Histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus

Summary I investigated the ultrastructural localization and histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus, by use of the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. HID-TCH-SP stain deposits were localized in the dental basal lamina and in the whole thickness of developing enameloid matrix, particularly closely associated with enameloid collagen fibrils. Most HID-TCH-SP stain deposits in the enameloid were susceptible to testicular hyaluronidase but some stain deposits survived. HID-TCH-SP stain deposits in the basal lamina resisted the enzymatic digestion, and were regularly localized to the internal and external sites of lamina densa at an early stage of development, subsequently tending to be randomly arranged with the increase in thickness of enameloid matrix layer. Furthermore, enzymatic digestion with heparitinase preferentially removed HID-TCH-SP stain deposits in the region of the basal lamina. Thus, it was confirmed that most HID-TCH-SP stain deposits in developing enameloid matrix are chondroitin 4-sulfate and/or 6-sulfate and that the stain deposits in the basal lamina represent heparan sulfate. The chondroitin sulfates tended to disappear with the advancement of enameloid mineralization.     

Ultrastructural distribution of sulfated glycosaminoglycans in epithelial-mesenchymal interface of developing rat tooth germs

We investigated the ultrastructural distribution of sulfated glycosaminoglycans in the epithelial-mesenchymal interface of tooth germs by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion method. At an early stage in odontoblast differentiation, HID-TCH-SP stain deposits were sparsely distributed in the basement membrane and in the intercellular spaces. Subsequently, as formation of the initial predentin matrix began, HID-TCH-SP stain deposits were densely distributed in the interfibrillar spaces and the basement membrane. Testicular hyaluronidase digested most of those in the progenitor pre-dentin, whereas those in the region of basal lamina resisted enzymatic digestion. Testicular hyaluronidase-resistant HID-TCH-SP stain deposits were susceptible to heparitinase, indicating that the sulfated glycosaminoglycan in the basal lamina is heparan sulfate. Furthermore, the heparan sulfate tended to be regularly arranged at the sites of internal and external lamina densa. However, as progenitor pre-dentin matrix formation proceeded, the numbers of stain deposits temporarily increased and their distribution pattern became irregular, finally tending to disappear with the disruption of basal lamina.     

Heterogeneity of distribution pattern at the electron microscopic level of heparan sulfate in various basement membranes.

Using the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique and enzymatic digestion, we investigated the ultrastructural distribution pattern of heparan sulfate side chains of heparan sulfate proteoglycan (HSPG) in various basement membranes (nerve, capillary, oral epithelial, muscle, and dental basement membranes). Four different distribution patterns of stain deposits were identified as heparan sulfate on the basis of enzymatic degradation by heparitinase. In some basement membranes associated with tooth germs and oral epithelium, HID-TCH-SP stain deposits were regularly located at both sides of the lamina densa, but few were observed in the lamina densa itself. In nerve, muscle, and capillary basement membranes, the stain deposits were localized at the external side of the lamina densa adjacent to the underlying connective tissue, but were not found in the laminae lucida and densa. In the internal basal lamina of junctional epithelium of gingiva, the stain deposits were detected mainly in the lamina lucida region. Finally, in some dental and oral epithelial basement membranes, the stain deposits were randomly distributed throughout both laminae lucida and densa. Thus, the present study demonstrated distinct differences in heparan sulfate distribution pattern among various basement membranes, suggesting their architectural heterogeneity.     

True enamel matrix of the newt,Triturus pyrrhogaster,contains no sulfated glycoconjugates

Summary The ultrastructural distibution and histochemical properties of sulfated glycoconjugates were investigated in the developing enamel of the adult newt, Triturus pyrrhogaster, by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. Development and ultrastructure of the enamel were also studied. After deposition of the mantle dentin matrix to a certain thickness, the first enamel matrix, globular in shape, appeared in juxtaposition to the dental basement membrane and tended to be intermixed with the previously deposited dentin matrix. Subsequently, enamel matrix was deposited outside (ameloblastic side) of the dental basal lamina and formed a true enamel layer. Thus, developing enamel of the newt consists of two layers: (1) an inner layer made up of a dentin-enamel mixed matrix and (2) an outer layer composed of only true enamel matrix. HID-TCH-SP precipitates resulting from the abovementioned studies were found in the mixed matrix and were identified as chondroitin sulfates; in contrast, the true enamel matrix contained no sulfated glycoconjugates.     

Ultrastructural cytochemistry and immunocytochemistry of proteoglycans associated with epiphyseal cartilage calcification

Proteoglycans (PGs) are closely associated with cartilage calcification. We have examined the hypertrophic zone of rat epiphyseal cartilage, in which calcification is occurring, using the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycosaminoglycans, an immunoferritin method specific for chondroitin sulfate A, and the tannic acid-ferric chloride (TA-Fe) method to stain cartilage matrix granules (MGs) presumed to be PG monomers. HID-TCH-SP produced stain deposits with a diameter of 11.2 +/- 3.2 nm (mean +/- SD; n = 200) in the MGs. However, HID-TCH-SP staining was not discernible in membrane-limited matrix vesicles (MVs). In areas of advanced calcification, partially disrupted MVs and globular bodies (GBs), derived in part from disrupted and/or degenerated MVs, contained a few too many small HID-TCH-SP stain deposits. Further down the epiphyseal cartilage, intact MVs markedly decreased and the GBs, containing many small HID-TCH-SP stain deposits, significantly increased in number. These GBs were found exclusively in the longitudinal septa rather than in the transverse septa. After enzyme digestion with testicular hyaluronidase, small (7.2 +/- 1.2 nm in diameter) stain deposits remained in the MGs and GBs, presumably localized to keratan sulfate. Immunoferritin localizing chondroitin sulfate strongly stained MGs, whereas MVs and GBs lacked staining. TA-Fe staining of glycoconjugates in the GBs demonstrated a striking decrease in the diameter of MGs associated with calcification in the GBs as compared with those in the noncalcifying area around the GBs. These results indicate that the GBs containing needle-like apatite crystals in morphologic preparations represent sites of chondroitin sulfate degradation. Testicular hyaluronidase-resistant sulfated glycosaminoglycans presumed to be keratan sulfate and partially degraded PGs selectively remain within the GBs as a probable requisite for expansion of the initial calcification in MVs.     

A single chondroitin 6-sulfate oligosaccharide unit at Ser-2730 of human thyroglobulin enhances hormone formation and limits proteolytic accessibility at the carboxyl terminus. Potential insights into thyroid homeostasis and autoimmunity

We localized the site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg). hTg was chromatographically separated into chondroitin 6-sulfate-containing (hTg-CS) and chondroitin 6-sulfate-devoid (hTg-CS0) molecules on the basis of their D-glucuronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6%. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondroitin 6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxyl-terminal region, starting at residue 2514. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as LTAGXGLRE (residues 2726-2734, X being Ser2730 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5 '-triiodothyronine (T3) (171%) and 3,5,3',5'-tetraiodothyronine (T4) (134%) than hTg-CS0. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, in particular, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Lys2714-Gly2715 from proteolysis, during the limited digestion of hTg-CS with trypsin. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg. The potential implications in the ability of hTg to function as an autoantigen and into the pathogenesis of thyroidal and extra-thyroidal manifestations of autoimmune thyroid disease are discussed.     

Cytochemical characterization of basement membranes in the enamel organ of the rat incisor

Ameloblasts are unique epithelial cells, in that once they have deposited the entire thickness of enamel and the process of maturation begins, they reform a basal lamina-like structure at their apical surface. In order to characterize further this basal lamina, its composition was analysed using (1) lectin-gold cytochemistry for glycoconjugates, (2) high-iron diamine (HID) staining for sulfated glycoconjugates and (3) immunogold labeling for collagen type IV and laminin. The labeling patterns were compared to that of other more typical basement membranes found in the enamel organ. Sections of rat incisor enamel organs embedded in Lowicryl K4M were stained with Helix pomatia agglutinin (HPA), Ricinus communis I agglutinin (RCA), wheat germ agglutinin (WGA) and Ulex europaeus I agglutinin (UEA). Samples from the late maturation stage were also reacted en bloc with lectins and embedded in Epon for transmission electron microscopic examination or prepared for scanning electron microscopy. Such samples were also stained with HID and conventionally processed for Epon embedding. Tissue sections were then reacted with thiocarbohydrazide-silver proteinate (TCH-SP). Analysis of the lectin labeling suggested that the region of extracellular matrix immediately adjacent to ameloblasts, where the basal lamina is situated, was intensely reactive with HPA and RCA, moderately reactive with WGA, and weakly reactive with UEA. In general, other basement membranes were mildly reactive with all lectins used. No HID-TCH-SP staining was observed directly over the basal lamina while numerous stain deposits were present over other basement membranes of the enamel organ. Immunolocalization of collagen type IV and laminin yielded a weak and variable labeling over the basal lamina. These results are consistent with the concept of basement membrane heterogeneity and, although the precise nature and composition of the basal lamina associated with maturation stage ameloblasts remain to be determined, they suggest that it may possibly function as a specialized basement membrane with particular compositional characteristics.     

Location and chemical composition of anionic sites in Bruch's membrane of the rat

The location and chemical composition of anionic sites in Bruch's membrane (BM) were examined using cationic probe molecules demonstrable in electron microscopic preparations and tissue digestion with specific degradative enzymes. Ruthenium red and native lysozyme revealed densities distributed at regular intervals in two major components of BM: the basal laminae of the retinal pigment epithelium (RPE) and choriocapillary endothelium (EN). Staining was not observed with succinylated lysozyme (anionic). Colloidal iron also failed to stain BM components. Following crude heparinase treatment at 43 degrees C (specific for heparan sulfate) anionic sites in the RPE basal lamina were not demonstrable with either ruthenium red or native lysozyme. Sites in the EN basal lamina were not affected. Chondroitinase treatment removed almost all of the ruthenium red-positive material in the EN basal lamina; lysozyme binding here was markedly reduced. No changes were observed in the RPE basal lamina after chondroitinase digestion. There was no morphological evidence for site removal by either neuraminidase or leech hyaluronidase, although a detachment of the RPE from BM often occurred after incubation of eye tissue in the latter. Pronase E removed all stainable material. These findings indicate that anionic sites in BM consist to a large extent of chondroitin sulfates and heparan sulfate.     

Histochemical identification of sulfation position in chondroitin sulfate in various cartilages

The ability of chondrocytes to synthesize chondroitin-4-sulfate (C4S) as opposed to chondroitin-6-sulfate (C6S) is a phylogenetically related phenomenon seen among adult higher vertebrates and developmentally during the embryogenesis of these vertebrates. While the embryonic cartilage may be initially a C6S matrix, C4S synthesis is seen to develop with time. We have histochemically localized these differences in sulfation with the cationic carbocyanine dye, Stains-all, in a spectrum of cartilages that vary in the sulfation position of their chondroitin sulfate. Cartilages from the rat and rabbit that are predominantly C4S stained magenta at pH 4.3, while the C6S-rich cartilage matrices from the regenerating rabbit ear and lamprey cranium stained blue. Embryonic chicken cartilages develop a gradient of magenta matrix with age, with increased concentration toward the articular surface. Both magenta and blue matrices were absent after pretreatment with chondroitinase ABC but were present after Streptomyces hyaluronidase digestion. The magenta staining was a property of the cartilage matrix as a whole, since isolated C4S and C6S stained blue. The differential staining was seen at pH 4.3, but not at pH 8.8, suggesting an interaction between the chondroitin sulfate and the adjacent tissue proteins.     

Fine structural and cytochemical observations of dental epithelial cells during the enameloid formation stages in red stingrays Dasyatis akajei

The fine structure and the localization of nonspecific acid phosphatase (ACPase), nonspecific alkaline phosphatase (ALPase), and calcium-dependent adenosine triphosphatase (Ca-ATPase) activities in the dental epithelial cells in tooth germs of Dasyatis akajei in the later stages of enameloid formation were investigated. Numerous invaginations of the distal cell membrane of the inner dental epithelial (IDE) cells were observed at the early stage of enameloid maturation. The invaginations contain many fine granular and filamentous substances; the lamina densa, which was thicker during the former stages, is obscure. Granules exhibiting defined ACPase activity were usually found in the IDE cells during the stages of enameloid mineralization and maturation. IDE cells are putatively involved in the removal of degenerated enameloid matrix during these stages. Marked ALPase activity was detected at the proximal and the lateral cell membranes of the IDE cells from the late stage of enameloid matrix formation to the early stage of enameloid maturation. Strong activity of Ca-ATPase was localized at the proximal and the lateral cell membranes of the IDE cells during the stages of enameloid mineralization and maturation. ALPase and Ca-ATPase activity is probably related to crystal formation in the enameloid and the removal of degenerated enameloid matrix from the enameloid.     

Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.     

Metastatic melanoma cell heparanase. Characterization of heparan sulfate degradation fragments produced by B16 melanoma endoglucuronidase

Heparan sulfate (HS), a prominent component of vascular endothelial basal lamina, is cleaved into large Mr fragments and solubilized from subendothelial basal lamina-like matrix by metastatic murine B16 melanoma cells. We have examined the degradation products of HS and other purified glycosaminoglycans produced by B16 cells. Glycosaminoglycans 3H-labeled at their reducing termini or metabolically labeled with [35S]sulfate were incubated with B16 cell extracts in the absence or presence of D-saccharic acid 1,4-lactone, a potent exo-beta-glucuronidase inhibitor, and glycosaminoglycan fragments were analyzed by high speed gel permeation chromatography. HS isolated from bovine lung, Engelbreth-Holm-Swarm sarcoma, and subendothelial matrix were degraded into fragments of characteristic Mr, in contrast to hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, keratan sulfate, and heparin which were essentially undegraded. Heparin, but not other glycosaminoglycans, inhibited HS degradation. The time dependence of HS degradation into particular Mr fragments indicated that HS was cleaved at specific intrachain sites. In order to determine specific HS cleavage points, HS prereduced with NaBH4 was incubated with a B16 cell extract and HS fragments were separated. The newly formed reducing termini of HS fragments were then reduced with NaB[3H]4, and the fragments hydrolyzed to monosaccharides by trifluoroacetic acid treatment and nitrous acid deamination. Since 3H-reduced terminal monosaccharides from HS fragments were overwhelmingly (greater than 90%) L-gulonic acid, the HS-degrading enzyme responsible is an endoglucuronidase (heparanase).     

Basal lamina of embryonic salivary epithelia. Production by the epithelium and role in maintaining lobular morphology

The role of the basal lamina in maintaining the normal morphology of mouse embryo submandibular epithelia was assessed by examining its production as well as the cellular and organ culture changes associated with its removal and replacement. The lamina was removed from epithelia isolated free of mesenchyme by brief treatment with testicular hyaluronidase in the absence of calcium. The treatment causes rounding- up of the cells, loss of cellular cohesion, appearance of microvilli, and changes in the organization of cytoskeletal structures. The lamina is not removed and the cellular alterations do not occur in the absence of hyaluronidase in calcium-free medium or when both enzyme and calcium are present, possibly because digestion of chondroitin sulfate, a component of the lamina, is inhibited by calcium. Within 2 h after treatment, in the absence of mesenchyme or biological substrata, the epithelia deposits a new lamina, which is identical by several criteria to the preexisting lamina, and reverses the cellular alterations. Epithelia treated with hyaluronidase lose lobular morphology during culture with mesenchyme. Delaying culture with mesenchyme, to allow restoration of the lamina and of normal cellular architecture, prevents the loss of lobular morphology. The results indicate that the basal lamina imposes morphologic stability on the epithelium, while the mesenchyme apparently affects processes involved in changes in morphology, possibly by selective degradation of the basal lamina.     

Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicer's hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiéry's periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.     

Immunocytochemical Localization of Chondroitin and Chondroitin 4- and 6-Sulfates in Developing Rat Cerebellum

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a chondroitin 4-sulfate proteoglycan carrier for heparin neutralizing activity (platelet factor 4) released from human blood platelets

Platelet heparin neutralizing activity (platelet factor 4) is released from human blood platelets by thrombin in the form of a high molecular weight proteoglycan-platelet factor 4 complex. This complex was partially purified by isoelectric precipitation and gel filtration. At high ionic strength (I = 0.75) the complex dissociates into the active component (mol. wt 29000) and the proteoglycan carrier. The components were separated by gel filtration and the proteoglycan further purified by Na₂SO₄ treatment. The molecular weight of the purified carrier was 59000. The carbohydrate moieties of the proteoglycan isolated after papain digestion and ion-echange chromatography were shown to consist of chondroitin 4-sulfate by chemical, physical and electrophoretic analysis. The multichain proteoglycan consists of four chondroitin 4-sulfate chains (mol. wt 12000) in covalent linkage to a single polypeptide. The molecular weight (350000) of the fully saturated proteoglycan carrier suggests that 4 moles of platelet factor 4 are bound per mole of proteoglycan and that the carrier occurs in the form of a dimer consisting of 8 moles of platelet factor 4 and 2 moles of proteoglycan. The isolated chondroitin 4-sulfate moieties combine with platelet factor 4 at a binding ratio of one mole of platelet factor 4 per carbohydrate chain. Heparin completely displaces platelet factor 4 from both the saturated proteoglycan and chondroitin 4-sulfate complexes. Heparitin sulfate, dermatan sulfate and chondroitin 6-sulfate also combine stoichiometrically with platelet factor 4 and are displaced by equimolar amounts of heparin. Hyaluronic acid did not combine with platelet factor 4. The relative binding capacities of glycosaminoglycans for platelet factor 4 were shown to be: heparin (100), heparitin sulfate (75), chondroitin 4-sulfate (50), dermatan sulfate (50), chondroitin 6-sulfate (50), and hyaluronic acid (o). Chondroitin 4-sulfate was identified as the major glycosaminoglycan in all platelet subcellular fractions; in addition, the soluble fraction contains a minor amount of hyaluronic acid. Subcellular distribution studies revealed that 55% of both the proteoglycan carrier and platelet factor 4 activity were localized in the “granule rich” fraction. This data together with the low recovery of both these components in the membrane fraction, suggest that they occur together as a complex within specific granules and are released in this form under physiologic conditions.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Epitope-specific changes in chondroitin sulfate/dermatan sulfate proteoglycans as markers in the lymphopoietic and granulopoietic compartments of developing bursae of Fabricius

The types and distributions of chondroitin sulfate proteoglycans within developing chick bursae of Fabricius were determined by indirect immunocytochemical analyses using mAb specific for chondroitin/dermatan sulfate epitopes. Analyses obtained from the use of well characterized mAb known to specifically identify chondroitin 4- and dermatan sulfates (antibody 2B6) and chondroitin 6-sulfate (antibody 3B3) were compared with those obtained from two additional mAb raised against chick chondroitin sulfates proteoglycans derived from hemopoietic tissue. The results indicate that chondroitin sulfate compositions of the adjacent lymphopoietic and granulopoietic compartments differ. Chondroitin 6-sulfate, notably absent from lymphopoietic regions, is a major chondroitin sulfate species in granulopoietic regions of day 13 bursae. Moreover, chondroitin 6-sulfate disappears from the granulopoietic compartment in a time course that corresponds to the decline in granulopoietic activity. Simultaneously, there is an apparent increase in chondroitin sulfates associated with developing medullary regions of lymphoid follicles. The content of chondroitin 4-/dermatan sulfates and, most significantly, of chondroitin/dermatan sulfates identified by antibodies raised against chick proteoglycans, increases within developing follicles. As a consequence, by day 18 of incubation, immunostained follicles become clearly demarcated from the connective tissue of the tunica propria. This study provides evidence that chondroitin sulfates are constituents of both lymphopoietic and granulopoietic microenvironments and that subtle changes occur within these proteoglycan structures during bursal development. These developmental changes in chondroitin sulfate compositions are consistent with these molecules playing a functional role in hemopoiesis.     

Developmental distribution of glycosaminoglycans in embryonic rat brain: Relationship to axonal tract formation

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system. © 1996 John Wiley & Sons, Inc.     

Selachian tooth development: III. Ultrastructural features of secretory amelogenesis in Squalus acanthias

Ultrastructural features of secretory amelogenesis during selachian tooth development show several similarities to mammalian amelogenesis. However, the following critical differences were noticed: 1) subcellular organelles associated with merocrine-type protein synthesis and secretion were located in both the infranuclear as well as supranuclear regions of the selachian ameloblasts; 2) no evidence for Tomes process formation was found; 3) the basal lamina was not removed during epithelial differentiation into ameloblasts in the selachian model, and the structural features of the basal lamina were significantly altered during amelogenesis in rows III, IV, and VI; and 4) no dentine-enameloid junction was detected. It is suggested that enameloid is an extracellular matrix which is derived from the selachian inner enamel epithelium and appears to be secreted from both the lateral and apical surfaces of ameloblasts.