Showing papers on "Granule (cell biology) published in 1990"

Layered structure of bacterial aggregates produced in an upflow anaerobic sludge bed and filter reactor.

Abstract: The ultrastructure of bacterial granules that were maintained in an upflow anaerobic sludge bed and filter reactor was examined. The reactor was fed a sucrose medium, and it was operated at 35 degrees C. Scanning and transmission electron microscopy revealed that the granular aggregates were three-layered structures. The exterior layer of the granule contained a very heterogeneous population that included rods, cocci, and filaments of various sizes. The middle layer consisted of a slightly less heterogeneous population than the exterior layer. A more ordered arrangement, made up predominantly of bacterial rods, was evident in this second layer. The third layer formed the internal core of the granules. It consisted of large numbers of Methanothrix-like cells. Large cavities, indicative of vigorous gas production, were evident in the third layer. On the basis of these ultrastructural results, a model that presents a possible explanation of granule development is offered. Images

Hydrolytic enzymes associated with the granular haemocytes of the marine mussel Mytilus edulis.

Abstract: The ultrastructural localization of a range of hydrolytic enzymes has been investigated in the granular haemocytes of the marine mussel Mytilus edulis. Arylsulphatase activity and immunocytochemical localization of beta-glucuronidase and elastase were demonstrated within the large granules of the haemocytes. Lysozyme and cathepsin B were both localized within all sizes of granule, however, at high dilutions the primary antibody against lysozyme was also restricted to the large granules. The labelling density for cathepsin B antibody tended to be very low. Antibodies for cathepsin G showed a clear, discrete labelling which was restricted to the granules of haemocytes containing small granules. The fact that antibodies raised against human proteinases recognize invertebrate enzymes suggests that there must be a certain degree of structural similarity between the human proteinases and the enzymes present in the mussel haemocytes indicating either convergence or conservation of the enzyme molecules. The presence of a range of hydrolytic enzymes including proteinases, glycosidases and sulphatases within the large granules shows that these granules are a form of lysosome. The reduction in activity of lysosomal enzymes in haemocytes following adhesion to glass is evidence for release of the enzymes from the granules (degranulation). The possibility of a serine protease being specifically associated with the small granules and its role as a cytolysin are discussed.

The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments.

Abstract: Cytolytic lymphocytes contain specialized lytic granules whose secretion during cell-mediated cytolysis results in target cell death. Using serial section EM of RNK-16, a natural killer cell line, we show that there are structurally distinct types of granules. Each type is composed of varying proportions of a dense core domain and a multivesicular cortical domain. The dense core domains contain secretory proteins thought to play a role in cytolysis, including cytolysin and chondroitin sulfate proteoglycan. In contrast, the multivesicular domains contain lysosomal proteins, including acid phosphatase, alpha-glucosidase, cathepsin D, and LGP-120. In addition to their protein content, the lytic granules have other properties in common with lysosomes. The multivesicular regions of the granules have an acidic pH, comparable to that of endosomes and lysosomes. The granules take up exogenous cationized ferritin with lysosome-like kinetics, and this uptake is blocked by weak bases and low temperature. The multivesicular domains of the granules are rich in the 270-kD mannose-6-phosphate receptor, a marker which is absent from mature lysosomes but present in earlier endocytic compartments. Thus, the natural killer granules represent an unusual dual-function organelle, where a regulated secretory compartment, the dense core, is contained within a pre-lysosomal compartment, the multivesicular domain.

Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules.

Abstract: Bactenecins are highly cationic polypeptides of bovine neutrophil granules and exert in vitro a potent antimicrobial activity. We have previously purified two bactenecins, designated in an abbreviated form Bac7 and Bac5 from their approximate molecular masses of 7 and 5 kD (Gennaro, R., B. Skerlavaj, and D. Romeo. 1989. Infect. Immun. 57:3142-3146). Here we have studied the biosynthesis, processing, and localization of precursors of Bac7 and Bac5 in bovine bone marrow cells of the myeloid lineage. In vitro translation directed by mRNA isolated from these cells has shown that the primary translation products are preprobactenecins of 23.5 and 21 kD, and are processed to polypeptides of 20 and 15.8 kD, respectively. The 20-kD polypeptide is the granule storage form of Bac7, or proBac7, as also demonstrated by Western blot analysis of lysates of peripheral neutrophils. Between 15 and 50 min from the beginning of its biosynthesis the 15.8-kD polypeptide is converted into the 15-kD granule storage form of Bac5, or proBac5. As shown by immunogold EM, proBac7 and proBac5 are sorted and targeted to the matrix of the so called large granules, which are the predominant organelles in the cytoplasm of bovine neutrophils and are the exclusive store of the nonoxidative antimicrobial system of these cells. Solubilization of granules with Triton X-100 with concomitant unmasking of proteases leads to cleavage of the proforms to Bac7 and Bac5. Experiments performed with protease inhibitors suggest that the proteolytic cleavage is catalyzed in detergent-solubilized neutrophils by neutral serine protease(s), very likely derived from the azurophil granules.

Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore.

Abstract: For fusion to occur the repulsive forces between two interacting phospholipid bilayers must be reduced. In model systems, this can be achieved by increasing the surface tension of at least one of the membranes. However, there has so far been no evidence that the secretory granule membrane is under tension. We have been studying exocytosis by using the patch-clamp technique to measure the surface area of the plasma membrane of degranulating mast cells. When a secretory granule fuses with the plasma membrane there is a step increase in the cell surface area. Some fusion events are reversible, in which case we have found that the backstep is larger than the initial step, indicating that there is a net decrease in the area of the plasma membrane. The decrease has the following properties: (i) the magnitude is strongly dependent on the lifetime of the fusion event and can be extensive, representing as much as 40% of the initial granule surface area; (ii) the rate of decrease is independent of granule size; and (iii) the decrease is not dependent on swelling of the secretory granule matrix. We conclude that the granule membrane is under tension and that this tension causes a net transfer of membrane from the plasma membrane to the secretory granule, while they are connected by the fusion pore. The high membrane tension in the secretory granule may be the critical stress necessary for bringing about exocytotic fusion.

Degradation of Native Starch Granules by Barley α-Glucosidases

Abstract: The initial hydrolysis of native (unboiled) starch granules in germinating cereal kernels is considered to be due to α-amylases. We report that barley (Hordeum vulgare L.) seed α-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant α-amylase isozymes. Two α-glucosidase charge isoforms were used individually and in combination with purified barley α-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl α-glucosidase was combined with either high or low pl α-amylase. Synergism was also found when low pl α-glucosidase was combined with α-amylases. Scanning electron micrographs revealed that starch granule degradation by α-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of α-glucosidases and α-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by α-amylase alone. As the presence of α-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the α-glucosidases to hydrolyze glucosidic bonds other than α-1,4- and α-1,6- that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by α-amylases. Since both α-glucosidase and α-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro.

Compound versus multigranular exocytosis in peritoneal mast cells

Abstract: We have used the whole-cell patch-pipette technique to measure the step increases in the cell membrane capacitance (equivalent to the membrane area) caused by the fusion of secretory granules in degranulating murine mast cells. We have observed that up to 30% of the total membrane expansion caused by degranulation results from large fusion events that cannot be explained by the fusion of single secretory granules. These large events are observed mainly in the initial phase of a degranulation. We have developed a simple mathematical model for a mast cell to test whether these large events are caused by a stimulus-induced, granule-to-granule fusion that occurs before their exocytosis (multigranular exocytosis). Our results suggest that the large fusion events are caused by the exocytosis of granule aggregates that existed before stimulation and that are located at the cell's periphery. We propose a novel mechanism by which granule aggregates can be formed at the periphery of the cell. This mechanism relies on the ability of a transiently fused granule ("flicker") to fuse with more internally located granules in a sequential manner. This pattern may result in the formation of larger peripheral granules that later on can fuse with the membrane. The formation of peripheral granule aggregates may potentiate a subsequent secretory response.

Deposition and role of thrombospondin in the histogenesis of the cerebellar cortex

Abstract: The patterns of deposition of thrombospondin (TSP), a trimeric extracellular matrix glycoprotein, were determined during the initial establishment of the external granule cell layer and the subsequent inward migration of granule cells forming the molecular and (internal) granule cell layers. The early homogeneous deposition of TSP became restricted to the rhombic lip in the region of granule cell exit from the neuroepithelium, and was present between migrating granule cells. During the later inward migration of granule cells, little TSP was associated with dividing granule cells; it was enriched in premigratory granule cells. With the cessation of migration, TSP was lost except in association with fasciculating axons in the molecular layer where staining persisted briefly. At the EM level, TSP was associated with the leading process of granule cells as they associated with Bergmann glial cells and migrated through the molecular layer. TSP was present within granule cell axons; Purkinje cells and their dendrites, as well as Bergmann glial fibers and endfeet were negative for TSP. When anti-TSP antibodies were added to explant cultures of cerebellar cortex during active granule cell migration, a dose-dependent inhibition of migration was observed. In control cultures, granule cells migrated into the (internal) granule cell layer, while granule cells exposed to anti-TSP antibodies were arrested within the external granule cell layer. These results suggest that TSP plays an important role in the histogenesis of the cerebellar cortex by influencing granule cell migration.

Manufacture of compacted granules for washing agents.

Abstract: Compacted granules of given particle size are obtained from preliminary mixtures of at least partly solid constituents of washing and cleaning agents by addition of water-soluble, water-dispersible or water-emulsifiable plasticizers and/or lubricants. The preliminary mixture is worked into a homogeneous mass and extruded at pressures of 25 to 200 bar through perforated moulds with apertures whose width is equal to the given granule size. The compacted extrudates are reduced to the given granule size. The process is suitable for manufacturing concentrates for domestic and industrial washing and cleaning agents in the form of bulk and pourable highly compacted granules. The concentrates possess a long storage life and show no tendency to agglomerate.

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

Abstract: Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.

Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins.

Abstract: PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.

Ultrastructure of esophageal glands and their secretory granules in the root-knot nematode meloidogyne incognita

Abstract: The dorsal and subventral esophageal glands and their secretory granules in the root-knot nematodeMeloidogyne incognita changed during parasitism of plants. The subventral esophageal glands shrank and the dorsal gland enlarged with the onset of parasitism. While secretory granules formed by both types of glands were spherical, membrane-bound, and Golgi derived, the granules differed in morphology and size between the two types of glands. Subventral gland extensions in preparasitic second-stage juveniles were packed with secretory granules which varied in diameter from 700–1,100 nm and had a finely granular matrix. Within the matrix of each subventral gland granule was an electron-transparent core that contained minute spherical vesicles. The size and position of the core varied within different granules. Few granules were present in the dorsal gland extension in preparasitic juveniles. The matrix of dorsal gland secretory granules formed during parasitism was homogeneous and more electron-dense than the matrix of subventral gland granules. Subventral gland secretory granules of parasitic juveniles and adult females appeared degenerate.

Co-localization of secretogranins/chromogranins with thyrotropin and luteinizing hormone in secretory granules of cow anterior pituitary.

Abstract: We investigated the co-localization in secretory granules of secretogranins/chromogranins, thyrotropin, and luteinizing hormone in ultra-thin frozen sections of cow anterior pituitary by double immunoelectron microscopy, using specific antibodies and protein A-gold particles of different sizes. The distribution of secretogranin II, chromogranin A, and chromogranin B (secretogranin I) was largely similar. In cells containing secretory granules of relatively small size (100-300 nm) and low electron density (identified as thyrotrophs and gonadotrophs by immunolabeling for the respective hormone) and in cells containing both small (170-250 nm) and large (300-500 nm) secretory granules of low electron density (also identified as gonadotrophs), all three secretogranins/chromogranins were detected in most if not all granules, being co-localized with the hormone. In cells containing both relatively large (400-550 nm), electron-dense granules and small, less electron-dense secretory granules (150-300 nm), identified as somatomammotrophs by double immunolabeling for growth hormone and prolactin, all three secretogranins/chromogranins were predominantly detected in the subpopulation of small, less electron-dense granules containing neither growth hormone nor prolactin. Interestingly, this granule subpopulation of somatomammotrophs was also immunoreactive for thyrotropin and luteinizing hormone. These data show that somatomammotrophs of cow anterior pituitary are highly multihormonal, in that the same cell can produce and store in secretory granules up to four different hormones and, in addition, the three secretogranins/chromogranins. Moreover, selective localization of the secretogranins/chromogranins together with thyrotropin and luteinizing hormone in a subpopulation of secretory granules of somatomammotrophs indicates the preferential co-packaging of the secretogranins/chromogranins and these hormones during secretory granule formation.

Ultrastructural characteristics of rat peritoneal mast cells undergoing differential release of serotonin without histamine and without degranulation

Abstract: Rat mast cells pretreated with the tricyclic antidepressant drug amitriptyline and stimulated with compound 48/80 secreted 60% of the total serotonin present in the cells, but only 15% of histamine, another amine stored in the same granules. Ultrastructural studies demonstrated that mast cells undergoing such differential release do not exhibit classical degranulation by compound sequential exocytosis. However, there were changes in granule shape and size, as well as alterations in many morphometric parameters consistent with secretion. Storage granules lost their homogeneity, exhibited greatly reorganized matrix and were surrounded by clear spaces which were often associated with small (0.1–0.01 μm) cytoplasmic vesicles, some of which contained electron-dense material. Secretory granules often had bud-like protrusions or were fused together in series. Quantitative autoradiography localized 3H-serotonin outside the storage granules, close to small vesicles, while staining with ruthenium red demonstrated that vesicular structures associated with differential release were not endocytotic. These results suggest that amitriptyline may inhibit regular exocytosis and permit at least serotonin to be moved selectively from storage granules to the cytosol or small vesicles from which it is eventually released.

Direct observation of internal structure in spray-dried alumina granules

Abstract: The internal structure of spray-dried alumina granules was characterized by optical microscopy by immersing them in a liquid having a refractive index close to that of alumina. This method provides a unique technique for the detailed analysis of the internal structure of spray-dried granules.

THE ORIGIN AND PHYSIOLOGICAL RELEVANCE OF α‐GRANULE ADHESIVE PROTEINS

Abstract: Platelet a-granules are the principal intracellular reservoirs of proteins destined for release during primary haemostasis at the site of vessel wall injury. Despite the identification of many adhesive proteins within the a-granules, the biological significance and origin of these moieties have been subject to much speculation, particularly since high concentrations are also present in the plasma and within extracellular matrices.

Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells.

Abstract: Using patch-clamp techniques, we have followed the attributes of the secretory granules of peritoneal mast cells obtained from rats of different ages. The granule attributes were determined by following the step increases in the cell surface membrane area caused by the exocytosis of the granules in GTP gamma S stimulated mast cells. Our data show that the amount of granule membrane available for exocytosis depends exponentially on the weight (age) of the donor rat, reaching a maximum at approximately 300 g. The data are consistent with an exponential growth in the number of granules contained by mast cells of maturing animals. Histograms of the sizes of the step increases in surface area caused by exocytosis of the granules showed at least four equally spaced peaks of similar variance where the position of the first peak and the spacing between peaks averaged 1.3 +/- 0.4 micron2. In all cells recorded, no more than seven peaks could be found, the higher order peaks having a lower probability of occurrence. The distribution of granule sizes did not change measurably between young and adult animals. This study suggests that at least two separate steps may determine the size of a secretory granule: granule to granule fusion that may account for the subunit composition of granule sizes and traffic of microvesicles through the maturing granules that may account for the variance observed in the granule sizes. This study also demonstrates a novel way to study granulo-genesis in living cells.

High integrity natural nitrogenous granules for agriculture

Abstract: A method of preparing high integrity natural nitrogenous granules for agriculture by heating natural nitrogenous materials under alkaline conditions until the materials develop adhesive properties, forming the materials into granules by mechanical means, and heating the natural nitrogenous granules until they harden; and the compositions formed by this method. The granules for agriculture include natural fertilizers, secondary nutrients, micronutrients, and natural animal feed protein supplements. The method also provides natural based plant food and animal feed supplement granules containing natural or synthetic additive substances which are useful in agriculture. The natural materials used in the method include poultry waste, poultry feather meal, hair meal, seafood meal, blood meal, bone meal, soybean meal, food waste, and grain by-products. The method provides natural nitrogenous granules which are free of disagreeable odor by admixing reactive aldehyde compounds with the natural nitrogenous materials prior to granule formation.

Sodium bicarbonate dialysate

Abstract: A sodium bicarbonate dialysate comprising an electrolyte granule A composed mainly of sodium chloride and containing no sodium bicarbonate and an electrolyte granule B containing sodium bicarbonate, wherein the granule B is granules of sodium bicarbonate primary particles having a particle size of at most 250 μm, and the particle size of the secondary particles after granulation is from 0.1 to 10 mm.

Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans.

Abstract: Murine natural killer (NK) cell-mediated inhibition of growth of a yeast-like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll-fractionated homogenates of rat LGL tumor cells demonstrated that the granule-containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti-cryptococcal activity; whereas only the cytoplasmic granule-containing fractions had cytotoxic activity against YAC-1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti-LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti-cryptococcal activity other than the granule peak, it is possible that the cytolysin-containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.