect of digestion with chondroitinases ABC and AC on the same reactions was examined in the same tissues for comparison. Digestion with keratanase diminished the intensity of all the reactions in the cartilage tissues to a variable extent; however, the diminutions in intensity of the reactions appeared to be less marked as compared with those following digestion with two chondroitinases. In the corneal stroma, all the reactions were markedly reduced in intensity following digestion with keratanase. In contrast, these reactions were only slightly or moderately diminished in intensity by digestion with the two chondroitinases. As glycosaminoglycans are known to be present in cartilage and corneal tissues and the substrate specificities of the three enzymes used are now well established, the present results are consistent with the concept that keratanase specifically degrades and releases keratan sulphates involved in the tissues.     

Insect mucosubstances. 3. Some mucosubstances of the nervous systems of the wax-moth (Galleria mellonella) and the stick insect (Carausius morosus)

Synopsis A mass of connective tissue, continuous with the neural lamella, develops on the dorsal side of the abdominal region of the nerve cord of Lepidoptera during the pupal stage. The mucosubstances of this tissue in the wax-moth,Galleria mellonella, have been characterized histochemically using various techniques involving Alcian Blue binding, periodic acid-Schiff and high iron diamine reactions, and enzyme digestions. The results indicate that this fibrous tissue contains chondroitin and dermatan sulphates and neutral glycoproteins.Thoracic ganglia of adult stick insects,Carausius morosus, were subjected to the same histochemical tests. The neural lamella possesses chondroitin, dermatan and keratan sulphates, while the glial lacunar system contains only hyaluronic acid.     

The histochemical specificity of Streptomyces hyaluronidase and chondroitinase ABC.

Synopsis Treatment of tissue sections with enzymes which degrade specific types of glycosaminoglycans should provide a means for localizing glycosaminoglycans in tissue sections. The feasibility of this technique was examined by utilizing endogenously labelled glycosaminoglycans in chick and quail embryos. Less than 8% of the total glycosaminoglycans appear to be lost non-specifically during fixation and dehydration. BothStreptomyces hyaluronidase and chondroitinase ABC degraded more than 90% of their respective substrates and demonstrated minimal non-specific extraction of other glycosaminoglycans. The selectivity of chondroitinase ABC for sulphated glycosaminoglycans was substantially increased by raising the pH of the incubation buffer to 8.6. At this pH, chondroitinase ABC degraded negligible amounts of hyaluronic acid. Use of bothStreptomyces hyaluronidase and chondroitinase ABC confirmed that embryonic hyaluronic acid binds Alcian Blue under conditions that were previously believed specific for sulphated glycosaminoglycans. We suggest that this may be due to the increased molecular weight of embryonic hyaluronic acid compared to the hyaluronic acid in adult tissues. The results presented suggest that treatment of adjacent sections with buffer, chondroitinase ABC at pH 8.6, andStreptomyces hyaluronidase and subsequent staining with Alcian Blue provides a method for localizing and quantitating glycosaminoglycans in tissue sections.     

Insect mucosubstances. II. The mucosubstances of the central nervous system

Synopsis The glycosaminoglycans of the glial lacunar system and neural lamella of cockroach and locust ganglia have been characterized histochemically, using primarily Alcian Blue binding methods at various pH levels and salt concentrations, the periodic acid-Schiff test together with recent modifications, the high iron diamine test, and enzymatic digestions. The results suggest the presence of hyaluronic acid in the glial lacunar system and of a mixture of chondroitin and dermatan sulphates, together with keratan sulphate in the neural lamella. The significance of the presence of these substances in the central nervous system of insects is discussed.     

Effects of novel (Streptomyces) hyaluronidase digestion upon some mucosaccharide stainings of the cartilages and aortas in the rabbit and rat

Summary Effects of novel (Streptomyces) hyaluronidase digestion upon the alcian blue (pH 1.0), alcian blue (pH 2.5), periodic acid-Schiff (PAS) and alcian blue-PAS stainings of mucopolysaccharides have been tested in the cartilage and aorta tissues of the rabbit and rat. These effects were compared with those of testicular hyaluronidase digestion upon the same stainings in the tissues. Evidence has been presented that a Streptomyces hyaluronidase digestible alcian blue (pH 2.5) reactive mucosubstance (hyaluronic acid) is present in the peripheral areas of the cartilage tissues and all the layers of the aorta tissues. Furthermore, the histochemical data obtained in this study appear to present a base line for establishing a promising enzyme digestion technique for the identification of hyaluronic acid in mucosaccharide histochemistry.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Synthesis of 1-(2,6,6-Trimethyl-4-hydroxy-1-cyclohexen-1-yl)-1,3-butanedione

Streptococcus dysgalactiae IID 678, belonging to group C of the streptococci, secreted a large amount of hyaluronidase (hyaluronate lyase, EC 4.2.2.1) into a culture medium containing hyaluronic acid. The purification procedures of hyaluronidase were 70% ammonium sulfate precipitation, ECTEOLA-cellulose chromatography, phospho-cellulose chromatography, and gel filtration on Sephacryl S-300. The hyaluronidase was purified approximately 27,000-fold from the culture filtrate. The purified enzyme was homogeneous by SDS-poIyacrylamide gel electrophoresis. The enzyme degradated only hyaluronic acid and chondroitin to zl 4,5-unsaturated disaccharides and did not act on other glycosaminoglycans containing sulfate groups, while the degradation rate of chondroitin was about 1/60 of that of hyaluronic acid. The optimum pH was wide, from pH 5.8 to pH 6.6, and the optimum temperature was 37°C. Fe^{2 +}, Cu^{2 +}, Pb^{2 +}, and Hg^{2 +} ions inhibited the activity strongly and Zn²⁺ inhibited it by half. The molecular weight of the enzyme was estimated to be 125,000 by gel filtration and 117,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was different immunochemically from the hyaluronidase from Streptococcus pyogenes belonging to group A.     

Insect mucosubstances. I. The mucosubstances of developing connective tissue in the locust,Locusta migratoria

Synopsis A layer of collagenous connective tissue develops around the ejaculatory duct of the male locust,Locusta migratoria, during the fourth and fifth instars and the first ten days of the adult stage. The mucosubstances associated with this tissue have been characterized by using a series of histochemical reactions, including Alcian Blue staining at different pH levels and salt concentrations the periodic acid-Schiff test and recent modifications of it, the high iron diamine test and enzymatic digestions. The results indicate that sulphated glycosaminoglycans accumulate during the developmental period, so that in the mature adult, the connective tissue probably contains chondroitin and dermatan sulphates, and possibly some keratan sulphate. Neutral glycoproteins also occur in the tissue.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS FROM BRAIN OF CHILDREN WITH PROTEIN-CALORIE MALNUTRITION

Abstract— The uronic acid containing glycosaminoglycans (GAGs) were isolated from the brains of 1-year-old and 4-year-old kwashiorkor children and characterised by constituent analyses. A marked reduction is the total GAG concentration of brain was noticed in both cases of kwashiorkor. In the 1-year-old kwashiorkor brain, hyaluronic acid is the most predominant GAG (73.5 per cent) whereas heparan sulphate, chondroitin sulphates and low sulphated chondroitin sulphate constituted less than 10 per cent. In the 4-year-old kwashiorkor brain, the proportion of hyaluronic acid was 27.5 per cent, low sulphated chondroitin sulphate 31.2 per cent, chondroitin sulphates 28.3 per cent and heparan sulphate 10 per cent. This marked reduction in the concentration as well as qualitative changes in GAG in protein-calorie malnutrition as compared to the normal is discussed in relation to brain function.     

Histochemical reactions of gastrointestinal mucosubstances with orcein,high iron diamine and alcian blue after prior oxidation of tissue sections

Summary The histochemical orcein reaction (orc) for mucosubstances in tissue samples from the human gastrointestinal tract was compared with PAS, high iron diamine (HID) and Alcian blue reactions at pH 1.0 or 2.5 (AB 1 and AB 2.5). Orc, HID and AB 1 reactions were performed also with prior oxidation of the tissue sections with potassium permanganate or performic acid (ox-orc, ox-HID and ox-AB reactions, respectively). Orc reaction stained mucosubstances similarly to HID and AB 1; only the brush border and goblet cells in the colon were stained. The reactions of the mucosubstances obtained with ox-orc differed from those with PAS, HID, AB 1 or AB 2.5 but were similar to those with ox-HID or ox-AB; the mucosubstances in the brush border and the goblet cells in the colon and small bowel and in the foveolar epithelium of the stomach were strongly stained. Pyloric and cardiac glands were stained faintly with ox-orc but not with ox-HID or ox-AB. Brunner's glands were negative with ox-orc, ox-HID and ox-AB reactions. It was assumed that the orc reaction stains, like HID or AB 1, sulphate groups in epithelial mucosubstances, and that sulphonic acid residues, resulting from oxidation of disulphide groups in the protein core of mucus glycoproteins, are responsible for the ox-orc as well as for the ox-HID and ox-AB reactions.The study was supported by grants from the Cancer Society of Finland, Foundation of Orion Corporation and from the Paulo's Foundation, Helsinki, Finland     

Tunicate Endostyles: Histochemistry of Acidic Glycoproteins Re-examined in Ciona intestinalis and Styela plicata

Previous reports of tunicate endostyles have suggested that they contain little or no acidic glycoproteins in the glandular zones. The endostyles of Ciona intestinalis and Styela plicata were examined after anhydrous fixation with cyanuric chloride. Polyanions were stained with alcian blue (AB) at pH 2.5 or azure A, while sulfomucins were stained with high-iron diamine (HID) or AB at pH 1.0. Endostyles were also tested for sensitivity to acid hydrolysis (AH) and saponification. In Ciona zones 2 and 4 sometimes demonstrated positive HID and AB 1.0 responses. Almost invariably zone 6 was AB+ at pH 2.5; zones 2 and 4 were frequently responsive to AB, but less intense. Each of these 3 zones, when AB+, was sensitive to AH. Responses by zones 3 and 5 to AB (pH 2.5), azure A and saponification suggest that these zones contain mostly nuclear material. In secretory zones 2, 4 and 6 histological responses are consistent with the histochemistry of sialomucins. Zones 1 and 8 had sulfated material in the apical edges in both animal groups. Among the fixatives used for Ciona, only anhydrous fixation demonstrated most of the positive responses to polyanion-sensitive stains.     

Binding of the basement-membrane glycoprotein laminin to glycosaminoglycans. An affinity-chromatography study.

The binding of the basement-membrane glycoprotein laminin to glycosaminoglycans (aggregating and non-aggregating subsets of heparan sulphates and dermatan sulphates, as well as heparin, chondroitin sulphates and hyaluronic acid) was studied by affinity chromatography. Partially periodate-oxidized chains of glycosaminoglycans were coupled to adipic acid dihydrazide-substituted agarose. Co-polymeric glycosaminoglycans reveal high affinity for laminin, whereas hyaluronic acid does not. Competitive-release experiments indicate that glycosaminoglycans share a common binding site on the laminin molecule.     

A chondroitin sulphate proteoglycan from human cultured glial cells aggregates with hyaluronic acid

Media harvested from cultures of glial cells grown in the presence of ³⁵S-sulphate were shown to contain ³⁵S-labelled proteoglycans. One of the components was a chondroitin sulphate proteoglycan that had an apparent monomer size similar to that of cartilage-derived chondroitin sulphate proteoglycan. The glial proteoglycan formed aggregates in the presence of hyaluronic acid; aggregation was abolished in the presence of deca- to tetradecasaccharides derived from hyaluronic acid or by previous reduction and alkylation of the proteoglycan. It is concluded that the ability to produce large chondroitin sulphate proteoglycan molecules capable of binding to hyaluronic acid is not confined to cartilage cells.     

Glycosaminoglycan synthesis in the chick gastrula

Explanted definitive primitive streak to four somite chick embryos were labeled with [H³]glucosamine or S³⁵O₄ and the glycosaminoglycans were isolated and characterized. On the basis of susceptibility to Streptomyces hyaluronidase, which specifically degrades hyaluronic acid, hyaluronic acid is the major glycosaminoglycan produced by these embryos (at least 84%). On the basis of electrophoretic mobility, about 10% of the [H³]glucosamine-labeled glycoaminoglycan is sulfated. At least 55% of the sulfate-labeled glycosaminoglycan is sensitive to testicular hyaluronidase, and 36–39% is resistant to testicular hyaluronidase, but sensitive to nitrous acid treatment. About 94% of the labeled glycosaminoglycans can be accounted for in ratios of 22:1:5:1 as hyaluronic acid:chondroitin sulfate:heparan sulfate. No stage-related changes were observed. It is suggested that hyaluronic acid synthesis at this time might be related to the appearance of extensive cell-free spaces.     

A new method for sequence analysis of glycosaminoglycans from heavily substituted proteoglycans reveals non-random positioning of 4- and 6-O-sulphated N-acetylgalactosamine in aggrecan-derived chondroitin sulphate.

We have developed a new procedure for the sequence analysis of glycosaminoglycans, which is particularly suitable for the analysis of chains from heavily substituted proteoglycans. The procedure has been applied to various aggrecan-derived chondroitin sulphates. The glycans are released from the core protein by alkaline scission of the xylose-serine bond, subjected to reductive amination using p-aminobenzoic acid and finally radioiodinated at an acidic pH. Sequence analysis is performed by using various enzymic degradations, partial or complete, followed by high-resolution polyacrylamide gel electrophoresis, blotting and autoradiography to identify segments extending from the labelled reducing end to the point of cleavage. By using chondroitin C lyase to identify the location of 6-O-sulphated hexosamines, we find that chondroitin sulphate from tracheal cartilage has its 6-O-sulphated repeats concentrated to the extreme non-reducing terminal portion of the chain. In chondroitin sulphates derived from intervertebral discs (nucleus pulposus), the 6-O-sulphated repeats have a biphasic distribution; they occur mostly near the linkage region (i.e. the reducing end), but also in the non-reducing portion of the chain. Chondroitin sulphate from nasal cartilage, which is mostly 4-O-sulphated, displays considerable heterogeneity in the linkage region. Three or possibly more charge variants are observed.     

Type, location and role of glycosaminoglycans in cloned differentiated chick retinal pigmented epithelium

In clonal culture, colonies of 3–4 week old chick retinal pigmented epithelial cells exhibit Alcian Blue positive extracellular matrix (ECM) material on the surface of the cells. Alcian blue positive ECM is located between undifferentiated cells at the edges of the disc-shaped colonies and beneath the differentiated cells in the colony center. The latter material is associated with the basement membrane. The staining properties suggest that glycosaminoglycans (GAG) are present in these regions. Extraction of GAG from homogenates of colonies, followed by electrophoresis on cellulose acetate strips, results in three bands with mobilities similar to those of hyaluronic acid, heparan sulfate, and chondroitin sulfate, respectively. All three bands label with [³H]glucosamine, and the last two also label with [³⁵S]sulfate. The composition appeared to differ when colonies were grown in different media. Digestion of the GAG preparations with various enzymes suggests that bands II and III represent heparan sulfate and chondroitin sulfate, respectively, in colonies grown in Ham's F10g medium. The composition of band I is as yet undetermined. In minimal Eagle's medium (MEM), bands I and III consisted of hyaluronic acid and chondroitin sulfate, respectively, while band II had properties suggestive of a copolymer of heparan sulfate and an unidentified GAG. Cells release only one [³H]glucosamine-labelled GAG into the medium. This material has a mobility similar to hyaluronic acid and is digested by Streptomyces hyaluronidase, suggesting that it is hyaluronic acid. Staining with Alcian Blue at different pH suggests that it may represent the material associated with the upper surface of the cells. Some of the ECM located between the undifferentiated cells and associated with the basement membrane in the differentiated regions of the colonies stains with Alcian Blue at pH 1.0 and 0.2 suggesting that it may contain GAGs found in bands I and II. Colonies treated with medium containing 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of GAG synthesis, for 48 hr showed a reduced Alcian Blue staining of the ECM in the undifferentiated regions. After 72 hr of treatment with DON, the undifferentiated cells had detached from the plate, whereas the differentiated cells remained intact. The results suggest that the GAG may be involved in cellular adhesion, particularly of the undifferentiated cells.     

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. II. The glycosaminoglycans

Summary The glycosaminoglycans secreted into the matrices associated with fractures of the rabbit tibia healing under stable and unstable mechanical conditions have been characterized histochemically using the dye Alcian Blue at pH 5.7 in the presence of increasing concentrations of magnesium chloride, and after enzymatic extractions. These results are compared with those of immunohistochemical experiments using monoclonal antibodies which recognize epitopes specific to various glycosaminoglycans.The results indicate that the fibrous tissues, including those of the cavities of the cancellous bone and periosteum, possess hyaluronate and chondroitin sulphate, but the amounts present are small. The glycosaminoglycans detected in the cortical bone are located mainly around the osteocyte lacunae where chondroitin and keratan sulphates are found. The developing trabeculae of cancellous bone in the callus contain chondroitin and keratan sulphates, but as the trabeculae mature, these glycosaminoglycans are no longer present throughout the matrix; they are found particularly around the osteocyte lacunae.The cartilage in the callus of mechanically unstable fractures contains chondroitin, chondroitin-4- and 6-sulphates and keratan sulphate, though their distribution is variable. The small, transient areas of cartilage in the callus of mechanically stable fractures also contain those glycosaminoglycans, but they appear to be less highly sulphated.The mechanical stability of the fractures appears to affect the amount and degree of sulphation of the glycosaminoglycans, rather than the types of glycosaminoglycan produced. The glycosaminoglycans produced during fracture healing are compared with those produced during embryonic development and other healing processes.     

Crystallization and some properties of chondroitinase from Arthrobacter aurescens.

A strain of Arthrobacter aurescens which secretes a large amount of chondroitinase into a culture broth, was isolated from soil. The chondroitinase was purified 380-fold over culture broth in 24% yield and crystallized. Some properties of the purified enzyme were studied and described: thermal stability (below 45 degrees), pH stability (pH 4.9 to 7.4), optimum temperature (50 degrees), and optimum pH (pH 6.0). Chrondroitin sulfate A and C, chondroitin, and hyaluronic acid were split by the enzyme but dermatan sulfate could not be. The initial rates of enzymic degradation of chondroitin sulfate C, chondroitin, and hyaluronic acid were 1.1, 1.95, and 3.2, respectively, compared to that of chondroitin sulfate A. When the enzyme was allowed to act on chondroitin sulfate A and C, the reducing power and the ultraviolet absorption at 232 nm increased proportionally to the decrease in viscosity of the substrate solution. Finally these substrates were degraded to the extent of 100% to disaccharides. By the enzyme action the main products from chondroitin sulfate A and C were deta 4,5-unsaturated disaccharides, which were identified as 2-acetamido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(Beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose by paper chromatography, ultraviolet absorption spectroscophy, and infrared spectroscopy. Thus it is suggested that the chondroitinase is a chondroitin sulfate A and C lyase, one of the hyaluronate lyases (EC 4.2.99.1).