Showing papers on "Granule (cell biology) published in 1989"

Molecules relevant for T cell-target cell interaction are present in cytolytic granules of human T lymphocytes.

Abstract: An ultrastructural analysis of human cytotoxic T lymphocyte-target cell (CTL-TC) interaction has been undertaken to enable a better understanding of the killing mechanism. Attention was focused on granules in the CTL, which are known to contain lethal compounds. Within the membrane-delimited cytotoxic granule an electron-dense core as well as numerous membrane vesicles were identified. In CTL-TC conjugates, specific membrane interactions take place, allowing the formation of intercellular clefts into which the granule cores and internal vesicles are released. T cell surface membrane molecules known to be involved in CTL-TC interaction (T cell receptor, CD3 and CD8) are present on the membranes of the granule cores and internal vesicles, facing outward. An explanation for this localization of the membrane may be found in the fact that the granule is connected with an endocytotic pathway. Moreover, the lumen of the granule is rich in the enzyme cathepsin D, which indicates an association with a lysosomal compartment. Exocytosed vesicles and cores are seen to adhere to the plasma membrane of the TC. Although the exact contents of the granule vesicles and core remain to be identified, we suggest that specific interaction of CTL membrane molecules on the cytolytic granule components with molecules on the plasma membrane of the TC may ensure the unidirectional delivery of the lethal hit.

Control of transmembrane lipid asymmetry in chromaffin granules by an ATP-dependent protein.

Abstract: THE Ca2+-dependent binding of annexin proteins to secretory granule membranes seems to be involved in the early stage of exocytosis1—3. Binding studies have shown that these proteins have a specificity for phosphatidylserine (PtdS) interfaces4,5. Further-more, aminolipids are necessary for contact and fusion between lipid vesicles6 or between liposomes and chromaffin granules7. Thus, PtdS must be present on the granule outer (cytoplasmic) monolayer. We report here that chromaffin granules possess a mechanism to maintain PtdS orientation, comparable to the ATP-dependent aminophospholipid translocase from human erythrocytes8-14. The translocase, in granules, selectively transports PtdS from the luminal to the cytoplasmic monolayer, provided the incubation medium contains ATP. As this protein shares several properties with the granule vanadate-sensitive ATPase II15,16, we infer that this ATPase, of relative molecular mass 115,000, is the protein responsible for aminophospholipid translocation. This is the first evidence for an ATP-dependent specific phospholipid 'flippase' in intracellular organelles.

The insulin secretory granule.

Abstract: The insulin secretory granule of the pancreatic B cell is a complex intracellular organelle comprised of a many proteins with different catalytic activities and messenger functions. With the advent of tumour models of the B cells and the application of immunological and molecular cloning techniques considerable progress has been made in recent years towards the elucidation of the structure and function of these granule proteins. A number of examples are selected here for review. Particular emphasis given to how the activities of quite different granule proteins are interdependent and how this contributes to the co-ordination and integration of the organelle's biological functions.

Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes.

Abstract: We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co-localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.

Ultrastructural study of the response of eosinophil granule cells to Aeromonas salmonicida extracellular products and histamine liberators in rainbow trout Salmo gairdneri richardson

Abstract: Intraperitoneal injections of Aeromonas salmonicida extracellular products (ECP), Compound 4880 and Concanavalin A were found to degranulate the eosinophil granule cells (EGC) in the lower intestine and rectum of the rainbow trout Salmo gairdneri Richardson. Ultrastructurally, the EGC response resembled the anaphylactic granule extrusion of mammalian mast cells. Varying degrees of granule vacuolation and loss of electron density occurred. Labyrinthine channels were observed at the peak of degranulation. EGC response however, differed from mammalian mast cells in two respects. Firstly, degranulation involved the release of intact electron lucent granules and the subsequent disintegration of the granule matrix extracellularly. Mammalian mast cells on the other hand, release their granules by direct exocytosis. Secondly, the 4880 and Con A-stimulated EGC degranulation was inhibited by antihistamines, promethazine and cimetidine. In mast cells, antihistamines do not prevent granule release but block histamine receptors in target cells. The degranulation of the EGCs was a non-cytotoxic event and the cells were capable of regeneration. As soon as the cells lost most of their granules, increased cytoplasmic activity was observed. This involved the expansion of the Golgi-endoplasmic reticulum complex.

Human neutrophil gelatinase is a component of specific granules.

Abstract: Previous investigators have proposed that gelatinase, a metalloproteinase found in neutrophils, is stored in a novel secretory compartment distinct from the two major granule populations, azurophilic and specific. To locate this proteinase in human neutrophils we reacted the cells for peroxidase and then applied monospecific polyclonal antibodies to human neutrophil gelatinase to immunolabel ultrathin frozen sections using an immunogold technique. Gelatinase was localized in a population of peroxidase-negative granules. Double-labeling experiments using antibodies against lactoferrin, a marker for specific granules, and gelatinase demonstrated colocalization of the two antigens in 80% of the specific granules. However, some granules immunostained with only the lactoferrin or gelatinase antibody. Similar techniques were used to examine precursor cells from bone marrow. In myelocytes both gelatinase and lactoferrin were present in large developing specific granules; however, some mature specific granules contained only lactoferrin. Thus, it is possible that lactoferrin synthesis begins earlier than gelatinase synthesis and that overlapping synthesis and segregation occurs during the myelocyte stage. These findings suggest that the main storage compartment of gelatinase is within the peroxidase-negative specific granules.

A specific interaction in vitro between pancreatic zymogen granules and plasma membranes: stimulation by G-protein activators but not by Ca2+.

Abstract: The molecular details of the final step in the process of regulated exocytosis, the fusion of the membrane of the secretory granule with the plasma membrane, are at present obscure. As a first step in an investigation of this membrane fusion event, we have developed a cell-free assay for the interaction between pancreatic zymogen granules and plasma membranes. We show here that plasma membranes are able to trigger the release of the granule contents, and that this effect is specific to pancreatic membranes, involves membrane fusion, requires membrane proteins, and is stimulated by activators of G-proteins but not by Ca2+. The assay is simple, reliable, and rapid, and should permit the identification of proteins that are involved in the exocytotic fusion event.

Pharmaceutical granules and tablets made therefrom

Abstract: A pharmaceutical granule comprising a core of active pharmaceutical agent and a coating of a polymer/microcrystalline cellulose is provided. These granules permit superior tableting of pharmaceutical agents whose physical properties in granule form render them unsuitable for conventional tableting.

Uptake and release of glycine in cerebellar granule cells and astrocytes in primary culture: potassium-stimulated release from granule cells is calcium-dependent.

Abstract: The properties of [3H]glycine uptake and release were studied with cerebellar granule cells, 7-9 days in vitro, (DIV) and astrocytes, 14-15 DIV, in primary cultures. The uptake of glycine in both cell types consisted of a saturable high-affinity transport and nonsaturable diffusion. The transport constant (Km) and maximal velocity (V) were significantly higher in granule cells than in astrocytes. Uptake was strictly Na+-dependent and also markedly diminished in low-Cl medium. The specificity of the uptake was similar in both cell types. The spontaneous release of glycine from granule cells and astrocytes was fast. Homoexchange with extracellularly added glycine in granule cells suggests that the efflux is at least partly mediated via membrane transport sites in these cells. Kainate stimulated the release more effectively in neurons than in glial cells, the effect apparently being mediated by specific kainate-sensitive receptors in both cell types. The release was enhanced by veratridine and by depolarization of cell membranes by high K (50 mM) in both neurons and astrocytes. The potassium-stimulated release was partially Ca-dependent in neurons but Ca-independent in glial cells. The results suggest a functional role for glycine in both cerebellar astrocytes and glutamatergic granule cells.

Artificial seed comprising a sustained-release sugar granule

Abstract: A sustained-release granule of sugar comprising a core comprising primarily one or more of a monosaccharide, a disaccharide and a trisaccharide and an outer film comprising 10-90% by weight of an ethylene-vinyl acetate copolymer and 90-10% by weight of a wax. This sustained-release granule of sugar may be prepared by coating a core comprising primarily one or more of a monosaccharide, a disaccharide and a trisaccharide with a coating material comprising 10-90% by weight of an ethylene-vinyl acetate copolymer and 90-10% by weight of a wax by spraying a solution of the coating material onto the core while tumbling or fluidizing the core. The sustained-release granule of sugar can be applied in an artificial seed comprising an active plant tissue and the sustained-release granule of sugar which are encapsulated in a gel matrix.

Phylogenetic considerations of neurosecretory granule contents: role of nucleotides and basic hormone/transmitter packaging mechanisms.

Abstract: The characteristics of neurosecretory granules include the presence of an acidic interior, a hyperosmolar concentration of granule solutes, the presence of chromogranin (CG) or CG-like soluble acidic proteins and a high content of nucleotides, predominantly ATP. The identification of “nucleotides” within the neuroendocrine “stem cells” of coelenterates (e. g. Hydra) has raised some interesting evolutionary questions as to the function of intragranular nucleotides.The chromaffin granules of adrenal medullary cells have been studied extensively, and are representative of the neurohormone/neurotransmitter packaging problems encountered in neurosecretory granules, in general. At the acid pH (5.7) of the interior of the chromaffin granule, ATP has three negative charges based on the pK value of the γ-phosphate group. ATP can therefore interact with positively charged amines, acetylcholine and divalent cations, forming binary and ternary complexes. The results of nuclear magnetic resonance (NMR) spectroscopy indicate that the hyperosmolar solutes within the chromaffin granule exist in a viscous, but fluid state; one function of ATP could be to help lower the osmotic pressure of the granule contents through extensive, but weak, intermolecular bonding. In addition, ATP is an excellent buffer to help maintain a pH of 5.7 within the interior of the chromaffin granule. An acidic milieu contributes to neurohormone/neurotransmitter packaging and granule stability. The presence of nucleotides within neurosecretory granules cannot, however, be explained on the basis of the ability of ATP to simply reduce osmotic pressure, since insulin molecules exist in a crystalline phase, a condition which, by itself, could substantially reduce osmotic pressure; nucleotides, nevertheless, co-exist in these insulin cores. ATP and ATP metabolites such as ADP, AMP and adenosine, formed as a result of the action of ectonucleotidases, can have extensive extracellular trophic and feedback effects after secretion. Extracellular nucleotides and adenosine can function as neuromodulators, agonists and antagonists to inflammatory cells, and regulators of blood flow, etc.It is possible that intragranular nucleotides were retained through a billion or more years of evolution because of the importance of these trophic and feedback effects. Parts of the neurosecretory granule, such as the F1 subunit of the proton-translocating ATPase, can be traced back to the aerobic bacteria, vacuolar amine transport to yeast and a CG-like acidic protein to protozoan secretory granules (i. e., the trichocysts of Paramecia).

Granules having core and their production

Abstract: Granules having a core are produced by spraying core granules with a dispersion of a low substituted hydroxypropylcellulose (L-HPC), and, if necessary, simultaneously applying a dusting powder. The granules having a core thus obtained exhibit increased granule strength and improved disintegrating property as compared with those produced by known methods. An active ingredient such as a drug can be contained in the dispersion, dusting powder or core granules.

Interactions of cytoplasmic granules with microtubules in human neutrophils.

Abstract: Ultrastructural and functional studies of degranulation responses by human neutrophils have suggested that microtubules (MTs) have a role in the intracellular transport of neutrophil granules. We have found that granule-MT complexes can be isolated from disrupted taxol-treated (1.0 microM) neutrophils, visualized by electron microscopy, and quantified in terms of granules per MT length. After incubation of neutrophils with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), granule-MT complex formation was found to be increased two- to threefold. Enhanced binding of granules to MTs was detectable within 30 s of fMLP stimulation and was dependent on the concentration of fMLP. Incubation of cells with dibutyryl cAMP inhibited this fMLP-stimulated granule-MT complex formation in a dose-responsive fashion. These granule-MT interactions could be reproduced in a cell-free system with neutrophil granules isolated by density gradient centrifugation and MTs polymerized from phosphocellulose-purified tubulin. Furthermore, reconstituted granule-MT interactions were found to be modulated by ATPase inhibitors. Sodium orthovanadate increased granule-MT interactions in a concentration-dependent manner, while AMP-PNP, a nonhydrolyzable ATP analogue, and N-ethylmaleimide decreased or eliminated these interactions. In addition, we found that a MT-activated ATPase could be recovered from intact neutrophil granules by salt extraction, and that extracts enriched in this ATPase contained a polypeptide of between 115 and 120 kD which binds ATP and is immunologically related to kinesin. These studies demonstrate that cytoplasmic granules interact with MTs in human neutrophils in a regulated stimulus-responsive manner, and they suggest that such interactions may involve an MT-based, ATPase-dependent, vesicle translocation system as has been demonstrated in other types of cells.

High molecular weight polymers block cortical granule exocytosis in sea urchin eggs at the level of granule matrix disassembly.

Abstract: Recently, we have shown that high molecular weight polymers inhibit cortical granule exocytosis at total osmolalities only slightly higher than that of sea water (Whitaker, M., and J. Zimmerberg. 1987. J. Physiol. 389:527-539). In this study, we visualize the step at which this inhibition occurs. Lytechinus pictus and Strongylocentrotus purpuratus eggs were exposed to 0.8 M stachyose or 40% (wt/vol) dextran (average molecular mass of 10 kD) in artificial sea water, activated with 60 microM of the calcium ionophore A23187, and then either fixed with glutaraldehyde and embedded or quick-frozen and freeze-fractured. Stachyose (2.6 osmol/kg) appears to inhibit cortical granule exocytosis by eliciting formation of a granule-free zone (GFZ) in the egg cortex which pushes granules away from the plasma membrane thus preventing their fusion. In contrast, 40% dextran (1.58 osmol/kg) does not result in a GFZ and cortical granules undergo fusion. In some specimens, the pores joining granule and plasma membranes are relatively small; in other cases, the exocytotic pocket has been stabilized in an omega configuration and the granule matrix remains intact. These observations suggest that high molecular weight polymers block exocytosis because of their inability to enter the granule matrix: they retard the water entry that is needed for matrix dispersal.

Subcellular distribution of 65,000 calmodulin-binding protein (p65) and synaptophysin (p38) in adrenal medulla.

Abstract: Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.

Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells.

Abstract: Human cloned 35S-labeled NK cells were disrupted by nitrogen cavitation, and their secretory granules were obtained by filtration through 5-micron and 3-micron membrane filters followed by Percoll density-gradient centrifugation. These granule preparations, which contained 35S-labeled chondroitin sulfate A proteoglycans, were sonicated and were analyzed for carboxypeptidase activity and tryptic serine esterase activity. A carboxypeptidase activity that digested angiotensin I to des-Leu-angiotensin I, Ile-His-Pro-Phe to Ile-His-Pro and Phe, and hippuryl-L-phenylalanine to hippuric acid and Phe was detected in the granules of these NK cells. As determined by cleavage of the tetrapeptide, the pH optimum of the carboxypeptidase was 7.0. As assessed by the cleavage of N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLTe), the granule preparations also contained a serine esterase with trypsin-like specificity that had a pH optimum of 8.5. When the isolated secretory granules were disrupted and chromatographed on columns of Sepharose CL-2B in PBS, greater than 60% of the BLTe serine esterase activity and essentially all of the carboxypeptidase activity filtered as a macromolecular complex with approximately 8% of the 35S-labeled proteoglycans. Whereas treatment with 4 M urea or nonionic detergent failed to disrupt the macromolecular complex, the serine esterase activity was dissociated from the macromolecular complex in the presence of 3 M NaCl, demonstrating an ionic interaction with the proteoglycans. No difference was observed in the disaccharide composition of the chondroitin sulfate glycosaminoglycans of the 35S-labeled proteoglycans that were complexed with the enzymes as compared to those that were not complexed. These studies indicate that the secretory granules of human NK cells contain serine esterase activity and carboxypeptidase activity, both of which have neutral pH optima, and both of which are bound to protease-resistant chondroitin sulfate proteoglycans.

Colocalization of elastase and myeloperoxidase in human blood and bone marrow neutrophils using a monoclonal antibody and immunogold.

Abstract: The authors have localized elastase in human blood and bone marrow neutrophils by immunoelectron microscopy using a monoclonal anti-human elastase antibody (NP 57) and compared its distribution with myeloperoxidase (MPO) and lactoferrin (LF), which mark primary and secondary neutrophil granule, respectively. Human bone marrow and blood polymorphonuclear leukocytes (PMN), either unstimulated or after phagocytosis of latex microbeads, were fixed in 4% paraformaldehyde. Ultrathin frozen sections were immunolabeled with NP 57, followed by an immunogold probe. In bone marrow granulocyte precursors elastase appeared simultaneously in the immature first granules of myeloblasts with MPO. As these granules became denser with maturation, labeling for both enzymes became weaker and sometimes negative (possibly due to masking of immunoreactivity). The ellipsoidal primary granules were strongly labeled by NP57. LF positive granules appeared later, at the myelocyte stage, and contained neither MPO nor elastase. In mature neutrophils, immunolabeling for elastase was found together with MPO in the large electron-dense primary granules and in a different granule population from the LF-positive secondary granules. Double labeling with two different-sized gold particles was used to compare the kinetics of degranulation of secondary and primary granules. The observation and the analysis of single phagosome content was made possible by this new technique. In conclusion, immunoelectron microscopy was used to show elastase in the primary granules of neutrophils, where it appears simultaneously with MPO. This technique has also allowed comparison of the kinetics of degranulation of both types of granules, and could be applied to different experimental and pathologic conditions.

Role of Ion Exchange in Release of Biogenic Amines

Abstract: Mast cells, chromaffin cells, and nerves contain granule materials that have the characteristics of weak ion exchangers. Biogenic amines are stored in and released from these granules acco...

A reassessment of polyphosphate granule composition in the ectomycorrhizal fungus Pisolithus tinctorius

Abstract: Comparison of the elemental composition of freeze-substituted and conventionally fixed phosphorus-containing granules of the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch, using energy dispersive X-ray microanalysis, shows that the cation composition is altered appreciably by the method of specimen preparation. Following conventional chemical fixation, the major cation detected in the granules together with phosphorus was calcium, while freeze-substituted granules most frequently contained phosphorus, potassium and sodium. We conclude that chemical fixation causes serious loss or redistribution of ions. The occurrence of monovalent cations in polyphosphate granules has not previously been demonstrated for mycorrhizal fungi and their presence should be considered in determining the role of the granules in mycorrhizas.

Localization of cyclo-oxygenase and prostaglandin E2 in the secretory granule of the mast cell.

Abstract: The application of anti-cyclo-oxygenase and anti-prostaglandin E2 immunoglobulins to A23187-stimulated rat connective tissue mast cells has permitted the localization of cyclooxygenase activity (prostaglandin H2 synthetase) and the site of prostaglandin E2 (PGE2) formation in the secretory granules. Because binding was carried out after stimulation but before dehydration and embedding, we have limited the loss of these antigens due to normal degradation and to aqueous and solvent washes. As this method permits labeling of exposed cell surfaces, only granules that have been exteriorized can be labeled. Contrary to what might have been expected, no labeling was associated with plasma membranes or with any portion of damaged cells. Antibodies to PGE2 were bound evenly over the surface of the granule matrix, whereas antibodies to cyclo-oxygenase appeared to be bound to strands of proteo-heparin projecting from the surface of the granule matrix. Where granule matrix had become unraveled and dispersed, label ap...

Ultrastructural and electron immunohistochemical features of medullary thyroid carcinoma.

Abstract: An ultrastructural study, both morphological and immunohistochemical, has been carried out on eight thyroglobulin-positive and nine thyroglobulin-negative medullary carcinomas of the thyroid. The morphometric analysis of granule size showed that all tumours contained cells with small granules and cells with medium size granules, whereas eight tumours had additional cells with large granules. The small granules had an electron dense core, while the medium and large sized granules were both pale-cored and dense-cored. The cells with small, medium or large secretory granules were all immunoreactive for calcitonin and CGRP. No ultrastructural differences were observed between thyroglobulin-positive and thyroglobulin-negative cases of medullary carcinoma of the thyroid.

Ultrastructural and immunocytochemical studies on the cytoskeleton in the anterior pituitary of rats with special regard to the relationship between actin filaments and secretory granules

Abstract: As previously reported, in anterior pituitary cells of the rat, secretory granules are linked with adjacent granules, cytoorganelles, microtubules, and plasma membrane by thin filaments, 4–10 nm in diameter. The quick-freeze, deep-etching method revealed that some of the filaments linking adjacent secretory granules show 5 nm-spaced striations on their surface which are known to be characteristic of actin. Immunocytochemistry showed that actin is localized in the cytoplasm beneath the plasma membrane, and around or between secretory granules. The heavy meromyosin decoration method demonstrated that actin filaments are mainly located in the cytoplasm beneath the plasma membrane, while some actin filaments are connected with the limiting membrane of the secretory granules. The actin filaments associated with the secretory granules are considered to be involved in the intracellular transport of the granules, while those localized in the peripheral cytoplasmic matrix might control the approach of the secretory granules to the plasma membrane and their release.