Ultracytochemical Methods Research Articles

Autophagy and vacuolar biogenesis during the nectary development.

Different autophagy pathways are a driver of vacuolar biogenesis and are development stage specific during the extrafloral nectary development in Citharexylum myrianthum. Plant autophagy plays an important role in various developmental processes such as seed germination, pollen maturation and leaf senescence. However, studies that address the evidence of autophagy and its role in the development of plant glands are scarce and largely restricted to laticifers. Regarding nectary, studies have repeatedly pointed to signs of degradation associated with the end of the secretory cycle, without exploring autophagy. Likewise, the relationship between autophagy and biogenesis of vacuoles remains an unexplored issue. In this study, using conventional and microwave fixation in association with ultracytochemical methods for transmission electron microscopy, we investigated the occurrence of autophagy and its implication in the differentiation of extrafloral nectary in Citharexylum myrianthum (Verbenaceae) under natural conditions, focusing on the vacuole biogenesis. We described a variety of vacuole types associated with the stage of nectary epidermis development, which differs with respect to origin, function and nature of the products to be stored. Three distinct autophagy pathways were detected: macroautophagy, microautophagy (both restricted to the undifferentiated epidermal cells, at the presecretory stage) and megaautophagy (circumscribed to the differentiated epidermal cells, at the postsecretory stage). Our study clearly demonstrated that the vacuole variety and autophagy processes in the nectary epidermal cells are development specific. This study highlights the role of autophagy in vacuole biogenesis and its implications for the development of nectary and opens new venues for future studies on regulation mechanisms for autophagy in plant secretory structures under normal conditions.

Ultracytochemical demonstration of glycoproteins in the canine knee synovium

By various ultracytochemical methods, glycoconjugates of the synoviocytes, the intercellular matrix and the wall of the small capillaries were studied in the synovial intimal tissues of the canine knee joint. Glycoconjugates with vicinal diol groups could be visualized in certain elements of the Golgi complex, lysosomes, vacuoles, the majority of intracellular cytomembranes, the surface coat of the plasma membrane and glycogen particles in type A cells. In type B cells, less-developed Golgi complexes, and fewer lysosomes and vacuoles were present in the cytoplasm than in that of type A cells. In contrast, a large number of cytoplasmic glycogen particles and abundant vicinal diol-containing groups in the surface coat of the plasma membrane became especially obvious in the B cells. Abundant neutral and acidic glycoproteins were observed in fibrous components in the intercellular matrix. In the small capillaries, strongly positive staining intensities for neutral and acidic glycoconjugates were observed in the basement membrane and perivascular connective tissue, as well as in the surface coat of the luminal plasma membrane of the endothelial cells, although to a somewhat weaker degree. Sialic acid, particularly, was notable in the surface coat of the latter cells. In addition, glycoproteins in the type A cells were shown by lectin ultracytochemistry to contain a variety of saccharide residues such as alpha-D-mannose, alpha-D-glucose, alpha-L-fucose, N-acetyl-beta-D-glucosamine, and N-acetyl-neuraminic acid, which were also found in the plasma membrane of the B cells. The properties of the glycoconjugates found are discussed in relation to the basic functions assigned to the synovial membrane of the canine knee joint.

Ultracytochemistry of glycosaminoglycans in the canine knee synovium

The accurate localization and nature of glycosaminoglycans (GAGs) in the canine knee synovium were studied by ultracytochemical methods that involved high or low iron diamine-thiocarbohydrazide-silver proteinate (physical development) staining in combination with enzyme digestion control procedures. The results obtained indicated that heparan sulfates and hyaluronan were present mainly in the plasma membrane of the B (fibroblast-like) cells. In contrast, the plasma membrane of the A (macrophage-like) cells showed negative reactions after the histochemical examination. Dermatan sulfates, chondroitin sulfates (A and/or C) and hyaluronan were localized in the extracellular matrix of the synovial intima, whereby dermatan sulfates were confined to the fibrous component, whereas chondroitin sulfates and hyaluronan were found in the interfibrous matrix. Heparan sulfate was the only notable GAG molecular species localized in the basement membrane of the capillary wall. It is obvious that differences in the quality and localization of glycosaminoglycans in the canine synovial tissue are of specific interest in understanding normal functions as well as pathological alterations of the knee synovium in mammals.

Ultracytochemistry of 3beta-hydroxysteroid dehydrogenase in Leydig cell precursors and vascular endothelial cells of the postnatal rat testis.

In the biosynthesis of steroid hormones 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is a key enzyme. The present report describes the subcellular localization of the enzyme in the fetal-type Leydig cells, the fibroblast-like precursors of adult-type Leydig cells and in endothelial cells of interstitial capillaries. Histochemical methods for light microscopy and ultracytochemical methods for electron microscopy were used on rat testes of postnatal day 15. 3beta-HSD reactivity was located at subcellular levels by means of the ferricyanide method. A specific, distinct localization of reaction product in the form of copper ferrocyanide precipitates was observed on the membranes of the smooth endoplasmic reticulum not only in the fetal-type Leydig cells and the fibroblast-like precursors of adult-type Leydig cells, but also focally in the endothelial cells of interstitial blood capillaries. Topographically, the 3beta-HSD-positive precursors were most often found in the outer layer of the boundary tissue and surrounding interstitial blood vessels. The capillaries with 3beta-HSD-positive endothelial cells were usually located in the vicinity of 3beta-HSD-positive Leydig cells. For the first time, 3beta-HSD has been located at the subcellular level in precursors of adult-type Leydig cells and focally in capillary endothelial cells associated with them. Due to the close association between 3beta-HSD-positive vascular endothelial cells and Leydig cells a paracrine relationship between the two cell types may be involved in the acute regulation of steroidogenesis by blood-borne luteinizing hormone.

Localization artefacts in ultracytochemical ion precipitation reactions.

The precipitation patterns of the following ultracytochemical methods in rat muscle cells were compared and examined critically: the potassium pyroantimonate method for calcium demonstration; the calcium phosphate technique for the Ca2+--ATPase reaction; the formazan reaction for the demonstration of creatine kinase activity (all performed on heart muscle); and the lead phosphate technique for the Mg2+--ATPase reaction in skeletal muscle. Using X-ray microanalysis, it was found that the antimonate precipitate contains only calcium as the precipitated ion in the vast majority of cases. Most probably it consists of pure calcium pyroantimonate. However, in myocytes showing the well-established precipitation pattern, the concentration of calcium was estimated to be about two orders of magnitude higher than the native concentration of total intracellular calcium. It is concluded that calcium ions diffuse freely from the extracellular space and from adjacent cells into cells containing antimonate and are precipitated mostly at sites where heterogeneous nucleation is facilitated by intracellular catalysts (biopolymers). As shown by the similar precipitation patterns for the four reactions compared, these catalysts are not specific to any of these reactions and are most probably neither calcium-binding sites nor sites of any one of the enzymes examined in the native cell.

Localization of nucleoside phosphatases (ATPase and 5′-nucleotidase) and nuclear ribonucleoproteins in the human endometric glandular cells during the secretory phase

Endometrial curettings from 3 healthy women between 16th and 19th d of the menstrual cycle were investigated employing ultracytochemical methods. In endometrial glandular cells ATPase and 5'-nucleotidase activities were demonstrated in the nuclei, nucleoli, and nuclear channel system (NCS), i.e., mainly in structures characterized ++as ribonucleoproteins according to the Bernhard method. The functional relationship between the NCS and the molecular biology of the endometrial glandular cell is discussed.

A histochemical study of variations in the localization of 5'-nucleotidase activity in the acinar cell of the rat exocrine pancreas over the twenty-four hour period.

The circadian variation of 5'-nucleotidase (AMPase) activity was studied in rat pancreatic exocrine cells. The localization of this enzyme, often associated with the plasmalemma, was studied by ultracytochemical methods at six time points over the 24-h period. The localization of AMPase activity exhibited a clear-cut circadian variation. During the light span strong activity was observed on the luminal plasmalemma, negative or weak activity on the baso-lateral plasmalemma and clearly visible activity on intracellular structures such as cytoplasmic vacuoles (fragmentation-like vesicles), dilated rims of the Golgi cisternae (or cisternal ends of the Golgi stacks), condensing vacuoles and lysosomal bodies. During the dark span the activity was detectable only on the baso-lateral plasmalemma. The fact that AMPase activity could not be found on the luminal plasmalemma during the dark span suggests that the luminal membranes may be replaced by the membranes of secretory granules, which do not display AMPase activity. The intracellular localization of AMPase activity during the light span, especially at 08.00 h, includes all cytoplasmic compartments which have hitherto been associated with the intracellular pathway for membrane retrieval from the plasmalemma. Moreover, the appearance of the activity in the dilated rims of the Golgi stack and condensing vacuoles indicates that these compartments may constitute a functional unit.

Phenolic ultracytochemistry of tobacco cells undergoing the hypersensitive reaction to Pseudomonas solanacearum

Tobacco leaf tissue was injected with avirulent (Bl) and virulent (K60) strains of Pseudomonas solanacearum . Avirulent bacteria induced a hypersensitive reaction (HR), whereas virulent bacteria caused a susceptible response. Ultracytochemical methods for localization of phenolics and polyphenoloxidase (PPO) over a 30-h period after inoculation showed cytoplasmic phenolic deposition at 10 h followed by PPO at 20 h during the HR. Polyphenol-oxidase was localized primarily in the grana of chloroplasts, but also occurred in the cytoplasm and in vesicles found between the plasmalemma and cell wall. Virulent bacteria caused only small increases in phenolic deposition and little observable PPO during the 30 h following inoculation.

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

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.

Ultracytochemical localization of Na+, K+-activated ATPase in the oocytes of Heterandria formosa Agassiz, 1853 (Pisces, Poeciliidae).

Using ultracytochemical methods, this paper studies the presence of Na+, K+-activated ATPase during oocyte development in Heterandria formosa. ATPase was discovered in all four oocyte stages, but its level was highest in stages II and III in which yolk formation took place. Na+, K+-activated ATPase was found on the membranes of both the yolk vesicles and the cortical granules and on the oocyte and follicular microvilli and the primary oocyte membrane, indicating that the microvilli were the sites of substance exchange between the follicle and the oocyte.

Adenosine Triphosphatases on Plasma Membrane and Membrane Surrounding Lipid Vacuoles in L1210 Lymphoid Leukemic Cells

The activity of adenosine triphosphatases in L1210 lymphoid leukemic cells was demonstrated by ultracytochemical methods. The activity of Na+, K+-ATPase was found only in the plasma membrane, but the activity of Mg2+-ATPase and Ca2+-ATPase was found in both the plasma membrane and in the membrane surrounding lipid vacuoles in the cytoplasm. The activity of Ca2+-ATPase predominated on the periphery of the lipid vacuoles. The localization of these enzymes is discussed in reference to its possible significance in transformed cells.

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

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 discussed.

Relative mobility of con A and anionic receptor sites on the cell surface of lymphocytes. A quantitative-cytochemical study.

Events following the binding of Concanavalin A and cationized ferritin to the surface of normal human peripheral blood lymphocytes were followed using ultracytochemical methods. In this paper, we described a technique involving microdensitometry, which was employed in order to make a comparative cytochemical quantitation of the dense horseradish peroxidase-diaminobenzidine reaction products representing the states of Concanavalin A receptors under various conditions of distribution and redistribution. The technique presents a direct approach to the analysis of various probes that are currently being employed for studying the nature and distribution of various cell surface components. An attempt was also made to analyze the distribution of anionic sites, and their lateral redistribution is compared with the mobility of Concanavalin A receptors. The data presented suggests an independent mobility of receptors for Concanavalin A and cationized ferritin.

Light-electron microscopic and cytochemical studies on the morphogenesis of familial medullary thyroid carcinoma.

Six cases of familial medullary thyroid carcinoma (MTC) were investigated by light and electron microscopy as well as by ultracytochemical methods. Light microscopic examination revealed multifocal C-cell proliferation in 5 subjects. These cells were mostly limited to thyroid follicles, but occasionally extended across the follicular capsule forming microscopic MTC. Electron microscopic examination showed that, in some follicles, the proliferating C-cells were still covered by a continuous layer of follicular cells, whereas in others the proliferation extended to the follicular center. C-cells were in direct contact with the colloid, and ultramicroinvasion of the follicular capsule was detected. These observations are consistent with the hypothesis that familial MTC seems to begin as multifocal C-cell proliferation, limited at first to thyroid follicles, between the capsule and the follicular epithelium. Later, the proliferation extends to the follicular center, and C-cells come in contact with the colloid, at which time an in situ carcinoma stage is reached. Some neoplastic cells invade the follicular capsule and, finally, multiple MTC appear and eventually conglomerate. Generally, there were no constant morphologic criteria for a dysplasia or neoplasia among the proliferating C-cells limited to thyroid follicles, when compared with normal or even malignant C-cells. For these reasons, a hyperplastic or dysplastic process preceding MTC cannot be clearly distinguished from a neoplastic process. Our study, however, shows that a light microscopic, apparently hyperplastic process may be a malignant one. Amyloid was present in the more voluminous MTC, associated with tumor cell necrosis, but it was not evident in small MTC and within the foci of C-cell proliferation. Ultracytochemical techniques revealed that the secretory granules of normal, proliferating and neoplastic C-cells contained polysaccharides and/or glycoproteins.

Prostatic Acid Phosphatase (PAP) Differentiated Ultracytochemically from Lysosomal Acid Phosphate in the Rat with a PAP/Specific Substrate, Phosphorylcholine

During the design and synthesis of new chemotherapeutic agents for prostatic carcinoma based on phosphorylated agents which might be enzyme-activated to cytotoxicity, phosphorylcholine, [(CH3)3+NCH2CH2OPO3Ca]Cl-, has been indicated to be a very specific substrate for prostatic acid phosphatase (PAP). This phenomenon has led to the development of specific histochemical and ultracytochemical methods for PAP using modifications of the Gomori lead method for acid phosphatase. Comparative histochemical results in prostate and kidney of the rat have been published earlier with phosphorylcholine (PC) and β-glycerophosphate (βGP). We now report the ultracytochemical results.Minced tissues were fixed in 3% glutaraldehyde-0.1 M phosphate buffered (pH 7.4) for 1.5 hr and rinsed overnight in several changes of 0.05 M phosphate buffer (pH 7.0) containing 7.5% sucrose. Tissues were incubated 30 min to 2 hr in Gomori acid phosphatase medium (2) containing 0.1 M substrate, either PC or βGP.

ULTRACYTOCHEMICAL DEMONSTRATION OF INTRACELLULAR AMINO GROUPS BY THE HYDROQUINONE-FERRICYANIDE SYSTEM

Ultracytochemical methods for the demonstration of reactive amino groups in the cell were presented. The method utilizes either the hydroquinoneferricyanide system or the hydroquinone-tetrazolium system.The reactive amino groups were observed in the mitochondrial matrix and probably in substances in Z in the cardiac muscle of the normal adult albino mice.