Ronald L. Austin

44-kDal bone phosphoprotein (osteopontin) antigenicity at ectopic sites in newborn rats: kidney and nervous tissues.

SummaryPrevious immunohistochemical data have shown that the 44-kDal bone phosphoprotein (44K BPP, also called sialoprotein I or oestopontin) recently isolated in our laboratory was synthesized by osteoblasts and osteocytes and was expressed early during differentiation of boneforming cells. We report here the presence of 44K BPP antigenicity at certain ectopic sites, namely, the proximal-convoluted tubule of the kidney, neurons, sensory and secretory cells in the internal ear. To insure specificity and reproducibility, different immunohistochemical methods were used and affinity-purified antibodies against two separate preparations of pure 44K BPP were tested. In the cells of the proximal-convoluted tubule, 44K BPP immunoreactivity was observed within apical endocytotic vacuoles and within lysosomes. This staining thus correlates with the degradation of the 44K BPP epitope which we previously demonstrated to occur in serum. On the other hand, in the neurons of the acoustic ganglion and the sensory cells of the macula, 44K BPP immunoreactivity was associated with the Golgi apparatus indicating synthesis and secretion by these cells. The finding that the 44K BPP (or a structurally related molecule) is synthesized by neurons and neuroepithelial cells deserves further investigation with respect to a possible embryologie relationship between neuroectodermal cells and the precursors of some bone forming-cells of the skull.

Ultrastructural cytochemistry and radioautography of hemoglobin—iron absorption☆

The subcellular route of hemoglobin-iron absorption by canine intestinal epithelial cells was investigated with cytochemical and radioautographic methods and compared to that observed for inorganic iron absorption. A solution of rabbit [59Fe]hemoglobin or an inorganic-iron solution was injected into closed duodenal loops of beagle dogs and mucosal biopsies were obtained 15, 60, and 120 min thereafter. Mucosal retention of radioactivity in dogs given hemoglobin iron was 6.9–10.2% after 120 min, and portal venous blood radioactivity was confined to the inorganic iron fraction. In the same animals diaminobenzidine (DAB)-reactive heme was visualized in microendocytic caveolae at the bases of microvilli and in membrane-bound tubulovesicular structures and granules of the apical cytoplasm. DAB-reactive heme was not identified in the lateral intercellular space or the basal extracellular space. In the same specimens, acid ferrocyanide stained ferric iron in a few apical cytoplasmic vesicles as well as along the outer lateral and basal plasmalemma but failed to identify inorganic iron in microvilli. Both the microvilli and the lateral plasmalemma appeared stained in specimens from animals given inorganic iron. In radioautographic specimens of animals given [59Fe]hemoglobin, silver grains were most frequently associated with the apical cytoplasm and the lateral plasmalemma. We conclude that at least one mechanism of hemoglobin-iron absorption involves endocytosis and degradation of hemoglobin in membrane-bound organelles with subsequent conversion of its iron to an inorganic form, which is then transported to the lateral intercellular space where subsequent processing is similar to that observed for absorbed inorganic iron.

Ultrastructural Cytochemistry and Radioautography of Complex Carbohydrates in Heterophil Granulocytes from Rabbit Bone Marrow

The subcellular route of incorporation of complex carbohydrates into rabbit heterophil primary granules and their subsequent intragranular distribution during granule maturation were studied with ultrastructural, cytochemical, and radioautographic methods. High iron diamine (HID) staining of sulfated glycoconjugates in primary granules was partially diminished after treatment with chondroitinase ABC or after removal of N-sulfate groups with nitrous acid, but was not altered by exposure to hyaluronidase, trypsin, or HCl. Subsequent thiocarbohydrazide-silver proteinate (TCH-SP) straining of thin sections increased the density of the HID reaction product. Golgi-derived spherules and very immature morular granules stained weakly with HID-TCH-SP and labeled intensely after a 10 min incubation with 35SO4. After a 60 min 35SO4 pulse and a 60 min chase, an increase in radiolabeling was observed in granules with HID stained, fused morular material, and some labeling was present in more mature rim stained granules. Fully mature granules lacked HID or HID-TCH-SP staining, but contained most of the 35SO4 labels after a 60 min pulse and 18 hr chase in vitro. Periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of unosmicated thin sections localized vicinal glycol-containing complex carbohydrates in Golgi-associated small vesicles. These vesicles lacked HID-TCH-SP staining and apparently contained neutral glycoprotein. They frequently bordered, in a rosette arrangement, the immature morular granules, but not the more mature primary granules. The PA-TCH-SP method localized complex carbohydrates in the rim of granules precursors and enclosed a spherule or morula, but failed to stain the sulfate-containing material in the morulas or spherules. PA-TCH-SP reactivity was diffusely distributed in moderately mature granules and was decreased in fully mature granules. These results indicate that heterophil primary granule contain several complex carbohydrates including O-sulfated and N-sulfated glycosaminoglycans, as well as vicinal glycol-containing glycoproteins. These complex carbohydrates are transported to immature primary granules by different Golgi-derived organelles. The complex carbohydrates are subsequently distributed differently within primary granules and become masked to staining as the granule matures.

Ultrastructural cytochemistry and immunocytochemistry of sulfated glycosaminoglycans in epiphyseal cartilage.

Cartilage proteoglycan monomers contain sulfated glycosaminoglycans (GAGs), which consist of chondroitin 4and 6-sulfate, and keratan sulfate. We have examined newborn rat epiphyseal cartilage to demonstrate the nature of reactive sites in the matrix granule with the high iron diamine (HID) and HID-thiocarbohydrazide-silver proteinate (HID-TCH-SP) methods for sulfated glycoconjugates and an immunoferritin method specific for chondroitin 4-sulfate. HID strongly stained matrix granules in osmicated specimens but weakly stained these granules in unosmicated specimens. HI D-TCH-SP staining produced two types of stain deposits, averaging 1 1 and 7 nm in diameter, in the matrix granule and was not influenced by postosmication. Testicular hyaluronidase digestion of specimens removed most of the 1 1 nm stain deposits in the matrix granule. The size of the HID-TCH-SP reaction products appeared to be related to molecular weight and! or sulfate content of the GAG chains, as evidenced by the

Ultrastructural cytochemistry of basophils in chronic myelocytic leukemia

Abstract Cytochemical methods were used to determine the ultrastructural distribution of antimonate precipitable cations, anionic complex carbohydrate, acid phosphatase, and peroxidase in blood basophils from patients with chronic myelocytic leukemia and markedly elevated basophil counts. The morphology of these basophils varied but was generally similar to that reported for normal basophils. Antimonate precipitable cations were sparse in specific granules but more abundant in extra-granular cytoplasm and in vesicular structures of many basophils and, although varying with the fixation method, were distributed atypically in nuclei in a pattern similar to that seen in mast cells. The dialyzed iron technique diffusely stained anionic complex carbohydrate in the specific granules of basophils, and high iron diamine staining disclosed a sulfated mucosubstance in these granules. Acid phosphatase and peroxidase were distributed in a pattern similar to that of the acid mucosubstance in the basophil granules, and their presence suggested a lysosomal function for these granules