Cytochemical demonstration of amine-storing vacuoles and lysosomes in the chicken thrombocytes

Summary A combined electron microscopic and cytochemical study of the thrombocytes of the chicken has clearly identified the amine-storing organelles and lysosomes. A chormaffin positive-reaction product was observed on the inner surface and the granules of the large electronlucent vacuoles. No acid phosphatase activity was localized in these amine-storing vacuoles. However, the acid phosphatase activity was observed in the small vesicles, the primary lysosomes, and in the large electron dense inclusions with myelin which may be secondary lysosomes. The results of this study suggest that the large empty vacuoles, with one or two very dense osmiophilic peripherally-situated granules, in the chicken thrombocytes are comparable to the vesicles with electron dense materials called dense bodies in mammalian thrombocytes.To whom offprint requests should be sent     

Cytochemical analysis of the content of chicken thrombocytes vacuoles.

The content of the large vacuoles present in chicken thrombocytes was analyzed by the use of cytochemical techniques which indicated the presence of basic proteins, unsaturated fatty acids, sugars rich in viccinal hydroxyl groups, linked to proteins or lipids and acid phosphatase. These substances perform one of the most conspicuous functions of these cells, which is phagocytosis. In addition, thrombocytes are committed to hemostasis. Both functions make these cells similar to human platelets, even though having different origins and morphologic characteristics. The large vacuoles described are connected to the open canalicular system (OCS) and together with other cellular structures they contribute to the endocytic system.     

Adenosine deaminase activity associated with phagocytic vacuoles. Cytochemical demonstration by electron microscopy

Adenosine deaminase activity has been localized within the cell membrane and it surrounds phagocytic vacuoles in mouse macrophages. Adenosine deaminase is thus strategically located to direct metabolic flux through the enzymes of the purine catabolic pathway. Xanthine oxidase, a key enzyme of this pathway, produces superoxide during its reaction with its substrates. Enzyme activity was visualized for electron microscopy by means of hydrolysis of 6-Chloropurine ribonucleoside to produce Cl-, which is precipitated with Ag+. The latter is converted into Ag0 by light, and the resulting deposit is visualized with the electron microscope.     

Immunomorphologic characterization of chicken thrombocytes

Summary Chicken thrombocytes were enriched for immunization by utilizing their strong capacity to adhere to plastic surfaces. The produced rabbit anti-thrombocyte serum ATS 3 reacted by means of the unlabeled antibody enzyme method (PAP) specifically with thrombocytes of fixed chicken-blood smears, but not with lymphocytes or other blood cells. When ATS 3 (substrate diaminobencidinetetrahydrochloride = DAB) and a 11 mixture of an anti-bursa serum and anti-thymus serum (ABS/ATS; substrate 4-chloro-1-naphthol = 4-Cl-1-N) were used simultaneously, thrombocytes revealed the brown color typical for DAB, whereas lymphocytes showed the blue stain of 4-Cl-1-N. The finding of a thrombocyte surface antigen not shared by lymphocytes is regarded as a further proof of the diversity of both cell systems, i.e., for the existence of a genuine thrombocyte system in chickens.     

Cytochemical and biochemical identification of lysosomes in cryptococcus neoformans

Normaski optics, fluorescence and electron microscopy were employed to demonstrate the occurrence of lysosomes in capsulated, enzymatically decapsulated, and dewalled cells of a human isolate ofCryptococcus neoformans. Fluorescent studies, using acridine orange as a lysosomal indicator, revealed the presence of variously sized, spherical, reddish-orange fluorescing bodies. Electron microscopy studies demonstrated the presence of acid phosphatase (AP), a lysosome marker enzyme, in single-membrane bound organelles. Lysosomes were removed from dewalled cells and separated by differential centrifugation on ficoll gradients. That fraction indicating the highest assay for AP was centrifuged at high speed, and the resulting pellet was fixed for electron microscopy and stained by the Gomori procedure for AP. Sections of the pellets revealed AP stained vesicles of the same size range as those within intact cells.     

Identification of fibronectin in chicken thrombocytes.

Cell attachment and spreading of chicken embryo fibroblasts and three mammalian cell lines were promoted by using thrombocyte secretion products (TSP) prepared from chicken thrombocytes which are analogous to mammalian platelets. The cell attachment activity following TSP treatment was interfered with by the addition of a synthetic peptide containing the RGD sequence from the cell attachment region of fibronectin (FN). Cell attachment on TSP-coated plates was inhibited by the addition of anti-chicken FN rabbit antiserum, but not by anti-chicken vitronectin (VN) rabbit antiserum. By immunoblotting, immunofluorescent test and sensitive enzyme linked immunosorbent assay using an anti-chicken FN antiserum, the presence of FN in thrombocytes and the release of FN from the cells were demonstrated. The release of FN from thrombocytes was partially induced by the in vitro cultivation of the cells and was further promoted by treatment of the cells with thrombin.     

Isolation and characterization of chicken liver lysosomes

The distribution patterns of chicken liver lysosomal enzymes were studied in iso-osmotic gradients of Percoll. The lysosomal enzymes separated by Percoll gradients showed three different types of distribution. In contrast with rat liver lysosomes, purified chicken liver lysosomes were very stable during storage at 4 degrees C.     

Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Summary Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.     

Cytochemical demonstration of phenoloxidase in peripheral blood leukocytes]

A comparative application of phenoloxidase-demonstration and Pappenheim staining allows a sure differentiation of phenoloxidase-positive cells to be made, which can be found in the peripheral blood with different activity and different pattern of response. While eosinophils, neurophils, monocytes and lymphocytes contain phenoloxidase, basophiles do not.     

Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.     

Cytochemical demonstration of glucose-6-phosphatase in human liver

To determine the cytochemical localization of glucose-6-phosphatase in the human hepatocyte, lead - based and cerium - based media were used. By studying the effects of systematic variation of the incubation medium components, the optimal experimental conditions were determined. The exclusive localization of the cytochemical reaction in the endoplasmic reticulum and nuclear envelope, together with the results of control experiments ensured that these findings could be correlated with the phosphohydrolase activity of the multicomponent glucose-6-phosphatase system.     

Cytochemical demonstration of adenylate cyclase activity with cerium

Cerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and cholera toxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.     

Cytochemical demonstration of adenylate cyclase activity with cerium

Summary Cerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and choleratoxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.This study was supported by the grant from Reino Lahtikari Foundation