Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow

Summary The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.     

Ultrastructural localization of glycogen in erythrocytes and developing erythrocytic cells in normal human bone marrow

Summary The periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) reaction was employed for the ultrastructural cytochemical localization of saliva-labile glycogen in the erythrocytic cells in normal human blood and bone marrow. Particulate glycogen was demonstrated in the cytoplasm of all developmental forms of erythrocytic cells from the proerythroblast through the reticulocyte; a few particles of glycogen also were present in mature erythrocytes even in the peripheral blood. Statistical evaluation of the number of glycogen particles in mid-plane cell sections at each morphological stage of development indicated a significant and stepwise decrease during cellular maturation. This change in glycogen content may reflect both cellular utilization and mitosis during the maturational sequence.Supported by Grant No. SR01AM 12084-15 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Anita Topson, Barbara Speakmon and Marjorie Griffith for their technical assistance and to Dr. Gerald King for performing the bone marrow aspirations.     

Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow

Developing neutrophil granulocytes of normal human bone marrow were investigated with the diaminobenzidine technique to determine the ultrastructural localization of peroxidase activity. Neutrophil granulocytes have three types of granule: nucleated, azurophil, and specific granules. These granules are produced consecutively during the eomyelocyte stage, the promyelocyte stage, and the myelocyte stage, respectively. The organelles involved in the production of granules, i.e., the nuclear envelope, rough endoplasmic reticulum, and Golgi apparatus, are peroxidase positive during the eomyelocyte and promyelocyte stages and peroxidase negative thereafter. This pattern differs for the granules themselves: nucleated granules are negative in the eomyelocyte and become positive in the promyelocyte. Azurophil granules become positive in the promyelocyte. Specific granules are negative. Our observations highly suggest that small Golgi-derived peroxidase-positive vesicles are involved in the maturation of both nucleated granules and azurophil granules.     

Ultrastructural localization of peroxidase activity in developing neutrophil granulocytes from human bone marrow

Summary Developing neutrophil granulocytes of normal human bone marrow were investigated with the diaminobenzidine technique to determine the ultrastructural localization of peroxidase activity. Neutrophil granulocytes have three types of granule: nucleated, azurophil, and specific granules. These granules are produced consecutively during the eomyelocyte stage, the promyelocyte stage, and the myelocyte stage, respectively.The organelles involved in the production of granules, i.e., the nuclear envelope, rough endoplasmic reticulum, and Golgi apparatus, are peroxidase positive during the eomyolocyte and promyelocyte stages and peroxidase negative thereafter. This pattern differs for the granules themselves: nucleated granules are negative in the eomyelocyte and become positive in the promyelocyte. Azurophil granules become positive in the promyelocyte. Specific granules are negative.Our observations highly suggest that small golgi-derived peroxidase-positive vesicles are involved in the maturation of both nucleated granules and azurophil granules.In honour of Prof. P. van Duijn     

Ultrastructural localization of peroxidase activity in normal human bone marrow cells

Summary The ultrastructural localization of peroxidase activity has been studied in the cells of normal human bone marrow using the diaminobenzidine peroxidase technique. Peroxidase activity has been localized within the primary (azurophil) granules of the neutrophilic series as well as in the cytoplasmic granules of eosinophils, basophils and monocytes. Peroxidase activity appears within the cisternal system (nuclear envelope, Golgi complex and rough endoplasmic reticulum) of these cells during the period of peroxidase-containing lysosome production. With the cessation of granulogenesis, peroxidase activity disappears from the cisternal system and does not reappear in subsequent developmental stages. In cells incubated in peroxide-free media, staining of granular components, but not of cisternae, is reduced. The inclusion of catalase in peroxide-free media eliminates all staining. This indicates that an endogenous peroxide is present within the cisternae and granules of these cell types.Supported by Grant No. AM-HE-12084-12 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Anita Topson and Barbara Jordan for their technical assistance and to Dr. Arthur Sagone who performed the marrow aspirations.     

Surface localization of an endogenous lectin in rabbit bone marrow

Summary A lectin, which may be involved in cell to cell adhesion during erythropoiesis in rabbit bone marrow, has been isolated and characterized. Several electron microscopical techniques have been used to investigate the cell surface distribution of this lectin in bone marrow utilizing colloidal gold conjugates of anti-lectin IgG or protein A. The lectin is present at the surface of erythroid cells at all stages of development but no lectin was detected on the surface of myeloid cells. The limitations and complementary nature of the techniques used are discussed.     

Ultrastructural localization of guanylate cyclase in bone cells.

Guanylyl imidodiphosphate (GMP-PNP) hydrolyzing enzyme activity as a means of detecting plasma membrane guanylate cyclase was demonstrated in osteoblasts of chicken tibial metaphysis using a lead citrate histochemical method at the electron microscopic level. Activity was not discerned in osteoclasts or osteocytes. The reaction product development was completely abolished when the sections were incubated with substrate-free or MnCl2-free medium. Guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP) was a less effective substrate than GMP-PNP, and Mn++ was a stronger stimulator than Mg++. No reaction product was observed on the plasma membrane of osteoblasts when beta-glycerophosphate or p-nitrophenylphosphate was used as substrate instead of GMP-PNP. The results implicate guanylate cyclase as a significant effector of osteoblast regulation at the site of the plasma membrane.     

Ultrastructural localization of fibronectin in bone marrow of the embryonic chick and its relationship to granulopoiesis

Summary Fibronectin was immunolocated in embryonic chick bone marrow by the use of both a direct peroxidase conjugated antiserum and an indirect Streptavidin bridge technique. Fibronectin is located in the extravascular granulopoietic compartment and, to a lesser extent, in the vascular, erythropoietic compartment. There is no evidence of fibronectin being associated with blood-stromal cell interactions involving either erythropoiesis or thrombopoiesis. However, mature thrombocytes display a substantial surface coat containing fibronectin. Much of the fibronectin appears to be situated on surfaces of those fibroblastic stromal cells which support granulopoiesis. Fibronectin containing extracellular material connects surfaces of developing granulocytes with surfaces of stromal cells. Fibronectin is a surface component of granulocytes as well as nearby stromal cells. However, there appear to be fewer ferritin particles per unit of surface on granulocytic cells. Many of the ferritin particles are not clearly associated with amorphous matrix material at cell surfaces. Immunocytochemical attempts to identify laminin were unsuccessful. These studies indicate that fibronectin is situated at sites where it could mediate adhesive interaction between granulopoietic cells and their stromal cells. Furthermore, cell surface-matrix interaction involving fibronectin could mediate migration of blood cells within the extravascular spaces.     

Cellular changes in bone marrow of malaria-infected mice. III. Chemotaxis of granulocytes

The number of bone marrow cells and their chemotactic activity was studied during malaria infection. Two days after infection of Balb/c mice with Plasmodium berghei, an increase in granulocyte number was observed in the blood. A modified Boyden chamber chemotaxis assay was employed to investigate the mechanism of granulocyte accumulation in the blood. Bone marrow cells from normal mice, from mice during a primary lethal infection and from immune mice after challenge were compared. The complement factor C5a showed chemotactic activity for bone marrow cells; a significant decrease of chemotaxis was only observed after 6 days of primary infection. Extracts of spleen, liver and infected erythrocytes lacked chemotactic activity, or caused inhibition of cell migration. Serum from mice with a 2-day primary infection contained chemotactic activity. The active component was heat labile, protease sensitive and had an estimated molecular weight of 250,000.     

Ultrastructural localization of basigin in normal human epidermis

Basigin is a glycosylated transmembrane protein belonging to the immunoglobulin superfamily. It is thought to play roles in intercellular recognition involved in cell differentiation. We previously demonstrated at the light microscope level a correlation between basigin expression and epidermal differentiation. In the present study, the ultrastructural localization of basigin in normal human epidermal keratinocytes was investigated by immunoelectron microscopy. The basigin labeling was strongest on membranes of basal cells, weaker on prickle cells, and absent in granular and horny cells. On the membrane of basal cells, labeling was observed on the apical and lateral sides but not on the dermal side. Gold particles were mostly observed on the surface of microvilli, especially on their tips. There were fewer on the intermicrovillous membrane and they were absent on the desmosome. These results are consistent with our previous report that basigin expression is correlated with differentiation of epidermal keratinocytes. Microvilli on basal and suprabasal keratinocytes might play roles in the differentiation of keratinocytes through basigin on the tips of microvilli.     

Oxygen metabolism in rabbit bone marrow and liver.

Oxygen metabolism has been quantified in rabbit bone marrow and liver. NADPH-Cytochrome $\text{c}$ reductase activity in bone marrow microsomal and cytosol fractions was about 40% of that found in liver. Superoxide anion and peroxide generation were found to be present in both liver and bone marrow. Catalase and superoxide dismutase activity were measured in liver and in marrow preparations free of erythrocytes; while liver catalase activity was approximately twice that of bone marrow, very low superoxide dismutase activity was observed in erythrocyte free bone marrow homogenates.     

Ultrastructural localization of heparan sulfate and chondroitin sulfates associated with granulopoiesis in embryonic chick bone marrow

Sulfated glycoconjugates were ultrastructurally localized within embryonic chick marrow by using the high iron diamine-silver proteinate stain. Stain was concentrated in the extravascular, granulopoietic compartment, indicating that granulopoiesis, but not erythropoiesis, proceeded in a highly sulfated environment. It was likely that most of the stainable material represented sulfated proteoglycans since staining was abrogated by predigesting tissue with enzymes and other treatments known to degrade specific glycosaminoglycan chains. Chondroitinase/hyaluronidase digestion resulted in the removal of most of the stainable material associated with the extracellular matrix and a portion of the stainable material associated with fibroblastic cell surfaces. Unaffected material lay in close proximity to fibroblastic cell membranes. Heparitinase/heparinase digestion had essentially the opposite effect. Sulfated material associated with matrix components was largely unaffected, but the fibroblastic plasmalemmal material was now absent. These results suggest that there are at least two categories of sulfated proteoglycans in the granulopoietic compartment, each differentially distributed. The plasmalemmal material likely represented heparan sulfate which in this tissue appeared to be associated in a uniform layer with fibroblastic stromal cell membranes and not with blood or endothelial cell membranes. Material identified as chondroitin sulfates was found within patches of amorphous matrix that was located on fibroblastic stromal cell surfaces and that was interspersed with fibrils in the extracellular matrix. Chondroitin sulfates were sparsely distributed on granulocytic cell surfaces.