High Iron Diamine-thiocarbohydrazide-silver Proteinate Research Articles

Differences in the ultrastructural localization of sulfated glycoconjugates between dentine and bone in the ganoid scales of Polypterus senegalus

ObjectivesSulfated glycoconjugates (S-GCs) are essential components of dentinogenesis and osteogenesis, and are involved in the regulation of the mineralization process. The scales of Polypterus senegalus, which are homologous to teeth and phylogenetically derived from ancestral dermal skeleton, comprise true enamel, dentine, and bone. As part of the phylogenic evolutional studies on mesenchyme-derived hard tissues, we investigated the ultrastructural distribution patterns and histochemical properties of S-GCs in the dentine and bone of scales. MethodsFor detection of S-GCs, a high iron diamine–thiocarbohydrazide–silver proteinate (HID–TCH–SP) staining technique was used. ResultsS-GCs were observed in unmineralized predentine and osteoid, mineralized dentine, osteocyte pericellular matrix, but not in mineralized bone and isopedine. Most S-GCs in the dentine and osteoid were susceptible to testicular hyaluronidase, indicating that they are chondroitin sulfates. ConclusionDistinct differences in the S-GC distribution pattern between the dentine and bone were observed. The S-GC distribution pattern associated with the dentinogenesis of scales is very similar to that of mammalian mantle dentine.

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

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.

Sulfated Glycoconjugates Demonstrated in Combination with High Iron Diamine Thiocarbohydrazide-Silver Proteinate and Silver Acetate Physical Development

Sulfated glycoconjugates in epithelial cells and mesenchymal cells were investigated after staining with high iron diamine-thiocarbohydrazide-silver proteinate. One purpose of the experiment was to apply a new physical developed to the staining. Instead of silver nitrate, silver lactate or silver bromide, we used silver acetate as an ion donor. This new method allowed physical development under normal lighting conditions, and resulted in the reduction of background staining even after amplification. As the developer did not contain gum arabic, troublesome treatment was not necessary. The time required for staining was very short and the electron density of the final reaction product was high and easily identifiable under the electron microscope. Fixing was not necessary. Very small amounts of reactive substance were detectable after physical development. This developmental procedure has been applied to both the preembedding staining and postembedding staining of sulfated glycoconjugates. The results obtained using this method are presented.

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.

Cytochemical detection of disulfide and sulfhyfryl groups in lamprey aortic microfibrils.

A cytochemical study was performed on the lamprey ventral aorta with special reference to disulfide and sulfhydryl groups of microfibrils, using the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method combined with several other types of treatment. The HID-TCH-SP staining observed was classified into three categories: 1) weak staining in the periphery of collagen fibrils, 2) moderate staining in the boundaries of collagen fibrils, microfibrils and smooth muscle cells, and 3) intense staining of microfibrils. The first and second categories of staining were considered to represent chondroitin and/or heparan sulfate because of sensitivity of the staining to chondroitinase ABC (ChABC) and its specific localization. By contrast, the third category of staining was considered to represent disulfide and sulfhydryl groups of microfibrillar glycoprotein, because it was disclosed only after Oxone oxidation or thiosulfation and was not removed by ChABC digestion. Although this staining reactivity was not apparently altered by SH blockade prior to oxidation or thiosulfation, it was markedly diminished or completely inhibited by S-S reduction followed by SH blockade. These results indicate that lamprey aortic microfibrils contain more S-S groups than SH groups.

Differential distribution and ultrastructural staining of oxytalan and elastic fibers in the periodontal ligament of Alligator mississippiensis

We have investigated ultrastructural cytochemical properties of elastic elements in Alligator periodontal ligaments decalcified with EDTA and stained with 1) the tannic acid-uranyl acetate (TA-UA) method for elastin in combination with elastase digestion; 2) the high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method with prior treatment of specimens with either monopersulphate or cupric-sulphite reagent for the localization of disulphide- and/or sulphydryl-containing material (i.e., oxytalan fibers); and 3) HID-TCH-SP alone for sulphated complex carbohydrates. Many microfibrils accumulated to form either large or small bundles. Large bundles having a diameter of 2.50 +/- 1.10 microns (mean +/- SD; n = 50) each showed an apico-occlusal distribution, although small bundles measuring 0.63 +/- 0.13 microns (mean +/- SD; n = 50) in diameter each were exclusively localized in interstitial areas rich in vessels and nerves. The former bundles always lacked TA-UA reactivity and represented oxytalan fibers; the latter bundles frequently contained TA-UA-reactive elastase digestible components and were similar in appearance to immature elastic fibers or elaunin fibers. HID-TCH-SP after oxidation strongly stained both the oxytalan and elastic fiber microfibrils but stained the amorphous elastin very weakly or not all. In nonoxidized specimens, there was no definite HID-TCH-SP staining of microfibrils and the amorphous elastin, although adjacent matrix proteoglycans stained consistently. These results indicate that although there is a marked difference in the distribution and size of oxytalan and elastic fibers in Alligator periodontal ligaments, their associated microfibrils lack stainable sulphate groups but are enriched with disulphide and/or sulphydryl groups, as has been described in mammals.

Histochemical localization at the electron microscopic level of sulfated glycosaminoglycans in the rat gingiva

Using the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining technique, we investigated ultrastructural localization of sulfated glycosaminoglycans (GAGs) in the rat gingiva shortly after eruption, especially those associated with internal and external basal laminae. In the apical portion of the internal basal lamina, HID-TCH-SP stain deposits were distributed mainly in the region of the lamina lucida located between the lamina densa and the distal surface membrane of the junctional epithelium and inside the depression of the distal surface membrane adjacent to the basal lamina. Stain deposits were also detected on the surface membrane of the cytoplasmic protrusion. Interestingly, the density of HID-TCH-SP stain deposits in the internal basal lamina was highest in the apical portion of the junctional epithelium and decreased in the coronal direction, finally tending to disappear completely. On the other hand, in the external basal lamina the deposits were localized in the whole region of the basal lamina or at both sites of the lamina densa. HID-TCH-SP stain deposits were also detected external to the lamina densa in the basement membrane associated with capillaries and in the connective tissue where they were distributed in close relation to collagen fibrils. Testicular hyaluronidase digested most HID-TCH-SP stain deposits in the connective tissue, whereas those in the region of basement membranes resisted this enzymatic digestion.

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

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 cytochemical properties of elastinassociated microfibrils and their relation to fibronectin

The characteristics of elastin-associated microfibrils were investigated in the tunica adventitia of mouse aortas at the ultrastructural cytochemical level. The high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method specific for sulphate groups was used with and without prior treatment ofen bloc specimens with either monopersulphate or cupric sulphite reagent. Amorphous elastin formed a clearly identifiable central core with microfibrils located both peripherally and interstitially. Sequential oxidation with monopersulphate and HID-TCH-SP demonstrated a characteristic staining for oxytalan fibres and intensely stained the microfibrils, whereas amorphous elastin stained weakly. Sequential thiosulphation with cupric sulphite and HID-TCH-SP for the demonstration of disulphide linkages and sulphydryl groups intensely stained microfibrils and weakly to moderately stained the amorphous elastin. This reactivity of the microfibrils was not altered by digestion with chondroitinase ABC, performed prior to or after treatment with either monopersulphate or cupric sulphite. In the specimens not exposed to either monopersulphate or cupric sulphite there was no definite HID-TCH-SP staining of microfibrils and amorphous elastin. Further, immunostaining with rabbit antibody specific for mouse fibronectin localized fibronectin in the microfibrils but not in the amorphous, elastin. These results indicate that elastin-associated microfibrils in mouse aorta lack stainable sulphate complex carbohydrates but are enriched with either disulphide or sulphydryl groups, or both, and further demonstrate the close correlation between these glycoproteins and fibronectin.

Lectin and ultrastructural cytochemistry of glycoconjugates in the cecal epithelium of the chicken

In the cecal epithelium of the chicken, glycoconjugates were studied by light microscopy with lectin-peroxidase and correlated procedures, and by electron microscopy with high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) and periodic acid (PA)-TCH-SP methods. Mucous granules of goblet cells and striated border of columnar cells at the basal cecum contained acidic and neutral glycoconjugates with sulfate ester, vicinal diol groupings and alpha-D-mannose, alpha-D-glucose, beta-D-galactose, N-acetyl-D-glucosamine, and sialic acid-galactose dimers. In addition, terminal galactose-(1-3)N-acetylgalactosamine disaccharides were detected. In the apical cecum, similar glycoconjugates involved in the mucous granules of goblet cells and the striated border of columnar cells were found to be smaller in amount. Terminal galactose-(1-3)N-acetylgalactosamine disaccharides together with glycogen particles were also visualized in columnar cells of the basal and apical cecum. The histophysiological significance of glycoconjugates involved in the chicken cecal epithelium was discussed.

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.

Light microscopic autoradiography and ultrastructural cytochemistry of sulfated proteoglycans in degenerated chondrocytes of rat epiphyseal cartilage.

Degenerating chondrocytes are often localized in the lower hypertrophic and calcifying zone of rat epiphyseal cartilage. The ability of these degenerating chondrocytes to synthesize and secrete sulfated proteoglycans was investigated using the light microscopic radiosulfate labeling method and the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated complex carbohydrates at the ultrastructural level. At 30 min following intraperitoneal (IP) injection of 35SO4, only intracellular autoradiographic labeling was observed, whereas both intracellular and extracellular radiosulfate labeling was seen at 1 h postinjection. A gradual increase in intracellular and extracellular labeling was seen at 3, 12 and 24h following IP injection of 35SO4. In ultrastructural cytochemical preparations, HID-TCH-SP weakly to moderately stained the chondrocyte Golgi vacuoles or immature and intermediate secretory granules but did not stain Golgi saccules. Mature secretory granules and extracellular matrix granules demonstrated strong staining. These results therefore indicate that proteoglycans are sulfated in the degenerating chondrocyte Golgi vacuoles, packaged in the secretory granules and then released into the extracellular matrix to form matrix granules.

An Electron Microscopic Histochemical Study of the Histogenesis of Major Salivary Gland Pleomorphic Adenoma

Six pleomorphic adenomas were studied histochemically by electron microscopy after staining with ruthenium red, high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP), and tannic acid-ferric chloride (TA-FeCl3); the latter two are quite specific for sulfated glycoconjugates. With ruthenium red staining, the proteoglycans of the myxochondroid stroma could be visualized as numerous extracellular 25 to 50-nm polygonal matrix granules with fine projecting filaments. Similar positive ruthenium red-stained intracellular granules were observed within the Golgi-derived vacuoles of the modified "myoepithelial" cells in the myxochondroid region. Secretion of these granules by exocytosis was occasionally observed. The modified myoepithelial cell in the myxoid region exhibited strong HID-TCH-SP- and TA-FeCl3-reactive sites within the Golgi-derived vacuoles. The intercellular space or microcyst of the cellular clusters and the extracellular matrix granules stained strongly positive with both HID-TCH-SP and TA-FeCl3. Some cells located at the periphery of the cellular clusters also showed similar but weaker reactive sites. No staining was obtained in neoplastic ductal epithelium. The above cytochemical observations indicate that the modified "myoepithelial" cell probably derives from the periphery of the cellular clusters and is the cellular source of the stromal matrix. Moreover, the observations are compatible with the histogenetic model recently proposed by Dardick et al. that the neoplastic, modified myoepithelial cell is the principal proliferating cell in pleomorphic adenoma.

Ultrastructural distribution of acidic glycosaminoglycans associated with matrix vesicle-mediated calcification in mouse progenitor predentine.

The ultrastructural localization of acidic glycosaminoglycans, presumed to be proteoglycans, was examined during initial matrix vesicle-mediated calcification in dentine, by using ruthenium red (RR) staining, high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining, and an enzymatic digestion method. Progenitor predentine 2-10 micron width of developing mouse molar tooth germs was used throughout the present study. The outer surface membrane of the intact matrix vesicles had a strong affinity for RR. The RR positive materials appeared beaded and extended perpendicularly from the vesicle membrane. They tended to disappear with the disruption of the vesicular membrane, which resulted from overextension due to needle-like, crystal-like structures. The HID-TCH-SP stain deposits, approximately 10 nm in diameter, were densely distributed around the intact matrix vesicles, though few were found inside them. Some matrix vesicles that were presumably disrupted, however, contained smaller stain deposits. On the outer surface membrane of the disrupted vesicles, HID-TCH-SP stain deposits were fewer in number. The results obtained from enzymatic degradation studies showed that the anionic materials on the outer surface membrane of the matrix vesicles were represented by chondroitin-4-sulfate and/or chondroitin-6-sulfate. We suggest that chondroitin sulfates attached to the outer leaflet of the vesicular membrane play an important role during the incipient stage of the matrix vesicle-mediated calcification process.

Ultrastructural distribution of sulfated complex carbohydrates in elastic cartilage of the young rabbit.

Sulfated glycosaminoglycans are an integral component of elastic cartilage. We have investigated the ultrastructural distribution of sulfated complex carbohydrates (CC) in the mature cartilage and the perichondrium of young rabbit auricles using the high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) and the tannic acid-ferric chloride (TA-Fe) methods. In the mature cartilage, HID-TCH-SP stained intracellular Golgi saccules of the mature face, secretory granules, and the extracellular matrix granules, but staining was not discernible in collagen fibrils and osmiophilic elastic fibers consisting of only amorphous elastin. The HID and TA-Fe staining were similarly observed in matrix granules, whereas the elastic fibers and collagen fibrils lacked the staining. The pericellular matrix granules had a diameter of 34 +/- 5 nm (mean +/- SD; n = 30). Thiéry's periodate-TCH-SP (PA-TCH-SP) method stained vicinal glycol-containing CC in collagen fibrils but failed to stain matrix granules and elastic fibers. In the perichondrium, HID-TCH-SP staining of the organelles was less intense in the flattened chondrocytes when compared with those in large mature chondrocytes. The extracellular HID and HID-TCH-SP staining were observed in the matrix granules. The diameter of pericellular matrix granules (19 +/- 4 nm, mean +/- SD; n = 30) was significantly smaller when compared to those in the mature cartilage (P less than 0.001). The HID-TCH-SP staining was closely associated with collagen fibrils. However, the staining was not seen in collagen fibrils and osmiophilic elastic fibers consisting of elastin and microfibrils. The PA-TCH-SP method stained collagen fibrils and microfibrils but did not stain the amorphous elastin. Thus these studies demonstrate that sulfated CC are packaged in chondrocyte secretory granules and are released into the extracellular matrix to form matrix granules, but are not incorporated into collagen fibrils and elastic fibers.

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.

Glycosaminoglycans in human neutrophils and leukemic myeloblasts: ultrastructural, cytochemical, immunologic, and biochemical characterization

Chondroitin sulfate is known to be present in normal and leukemic myeloid cells; however, its definitive subcellular location and association with other glycosaminoglycans (GAGs) has not been demonstrated. We have studied the type and distribution of GAGs in neutrophil granule subpopulations of normal and leukemic myeloid cells using ultrastructural, cytochemical, immunologic, and biochemical methods. At the ultrastructural level, high-iron diamine- thiocarbohydrazide-silver proteinate (HID-TCH-SP) stained sulfated glycoconjugates selectively in immature primary granules of normal promyelocytes and Auer rods and immature granules of leukemic myeloblasts. Staining was weak or absent in mature primary granules, whereas tertiary granules stained moderately. Primary granule staining with HID-TCH-SP was greatly diminished by prior treatment of the specimens with chondroitinase ABC and/or nitrous acid, indicating the presence of chondroitin sulfate and N-sulfated glycosaminoglycan. Immunostaining of myeloid cells with a rabbit antichondroitin 4-sulfate and ferritin-conjugated goat anti-rabbit IgG sequence resulted in staining of most primary granules. Biochemical analysis of GAGs from leukemic myeloblasts containing primary granules and Auer rods, but lacking secondary and tertiary granules, revealed 8 x 10(-17) mole of uronic acid/cell and electrophoretic and sulfaminohexose analysis showed 60%-70% chondroitin sulfate AC of heterogeneous molecular weight, 20%-30% of a GAG that most closely resembled heparan sulfate, and 10% dermatan sulfate. The lack of significant HID-TCH-SP staining of sulfate iin sites other than Auer rods and primary granules in leukemic myeloblasts indicates that these granules contain the chondroitin, dermatan, and heparan sulfate isolated from the same specimen. Similar GAGs are present in primary granules of normal cells as evidenced by their cytochemical and immunostaining properties. Thus, these studies demonstrate a heterogeneous population of GAGs not previously identified and localize these substances to the primary granule of leukemic and normal cells.

Glycosaminoglycans in Human Neutrophils and Leukemic Myeloblasts: Ultrastructural, Cytochemical, Immunologic, and Biochemical Characterization

Chondroitin sulfate is known to be present in normal and leukemic myeloid cells; however, its definitive subcellular location and association with other glycosaminoglycans (GAGs) has not been demonstrated. We have studied the type and distribution of GAGs in neutrophil granule subpopulations of normal and leukemic myeloid cells using ultrastructural, cytochemical, immunologic, and biochemical methods. At the ultrastructural level, high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) stained sulfated glycoconjugates selectively in immature primary granules of normal promyelocytes and Auer rods and immature granules of leukemic myeloblasts. Staining was weak or absent in mature primary granules, whereas tertiary granules stained moderately. Primary granule staining with HID-TCH-SP was greatly diminished by prior treatment of the specimens with chondroitinase ABC and/or nitrous acid, indicating the presence of chondroitin sulfate and N-sulfated glycosaminoglycan. Immunostaining of myeloid cells with a rabbit antichondroitin 4-sulfate and ferritin-conjugated goat anti-rabbit IgG sequence resulted in staining of most primary granules. Biochemical analysis of GAGs from leukemic myeloblasts containing primary granules and Auer rods, but lacking secondary and tertiary granules, revealed 8 x 10(-17) mole of uronic acid/cell and electrophoretic and sulfaminohexose analysis showed 60%-70% chondroitin sulfate AC of heterogeneous molecular weight, 20%-30% of a GAG that most closely resembled heparan sulfate, and 10% dermatan sulfate. The lack of significant HID-TCH-SP staining of sulfate iin sites other than Auer rods and primary granules in leukemic myeloblasts indicates that these granules contain the chondroitin, dermatan, and heparan sulfate isolated from the same specimen. Similar GAGs are present in primary granules of normal cells as evidenced by their cytochemical and immunostaining properties. Thus, these studies demonstrate a heterogeneous population of GAGs not previously identified and localize these substances to the primary granule of leukemic and normal cells.

Ultrastructural visualization of complex carbohydrates in eosinophilic leukocytes.

Complex carbohydrates in eosinophils from human, rabbit, and rat marrow were identified and localized by cytochemical and radioautographic methods. The high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP), low iron diamine (LID)-TCH-SP, and periodate (PA)-TCH-SP methods were used for the localization of sulfate, sulfate and carboxyl, and vicinal glycol- containing complex carbohydrates, respectively. Golgi vesicles and small precursor granules (0.2-0.4 micrometer in diameter) demonstrated strong HID-TCH-SP staining and labeled intensely after a 10-minute pulse with 35SO4(2). Crystalloid-free or immature specific granules (0.5-0.9 micrometers in diameter) labeled heavily after a 60-minute incubation and 60-minute chase with 35SO4(2). Immature granules were graded according to their HID-TCH-SP staining. Type I granules demonstrated strong rim staining and similar or somewhat less central staining, whereas type 2 granules only demonstrated rim staining, and type 3 granules lacked staining. Fully mature crystalloid-containing granules lacked staining. LID-TCH-SP similarly stained the HID-positive sulfated material in cytoplasmic granules. PA-TCH-SP stained some Golgi vesicles and diffusely stained all precursor granules and type 1 granules. Weaker staining was observed in type 2 granules and staining was very weak or absent in type 3 and crystalloid-containing granules. In early eosinophils, tubulovesicular structures (TVS) were observed rosetting and contacting precursor and type 1 granules. These TVS contained material with strong PA-TCH-SP staining but lacked HID-TCH-SP or LID-TCH-SP-reactive acidic glycoconjugates. Flattened Golgi saccules or early eosinophils stained weakly or not at all with the PA-TCH-SP method. Small granules and TVS in late (bilobed) eosinophils displayed PA-TCH-SP reactivity and lacked HID-TCH-SP staining but differed from TVS in early eosinophils in that they were not associated as rosettes with specific granules. These results indicate that sulfated and vicinal glycol-containing complex carbohydrates are differently disturbed in immature specific granules of eosinophils and presumably become masked to staining as the granule matures.