Perinuclear Granules Research Articles

The effect of intermediate filament inhibitors on steroidogenesis and cytoskeleton in Y-1 mouse adrenal tumor cells

When Y-1 mouse adrenal tumor cells were treated with sodium orthovanadate, an agent disrupting BHK21-F cell microtubule-intermediate filament (IF) interactions, there was no change in the amount of 20-dihydroprogesterone produced. A neurofilament-microtubule perturbing agent, beta, beta-iminodipropionitrile (IDPN), enhanced the ability of Y-1 cells to produce steroid in response to ACTH by acting on the plasma membrane. Electron microscopy of Y-1 cells extracted with Triton X-100 revealed that both vanadate and IDPN caused the aggregation of granular structures in the perinuclear area. Based on the steroidogenic effects of IDPN, perinuclear granule aggregation may result from an altered interaction between intermediate filaments, microtubules and the plasma membrane. The reason for the ultrastructural changes caused by vanadate is unknown.

Use of the avidin-biotin-peroxidase complex (ABC) method for the localization of rabbit cathepsin B in cells and tissues.

Visualization of rabbit cathepsin B was achieved utilizing monospecific sheep antibodies and the avidin-biotin-peroxidase complex (ABC) method. This technique was applied to stain 1) paraffin sections of the liver, 2) fixed fibroblasts from tissue culture, and 3) fixed mesenteries. Cathepsin B was found to be localized within cells of the lining of the liver sinusoids (most probably Kupffer cells), in perinuclear granules of cultured fibroblasts, and within histiocytes of the mesentery. The results demonstrate that the method permits precise and highly sensitive localization of cathepsin B within cells and tissues. Compared to fluorescent staining of cathepsin B, the ABC method has the advantage that routine paraffin sections can be stained, and that all the orthodox histological staining procedures can still be carried out.

IgM paraproteins with immunological specificity for a Schwann cell component and peripheral nerve myelin in patients with polyneuropathy

The sera of 9 patients with benign IgM paraproteinaemia and chronic sensorimotor neuropathy were tested for reactivity to human peripheral nerves by the indirect immunoperoxidase method. They reacted in very high titre (10 −3–10 −6) with a cytoplasmic Schwann cell component, and to lesser degree, with peripheral nerve myelin (10 0–10 −3. The Schwann cell staining was in the form of perinuclear cytoplasmic granules and was only seen with adult nerve. The distribution of the antigen was similar to that of the metachromatically staining Pi-granules of Reich. which accumulate in the peripheral nerves with age. Specific activity was present in the IgM and F(ab) 2 fractions and could be absorbed out with peripheral nerve tissue, but not with liver. Reactivity is not a simple function of the IgM level, since many IgM paraproteins do not react. The antibody is species specific and binds to human, but not to any component of rabbit, rat or guinea pig sciatic nerves. Antigenicity is removed by pretreatment of the nerve with chloroform-methanol or periodate, but not protease or trypsin. Reactivity is restored, after periodate treatment, by exposure to sodium borohydride. It is suggested that some IgM paraproteins have a specificity for a myelin glycolipid or glycoprotein, which normally accumulates in the Pi-granules of the Schwann cell cytoplasm as a function of age.

Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D.

1. N-Pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane, a potential active-site-directed localization reagent for cathepsin D, was found to bind non-specifically to immuno-precipitates containing cathepsin D. 2. Three new water-soluble localization reagents were synthesized, by using NN'-bis-(3-aminopropyl)piperazine, 3-oxa-1,5-diamino-pentane or 3,6-dioxa-1,8-diamino-octane, as spacer arms between the pepstatin and dinitrophenyl moieties. 3. The hydrophilic dinitrophenyl-pepstatins were all tight-binding inhibitors of cathepsin D at pH 3.5, but showed little or no binding to immuno-precipitates containing the inactive enzyme at pH 7.4. 4. Gel-chromatographic experiments showed that, at pH 5.0, all the dinitrophenyl-pepstatins were bifunctional reagents able to bind cathepsin D and anti-dinitrophenyl antibody at the same time. Enzyme-inhibitor-antibody complexes were not formed at pH 7.4, thus confirming that the reagents were active-site-directed. 5. Cultured human synovial cells were fixed and incubated with the dinitrophenyl-pepstatins at pH 5.0 or pH 7.4. After washing briefly, the cells were incubated at the appropriate pH value with anti-dinitrophenyl antibody labelled with fluorescein. When examined by fluorescence microscopy the cells stained at pH 5.0 showed fluorescent perinuclear granules, which were not seen in the cells treated at pH 7.4. The distribution of cathepsin D, determined by indirect immuno-fluorescence at pH 7.4, closely resembled that revealed by the dinitrophenyl-pepstatins at pH 5.0. 7. NN'-(3-Pepstatinylaminopropyl-3'-dinitrophenylaminopropyl)piperazine gave the most intense lysosomal staining and showed no non-specific binding. We conclude that this reagent is suitable for the subcellular localization of the active conformation of cathepsin D.

Culture of human laryngeal papilloma cells in vitro.

Human laryngeal epithelial cells have been grown in tissue culture in a hydrated collagen gel containing Nutrient Mixture F12 (Gibco) supplemented with 15% fetal calf serum and 10 micrograms/mL hydrocortisone. Primary cells often remain viable in culture for more than six months. They can be serially transferred two to four times before senescence. Cells derived both from normal epithelium and from laryngeal papilloma have been successfully cultured. Papilloma cells appear to contain more perinuclear granules and form fewer tight junctions than normal cells.

Sideroblasts and haemosiderin in thyrotoxicosis.

Bone marrow sideroblasts and haemosiderin were studied in 19 thyrotoxic patients before therapy and in the euthyroid state. The proportion of sideroblasts and the amount of haemosiderin were significantly higher in the hyperthyroid than in the euthyroid phase. Pathological sideroblasts with coarse perinuclear iron granules were found before therapy but not in the euthyroid phase. It is concluded that during thyrotoxicosis the supply of iron into erythroblasts is greater than the amount used for haemoglobin synthesis.

The effect of sexual hormones on the lipid and proteinaceous secretion of the rat preputial sebaceous gland

The diameters of Sudan Black B stained sebum vesicles and acid hematein stained perinuclear granules, and the numerical density of the latter, were determined in mature sebaceous preputial cells from normal male and female rats, testosterone-treated female rats and estradiol-treated male rats. Statistical analysis by Student's t-test showed that the diameter of lipid droplets was significantly higher in male than in female controls and that testosterone increased female values up to make control levels. Estradiol treatment decreased male values to levels below those of normal male and female controls and of testosterone-treated female rats. Diameters of perinuclear granules did not vary among animal groups but their numerical density was larger in testosterone treated than in normal female rats or estradiol treated male rats. Lipid droplet sizes and perinuclear granule numbers are thus increased by androgens and decreased by estrogens, which was interpreted an meaning that sexual hormones do not only act on sebaceous cell multiplication or turnover time as previously known, but also on the production of lipid and the output of beta-glucuronidase containing granules in this gland.

Characterization of storage material in cultured fibroblasts by specific lectin binding in lysosomal storage diseases.

The lysosomal storage material in cultured fibroblasts from patients with various lysosomal storage diseases was characterized by fluorescence microscopy using lectins specific for different saccharide moieties. In normal fibroblasts and cultured amniotic fluid cells lectins specific for mannosyl and glucosyl moieties, Con A and LcA gave a bright perinuclear cytoplasmic staining corresponding to the localization of endoplasmic reticulum in the cells. All other lectins stained the Golgi apparatus as a juxtanuclear reticular structure. In fucosidosis fibroblasts, only lectins specific for fucosyl groups LTA and UEA, distinctly stained the lysosomal inclusions. The lysosomes in mannosidosis fibroblasts did not react with Con A and LcA, both specific for mannosyl moieties of glycoconjugates, but were brightly labeled with WGA, a lectin specific for N-acetyl glucosaminyl moieties. In I-cell fibroblasts, the numerous perinuclear phase-dense granules, representing abnormal lysosomes, were labeled with every lectin used. In fibroblasts from patients with Salla disease, a newly discovered lysosomal storage disorder, the lysosomes were brightly stained only with LPA, indicating the presence of increased amounts of sialic acid residues in the lysosomal inclusions.

In Vitro Studies on The Protoporphyrin Uptake and Photosensitivity of Normal Skin Fibroblasts and Fibroblasts From Patients With Erythropoietic Protoporphyria

Fibroblasts derived from patients with erythropoietic protoporphyria (EPP) are not sensitive to violet light and do not contain an excess of protoporphyrin (PP). Fibroblasts from both normal individuals and patients with EPP can take up very low concentrations of PP from culture medium. Cells grown in PP-containing medium showed a gradually increasing but limited uptake of PP and also an increased sensitivity to light. A sensitive scanning microfluorometric method has made it possible to demonstrate that the PP is mainly localized in the perinuclear granules. After exposure to violet light, cells photosensitized by PP in a culture medium showed increased membrane permeability as well as reduced reproductive capacity. Both of these photodamage effects can be repaired during postirradiation incubation in culture medium at 37 degrees C.

The fine structure of the yolk nucleus in oocytes of Antedon bifida (Pennant) (Echinodermata: Crinoidea)

The yolk nucleus of Antedon bifida is a large, intensely basophilic structure located in the cytoplasm of small to medium-sized oocytes. Electron microscopy reveals that this yolk nucleus is a conglomeration of hundreds of dense clumps. Each clump, which is a micrometer or two in diameter, consists of finely fibrogranular material in which are embedded ribosome-like particles. Also present in the oocyte cytoplasm are perinuclear dense granules, which may well be the precursors of the clumps making up the yolk nucleus. The yolk nucleus does not appear to participate directly in the elaboration of the yolk granules.

Acid hydrolases in the perinuclear secretion granules of the rat preputial gland. a light and electron microscope study.

Four acid hydrolases in the secretory cells and the sebum of the preputial sebaceous gland of the rat were incestigated cytochemically. A strong β-glucuronidase activity was found to occur in the matrix of the perinuclear secretion granules, whereas the granule crystalloids were unreactive. The distribution of acid phosphatase at the light microscope level was similar, though the intensity of the reaction was lower and the number of positive granules smaller. By electron microscopy, the final reaction product of acid phosphatase occurred in patches at the periphery of the granule matrix, as well as in the vesicles adjoining the Golgi stacks, from which the perinuclear granules seemed to arise. In the sebum, the two hydrolases occurred in the background material between the unstained crystalloid masses. There was noN-acetyl-β-glucosaminidase or aryl sulphatase activity in the gland. The perinuclear granules appear to be secretory lysosomes which, after discharge from the disaggregating cell, release their acid hydrolases into the sebum.

The perinuclear secretion granules of the rat preputial gland an electron microscope cytochemical study

The perinuclear secretion granules of the rat preputial gland are membrane-bound and contain a light or dense matrix, within which there may be a crystalloid body. The light matrix contains glycoproteins, being PA-silver-methenamine- and PAS-positive. The dense matrix, in addition, possesses phospholipids since it is osmiophilic, uranophilic, and Baker-positive. Crystalloids contain phospholipids but lack glycoproteins. All three components have proteins, being positive to the coupled-tetrazonium-reaction and Pronase-labile. Granules with light matrix (type A), sometimes with a crystalloid (type B), occur in differentiating cells; those with dense matrix (type C), sometimes with one or more crystalloids (type D), predominate in differentiated cells. The perinuclear granules thus seem to have only glycoproteins at first, to which phospholipids are later added. After cell disaggregation, crystalloids are released intact, while granule matrix glycoprotein diffuses around.

Brunner's glands of the duckbilled platypus (Ornithorhynchus anatinus).

AbstractBrunner's glands in the platypus form a lobulated, glandular collar confined to the submucosa of the most distal portion of the stomach. The glands end immediately proximal to the gastrointestinal junction and excretory ducts empty in the region where the stratified squamous epithelium lining the stomach changes abruptly to the intestinal lining epithelium of the duodenum. An individual gland of Brunner is composed of several elongate lobules drained by intralobular ducts which often join to form a single excretory duct.Light and electron microscopic studies have shown the secretory tubules to be comprised of large, pyramidal cells limited basally by a delicate basal lamina. The ergastoplasm, cisternae of which are dilated and contain amorphous material, is associated with numerous ribosomes. In basal and perinuclear regions intercisternal granules and smooth surfaced vesicles are found. Numerous small vesicles found in supranuclear areas apparently form from the smooth membrane portions of ergastoplasm located adjacent to Golgi complexes. Membrane‐bound amorphous granules of varying electron density occupy the apical cytoplasm and show a tendency to coalesce before emptying their contents into the adjacent lumen.The intralobular duct system is lined initially by a columnar epithelium which changes to a simple squamous form before the ducts combine to form a short excretory duct lined by stratified squamous epithelium. The epithelium lining the duct system contains relatively few organelles but appears to be engaged in a limited amount of synthesis and release of secretory material.Histochemical studies indicate that both the secretory tubules and the duct system elaborate a neutral mucopolysaccharide.

Electron Microscopy and Histochemical Demonstration of Lysosomal Structures in Chicken Thrombocytes

Lucas and Jamroz (5) described the chicken thrombocyte as an elongated blood cell, slightly larger than an erythrocyte, with an oval nucleus and a cytoplasm composed of a framework with large spaces. Those workers deduce that the spaces are not vacuoles, because they lack discretion or regularity of conformation, and suggest that the characteristic feature of the stained thrombocyte is the specific perinuclear cytoplasmic granules, which vary in number, size, and intensity of color. The avian thrombocyte is considered to perform the same function as the mammalian platelet in hemostasis (5), which is substantiated by observations of Stalsberg and Prydz (11) that thrombocytes were the only cellular elements in hemostatis plugs in the extraembryonal circulation of chick embryos. Very little histochemistry or electron microscopy on avian thrombocytes has been reported. Atwal and McFarland (1), in a cytochemical study of the whole blood of Coturnix coturnix japonica, observed acid phosphatase activity only in monocytes and a few lymphocytes. Merkal and Mora (7) demonstrated a weak, acid phosphatase activity in immature chicken thrombocytes that decreased as the cells aged. Schumacher (10) published an ultrastructural study of Gallus domesticus thrombocytes, and noted a paucity of cytoplasmic organelles, including few mitochondria and little golgi. He also noted the presence of large cytoplasmic vesicles, but described no other cytoplasmic granules or particles.

The uptake and digestion of iodinated human serum albumin by macrophages in vitro.

Mouse peritoneal macrophages take up I*-HSA from their medium during in vitro cultivation. Conditions which promote I*-HSA uptake are the same as those which stimulate formation of pinocytic vesicles. Autoradiography of cells pulsed with (125)I-HSA showed that intracellular isotope is localized in perinuclear granules, or secondary lysosomes. Following a pulse of (125)I-HSA, intracellular radioactivity decreases and the amount of TCA-soluble isotope in the medium increases correspondingly. About 50% of the intracellular isotope is lost in 5 hr. The release of isotope from pulsed cells is not inhibited by parafluorophenylalanine, 2,4-dinitrophenol or by a reduction of the serum concentration of the medium. However, the processing of ingested (125)I-HSA is reversibly inhibited by reduced temperature. The TCA-soluble radioactive material excreted by pulsed macrophages was identified as monoiodotyrosine.

Hexamastix dobelli n.sp., a New Trichomonad Parasitic in the Starred Tortoise

Hexamastix dobelli n. sp. (Trichomonadidae) is described from the large intestine of the starred tortoise, Testu‐do elegans Schoepff. This is the first species of Hexamastix to be reported from a chelonian. The organism is pyriform, oval, or round in shape. There are five anterior flagella, of which two are long and three short and a single recurrent flagellum, which equals the short anterior flagella in length. The axostyle is very slender and projects for some distance outside the body. The nucleus contains three to six granules and has no endosome. The pelta is crescent‐shaped and the parabasal body is rod‐like. Perinuclear granules are present in the anterior half of the body. The parasite is 6.0 to 16.5 μ long, 2.0 to 10.3 μ wide and the diameter of nucleus is 2.5 μ.

The effect of pH on growth, protein synthesis, and lipid-rich particles of cultured mammalian cells.

A simple procedure has been established for controlling and measuring the pH of media in which the bicarbonate-carbonic acid system is the predominant buffer. The HCO(-) (3) concentration was maintained at 22.5 mM and the H(2)CO(3) concentration was varied by equilibrating the media with 0.5 to 40 per cent CO(2) in air. The curve relating extracellular pH to 3 day cell growth was similar for glass-attached HeLa and Chang liver cells. Maximum growth occurred over a pH range of 7.38 to 7.87. Cell growth declined precipitously on the alkaline side and more gradually on the acid side of the optimal pH range. Comparable pH growth curves were also obtained with newly isolated cells from rat liver and skeletal muscle. It was shown that the effect of pH on growth was independent of the CO(2) concentration and that the essential nutrients in the medium were stable over the pH range studied. Although alkalosis depressed the 3 day cell population, cells exposed to a pH of 8.0 to 8.2 grew at the maximal rate for the first 12 to 24 hours. Growth then ceased abruptly and the cells entered a steady state with respect to net protein synthesis. This was followed by cytoplasmic retraction and cell death. Increasing the concentrations of calcium or magnesium in the medium failed to prevent the effects of alkalosis. Moreover, the increase in CO(-) (3) concentration of the media and the concomitant decrease in Ca(++) ion concentration that occur at high pH were eliminated as determining factors in the growth failure and death. While acidosis had a less pronounced effect on the 3 day cell population, its effect on the growth rate was immediate. The increase in cell generation time was proportional to the H(+) ion concentration. In each of the cell lines studied, acidosis was accompanied by a striking increase in the number of cytoplasmic perinuclear granules. These granules which stain supravitally with Janus green are extracted from fixed cells with lipid solvents. They maintain their identity in cell homogenates and may be isolated from the other subcellular structures by differential centrifugation; at 100,000 g they form a distinct layer at the top of the supernatant fraction. On the basis of their physical and chemical properties, these granules have been called lipid-rich particles. The accumulation of lipid-rich particles in acidosis was independent of the growth rate and the CO(2) concentration.

Cellula apicale e metabolismo degli acidi nucleici nella spermatogenesi degli Ortotteri(Acrididae e Locustidae)

(1) The apical or Verson cell of the testis ofOrthoptera is a connective tissue element having a trophic function. Its secretion is rich in ribonucleic acid (RNA), which is absorbed by the germ cells. This is a primary source of RNA for spermatogony. (2) Nuclear chromatin is probably concerned in RNA elaboration. The variation in amount and distribution of the perinuclear RNA granules suggest the existence of a secretory cycle analogous to that described inAsellus. (3) In the connective tissue cells of the zone below the apical complex, degenerative processes are observed, the end-product of which is essentially RNA. The spherules which stain green or violet withUnna's mixture and are Feulgen-positive, are interpreted as pyknotic nuclei. Their chromatin is transformed into RNA and constitutes spherules and small granules positive to pyronine, which are absorbed by spermatocytes in meiotic, prophase. Thus, during spermatogenesis in the investigatedOrthoptera, both the apical cell and some degenerating connective tissue elements produce RNA, which is absorbed by the developing germ cells.

Comparative Morphology of the Trichomonad Flagellates of Man

Summary Morphologically, the three species of Trichomonas of man are distinct, although T. vaginalis and T. tenax resemble each other more than either resembles T. hominis. T. vaginalis is much the larger, the other two being about the same length. T. vaginalis is more robust in shape and T. tenax is more slender. T. vaginalis and T. tenax normally have a single compound blepharoplast which may occasionally appear as two approximately equal elements. In T. hominis there are characteristically two blepharoplasts of unequal size, the smaller being ventral in position. The oral and vaginal species have regularly four anterior flagella. T. hominis has a variable number, usually four or five, but more characteristically five. One of these is attached to the smaller ventral blepharoplast and the other four to the larger one. The undulating membrane of T. tenax is relatively longer than that of T. vaginalis, although typically less than body length in both and without an extension of the marginal filament into a trailing flagellum. T. hominis has a full-length undulating membrane with a trailing flagellum. At the margin of the undulating membrane each of the three species has an accessory filament which is more slender than the posterior flagellum. The costa of T. vaginalis and T. tenax is very slender and of uniform diameter while that of T. hominis is coarser, and thicker in its midregion. Both T. vaginalis and T. tenax have a well-developed parabasal apparatus, but this is apparently absent in T. hominis. The axostyle of T. hominis is relatively somewhat coarser than that of the other two species. In large individuals of T. vaginalis it tends to split into a number of fibrils. The nucleus of T. vaginalis is typically much elongated while in the other two species it is more commonly rounded or oval. In T. vaginalis and T. tenax the chromatin is usually aggregated into granules which are uniformly distributed; these are larger and fewer in T. tenax. In T. hominis the chromatin is less often aggregated into granules and commonly forms a layer against the nuclear membrane. The siderophilic cytoplasmic granules of T. vaginalis are numerous and scattered through the cytoplasm but tend to collect along the axostyle and costa. In T. tenax they are fewer, forming a row along the costa but not along the axostyle. In T. hominis there is a row of paracostal granules at the base of the undulating membrane and a zone of perinuclear granules or a chromatic cloud in the vicinity of the nucleus. The cytostome is inconspicuous in T. vaginalis but more evident in the other two species, especially in T. hominis. In fresh material T. vaginalis usually shows no food inclusions except a leukocyte in an occasional individual while T. hominis usually shows a variety of food bodies. T. vaginalis forms pseudopodia more often than the other two species, is more sluggish, and shows greater tendency to adhere to solid objects and to each other at the posterior ends. Survey data show that T. vaginalis and T. tenax are much more prevalent than T. hominis even in the same group of persons, indicating that the latter species is not transferable to other parts of the body. Available literature provides abundant evidence that, in the matters of culture conditions, inoculation into various animals and unsuccessful transmission of intestinal and oral trichomonads to the human vagina, these three species are physiologically as well as morphologically distinct. Trichomonas vaginalis Donne is the type species of the genus and presents no taxonomic problem. T. tenax (O.F.M.) has priority for the oral species. The intestinal form appears to have a variable number of flagella, characteristically five. For the present, T. hominis (Davaine) is retained for it. Tritrichomonas fecalis Cleveland is shown to be distinct from T. hominis and very similar to, if not identical with, a species from the intestine of frogs and toads.