Ultracytochemistry of cyclic 3′,5′-nucleotide phosphodiesterase activity in the planarian Dugesia lugubris (O. Schmidt)

Summary The enzyme 3,5-nucleotide phosphodiesterase was localized in certain tissues of the planarian Dugesia lugubris (O. Schmidt) by means of ultracytochemical methods. This enzyme was found to be active in epithelium, muscles, nerve tissue and in rhabdite-forming cells. The active enzyme was present at the outer or inner side of the membrane, and even in the cytoplasm. Problems of the ultracytochemical localization of PDE are discused.     

Localization of adenylate cyclase activity in the tissues of an intact planarian Dugesia lugubris (O. Schmidt)

Summary Adenylate cyclase was localized in tissues of an intact planarian Dugesia lugubris (O. Schmidt) by ultracytochemical methods. The enzyme was found in epithelium, muscles, secretory cells (mucous), and rhabdites forming cells and neoblasts. Adenylate cyclase occurred on the external side of cell membranes in cisterns of the endoplasmic reticulum, nuclear envelope and mitochondria. The problems of ultracytochemical localization of AC are discussed.     

Perineuronal glial system in the cerebral ganglion of active and hibernating Helix aspersa

The electron microscopical changes in the glial lacunar network that surrounds the large neurons of meso- and metacerebrum of land snail cerebral ganglia were considered, in order to get an insight into the functional role of this peculiar structure in invertebrates. Compared with snails during the active period, in the hibernating animals the extension of glial processes was reduced and the glial processes appeared more regular and stacked around neurons. Moreover, they did not form deep, long interdigitations with neuronal infoldings as during the active period. In particular, data on the ultracytochemical detection of alkaline phosphatase and Ca(2+)/Mg(2+)-ATPase enzyme activities, point to a correlation between the extension of the glial system and its function in the regulation of the extracellular environment. In fact, in hibernating snails, lower reactivity was found on the glial membranes, including those of the trophospongium.     

Fine localization of the acetylcholinesterase activity in somatic and germ cells during the morphogenesis of chick ovarian cortex

The ultracytochemical localization of the enzyme acetylcholinesterase (AChE) was studied in the germinal epithelium and cortex during chick ovarian morphogenesis at the 7, 12.5, 14 and 19 day of incubation. The results evidenced at day 7 the presence of the enzyme in some somatic cells, both "dark" and "light" and in some gonocytes. The reaction appears also in some tracts between these types of cells. At day 12.5 the reaction is present in some somatic cells only towards the deepest zone of the cortex, in many oogonia and oocytes and in some tracts between germ and somatic cells. At day 14 and day 19 the various cell categories of the cortex are negative for the reaction. The significance of the presence of the enzyme is discussed in relation to an embryonic cholinergic system active during morphogenesis. Regarding the presence of the enzyme in germ cells, the positivity in endoplasmic reticulum cisternae associated to mitochondria may suggest an implication of the enzymatic activity in the proliferation and transformation of these organelles.     

Ultracytochemical localization of 3β-hydroxy-steroid ferricyanide reductase activity in the fetal mouse ovary

It has been suggested that the production of estrogens by the fetal ovary may modulate the entry or progression of meiosis in the female mammalian fetus. In the present study the possibility that the site of this steroid synthesis is the rete ovarii system was explored in the fetal mouse of gestational ages 12.5 to 18 days. The method of ultracytochemical localization of 3β hydroxy-steroid ferricyanide was used. Reaction product was found in the cytoplasm of the rete ovarii (prefollicular) cells as early as day 14 with increasing amounts seen at later gestational ages. The presence of this essential enzyme system in cells closely applied to oogonia and oocytes during an active meiotic period must be considered in developing concepts of meiotic entry.     

Ultracytochemical detection of nucleoside phosphatase activity in human peripheral blood cells

The authors elaborated and described the optimum conditions for fixation, incubation and preparation of human blood cell samples in minimum quantities for ultrastructural and ultracytochemical investigations of 5'-nucleotidase and ATPase activities. The best preservation of the blood cell ultrastructure was obtained after fixation with buffered 1% glutaraldehyde solution followed by postfixation in buffered 1% OsO4 solution. The best ultracytochemical demonstration of 5'-nucleotidase and ATPase activities was achieved after fixation in buffered 2% formaldehyde prior to cytochemical incubation. DMSO added to either fixation or incubation media was shown to damage the plasmalemma and glycocalyx structure in cell suspensions. ATPase in 5'-nucleotidase activities were revealed in plasmalemma, cytoplasmic reticulum, Golgi complex, mitochondria and in the nuclei, in particular, in the perinuclear space, nucleolus and chromatin. With respect to the localization and activity of nucleosidephosphatases, lymphocytes proved to be most heterogenic, with the enzyme activity level directly depending on the rate of ultrastructural differentiation in lymphocytes.     

An ultracytochemical study of the hydrogenosomes of Trichomonas vaginalis

The ultracytochemical study of cultural T. vaginalis has been carried out. The activity of magnesium-dependent ATPase, transport (sodium-potassium-dependent) ATPase and adenylate cyclase was detected in dense corpuscles, located on the membranes of hydrogenosomes having a rounded form. The authors suggest that the dense corpuscles are the zones of the active transport of ions through the membranes of hydrogenosomes.     

An electron microscopic study of the localization of the activity of the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase component of the Na, K-ATPase complex in the rat brain

The localization of an ouabain-sensitive potassium-dependent p-nitrophenylphosphatase part of the Na+,K(+)-ATPase complex in the white rat's brain has been studied at the ultrastructural level. The physiological pH of incubation medium highly increases the specificity of ultracytochemical enzyme demonstration. The main characteristics of the enzymatic p-NPP hydrolysis used for this methodological technique are discussed.     

Ultracytochemical localization of guanylate cyclase activity in guinea-pig peritoneal macrophages under different physiological conditions

Summary Guanylate cyclase activity was investigated in guinea-pig peritoneal macrophages under different physiological conditions (such as adhesion and phagocytosis) with an ultracytochemical method using guanylyl-imidodiphosphate as a substrate. The enzyme was detected on the perinuclear envelope, endoplasmic reticulum, Golgi complex and mitochondria of adherent and phagocytozing macrophages. No reaction product was present around phagocytozed polystyrene particles. The amount of final reaction product was increased by the addition of sodium azide to the incubation medium and no staining was observed when the substrate was omitted from the medium.     

Ultracytochemical studies of capillary endothelial cells in the rat central nervous system

The ultracytochemical localization of eight hydrolytic enzymes (TMPase, 5'-NPase, TPPase, TTPase, Mg++-ATPase, Ca++-ATPase, ALPase and K+-NPPase) and one oxidative enzyme (MAO) was determined in rat brain capillary endothelial cells. In the somal plasma membrane, the enzymatic activity was mainly located in the antiluminal plasma membrane. This finding was appropriate for enzymes possessing the optimal pH at alkaline ranges, except for alkaline phosphatase. Most enzymes investigated showed a positive reaction on the pinocytotic vesicles of capillary endothelial cells. Differences in the intensity of the enzyme activities of the luminal and antiluminal plasma membranes may reflect the polarity in the capillary endothelial cells and relate to blood-brain barrier mechanisms.     

Ultrastructural localization of acid phosphatase in germ cells of chick embryo left ovary

The ultracytochemical localization of acid phosphatase was studied in oogonia and oocytes of the chick embryo left ovary. The reaction products are evident in lysosomes of various types and, in some cells, in the GERL as well. Furthermore, from the onset of the meiotic prophase, the enzymatic reaction also appears in the rough endoplasmic reticulum. Non-incubated sections of the same stages were observed, with the aim of identifying and describing the structure of the organelles, in particular lysosomes which appeared positive in incubated sections. The significance of the presence of the enzyme is discussed.     

Electron microscopic study of intracellular digestion in intestinal cells of the ixodid tick Hyalomma asiaticum. I. Formation of primary and secondary lysosomes]

The formation of primary and secondary lysosomes in digestive cells of midgut of the tick H. asiaticum was investigated using ultracytochemical methods for acid phosphatase. This enzyme is synthesized in the rough endoplasmic reticulum cisternae to be concentrated in the Golgi complex. Vesicles 0.1-0.15 mum in diameter filled with the enzyme are propagated from the distal Golgi cisternae which are primary lysosomes. Secondary lysosomes are produced in result of fusion of primary lysosomes with heterophagosomes that appear during endocytosis. Another type of structures responsible for transport of lysosomal enzymes into heterophagosomes is represented by dense bodies 0.3-0.5 mum in size. These are rich in acid phosphatase being different stages of heterophagolysosomes and telolysosomes.     

Ultrastructural localisation of alkaline phosphatase in the intertubular tissue of the testis in the domestic fowl

Alkaline phosphatase activity in the intertubular tissue of the testes of the domestic fowl was examined using an ultracytochemical technique based on the lead capture method. In the interstitial tissue, the Leydig cells, transitional cells and the fibroblasts displayed enzyme activity on their cell membranes. Vacuoles located in the transitional cells were lined by reaction products of enzyme activity, whereas the vacuoles representing extracted lipid droplets and present mainly in the Leydig cells were free of enzyme activity. In the peritubular tissue the cell processes of fibroblasts showed enzyme activity on the cell membranes and in pinocytotic vesicles. Cell processes lying adjacent to blood vessels showed pronounced activity. In the blood vessel itself some activity was present in the basement membrane and the endothelium. The surface of the red blood cell showed moderate activity. The possible role of alkaline phosphatase in the transfer of hormone from the Leydig cells to the seminiferous tubules and from the seminiferous tubules to the interstitium is discussed. The myoid cells and their processes were devoid of enzyme activity.     

Electron microscopy and ultracytochemistry of blood lymphocytes containing Gall bodies in healthy individuals

The authors have conducted electron microscopic and ultracytochemical studies of blood lymphocytes of healthy individuals containing Gall bodies (GB) which are typical for CD4+ cell subpopulation. It has been established that GB have quite a complex submicroscopic structure and together with its derivates, granules-satellites, as well as mitochondria, microfilaments, rough endoplasmic reticulum canaliculi, and Golgi complex it forms a unique active functional complex. GB show a high activity of acid phosphatase and positive reaction to glycogen. The data obtained suggest the leading role of GB in the production of cytokins and other biologically active substances.     

Enzyme ultracytochemical demonstration of Ca(++)-ATPase in the rat cerebral cortex.

The localization of Ca(++)-ATPase activity was investigated in the rat cerebral cortex using an ultracytochemical method. Our cytochemical procedure for the detection of enzyme activity is based on an incubation medium consisting of tricine buffer, ATP-disodium salt as substrate, cerium chloride as capturing agent, CaCl2, and levamisole as a nonspecific alkaline phosphatase inhibitor. Final pH was 7.4. Reaction products showing Ca(++)-ATPase activity were localised on the pre- and postsynaptic plasma membrane in association with synaptic vesicles, postsynaptic dendrites and on the axolemma in myelinated nerve fibres. The verification of the main enzymatic properties of Ca(++)-ATPase localization activity is the subject of the following report.     

ULTRACYTOCHEMICAL AND BIOCHEMICAL STUDIES OF PLASMA MEMBRANE ATPASE IN THE INDIVIDUAL PARTS OF THE MATURE,DORMANT SEED OF PISUM SATIVUM

The location and activity of a K⁺-ATPase in mature, dormant peas were investigated using two ultracytochemical techniques, as well as biochemical assays of plasma membrane fractions from separate seed parts. Both the Wachstein and Meisel (1957) and the Ernst (1972) cytochemical methods showed plasma membrane-associated reaction product located primarily on the exterior surfaces of the entire pea embryo, except for the stem apex and tip-most cells of the radicle. No plasma membrane-assocated reaction product was found in the seed coat, which typically consists of cells with degenerating protoplasts. Biochemical results showed the highest specific K⁺-ATPase activity in the radicles (775 nmol Pi/mg protein/hr), followed by epicotyls (168 nmol Pi/mg protein/hr) and cotyledons (147 nmol Pi/mg protein/hr). It is proposed that the entire pea embryo may function in the active absorption of nutrients during the initial phases of germination. Additional functions of the enzyme may include cell wall loosening prior to cell elongation, regulation of cytoplasmic pH, and the generation of turgor.     

Alkaline phosphatase in human lymphocytes. II. A method for ultracytochemical detection of alkaline phosphatase in lymphocytes

For the ultracytochemical identification of alkaline phosphatase in lymphocytes gained from the peripheral blood of healthy individuals a sensitive method is described which allows the low enzyme activity of these cells to be determined. This was possible because the authors succeeded in stabilizing lead ions in the alkaline medium by forming a complex directly between tris-(hydroxymethyl) aminomethan and lead (II) citrate. AP localized ultrachemically in lymphocytes in particular formations similar to phosphasomes of neutrophilic granulocytes. In those lymphocytes stimulated by lipopolysaccharides a high enzyme activity could be observed and, in addition to phosphasomes, the product of response can also be found in canal-like structures of the endoplasmatic reticulum. These findings contribute to clarify the ultrastructural localization of alkaline phosphatase in lymphocytes and may be regarded as an aid in discovering the importance of the enzyme in the biology of lymphocytes or in its activation, respectively.     

Particulate guanylate cyclase and adenylate cyclase activities after activation with various agents in rabbit platelets. An ultracytochemical study

Summary The cytochemical localization of particulate guanylate cyclase and adenylate cyclase activities in rabbit platelets were studied after stimulation with various agents, at the electron microscope level. In the presence of platelet aggregating agents such as thrombin and ADP, the particulate reaction product of guanylate cyclase activity was detectable on plasma membrane and on membranes of the open canalicular system. In contrast, samples incubated with platelet-activating factor showed no activation of the cyclase activity. Atrial natriuretic factor stimulated the particulate guanylate cyclase. The ultracytochemical localization of this activated cyclase was the same as that of thrombin-or ADP-stimulated guanylate cyclase. Adenylate cyclase activity was studied in platelets incubated with prostaglandin E₁ plus or minus insulin. The enzyme reaction product was found at the same sites where guanylate cyclase was detected. Therefore guanylate and adenylate cyclase activities do not seem to be preferentially localised in platelet membranes.     

Electron Microscopic Cytochemical Study of Cyclic Adenosine Monophosphate Phosphodiesterase Activity During Gold Acclimation of Wheat Seedlings

Comparative studies on localization and distribution of cyclic adenosine monophosphate phosphodiesterase (cAMP-PDEase) activity in the young leaf cells of two different cold resistant wheat (Triticum aestivurn L.) varieties during cold acclimation were carried out by means of ultracytochemical method. The results indicated that the reaction products of cAMP- PDEase activity in two varieties of the seedlings grown at optimum temperature (20–25℃) were mainly localized at plasmalemma, nucleoli and chromatin. When the wheat seedlings were subjected to low temperature acclimation, cAM P-PDEase activity in the strong cold resistant variety Yanda 1817 seedlings was markedly decreased; however, little alteration of this enzyme activity was observed in the weak cold resistant variety Zhengzhou 39-1. cAMP-PDEase activity was recovered after deacclimation. The results suggested that changes in cAMP-PDEase activity during cold acclimation were closely related to the development of plant cold hardiness.     

Ultrastructural localization of endogenous mammary gland peroxidase during lactogenesis in the rat results after tannic acid-formaldehyde-glutaraldehyde fixation.

Endogenous mannary gland peroxidase in acinar cells of prelactating and lactating rats is revealed in tannic acid-formaldehyde-glutaraldehyde-fixed tissue by means of the standard diaminobenzidine procedure. Diaminobenzidine cytochemical reaction product is present in perinuclear cisternae, in the granular endoplasmic reticulum and in Golgi apparatus of functionally differentiated secretory cells. The mammary gland peroxidase is thought to represent lactoperoxidase. Peroxidase staining is diminished or absent in acinar cells of hypophysectomized and ovariectomized rats, in normal rats during early pregnancy and in nonpregnant mature females. Endogenous peroxidase or a heme protein with peroxidatic activity may be considered an ultracytochemical marker enzyme for acinar cells actively engaged in lactogenesis.