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PubMed2002

Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression.

Absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein, is responsible for the Fragile X syndrome, the most common form of inherited mental retardation. FMRP is a cytoplasmic protein associated with mRNP complexes containing poly(A)+mRNA. As a step towards understanding FMRP function(s), we have established the immortal STEK Fmr1 KO cell line and showed by transfection assays with FMR1-expressing vectors that newly synthesized FMRP accumulates into cytoplasmic granules. These structures contain mRNAs and several other RNA-binding proteins. The formation of these cytoplasmic granules is dependent on determinants located in the RGG domain. We also provide evidence that FMRP acts as a translation repressor following co-transfection with reporter genes. The FMRP-containing mRNPs are dynamic structures that oscillate between polyribosomes and cytoplasmic granules reminiscent of the Stress Granules that contain repressed mRNAs. We speculate that, in neurons, FMRP plays a role as a mRNA repressor in incompetent mRNP granules that have to be translocated from the cell body to distal locations such as dendritic spines and synaptosomes.

Cytoplasmic Granules↗

PubMed1980

Ultrastructural localization of basic proteins in cytoplasmic granules of rat eosinophils and mast cells.

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (EPTA) methods were used to localize basic protein at the ultrastructural level in cytoplasmic granules of rat eosinophils and mast cells isolated using a Metrizamide gradient. Intense reaction was seen in the granules of EPTA-treated eosinophils. Following incubation of the cells for 2 hr in EPTA alone, the matrix was stained. After longer incubation (10 hr), however, both the matrix and core were stained. Cytoplasmic granules of the mast cell show a slight or negative reaction with EPTA. With the AS technique, a large number of silver particles were seen in the nucleus of both eosinophils and mast cells. The mast cell cytoplasmic granules showed intense reaction, while those from eosinophils showed no clear reaction. Acetylation of the cells under conditions sufficient to block most free amino groups prio to EPTA or AS treatment greatly reduced (EPTA) or abolished (AS) the reaction. The results indicate 1) that eosinophil granules contain basic proteins both in the matrix and the core, 2) that the mast cell granules contain a basic protein (probably the alpha-chymotrypsin-like enzyme), which reacts strongly with AS, and 3) that the AS and EPTA methods have different specificities.

Animals↗

PubMed1977

Isolation of the cytoplasmic granules of guinea pig basophilic leukocytes: identification of esterase and protease activities.

Procedures were developed for isolating highly purified cytoplasmic granules of basophilic leukocytes from guinea pig peripheral blood. The methods involved disruption of cells in 0.34 M sucrose followed by a series of membrane filtrations and fractionation on sucrose density gradients. These preparations, up to 95% pure basophil granules by electron microscopy, contained a mixture of neutral esterases-proteases including caseinolytic activity; both trypsin- and chymotrypsin-like serine hydrolases were identified by means of appropriate inhibitors. Localization of at least one such activity to the basophil granule was confirmed by a cytochemical method; this activity was absent in contaminating lymphocytes and eosinophils. By contrast, several lysosomal enzymes, lactic dehydrogenase, and plasminogen activator activity, present in cell homogenates, were absent from purified granules. The granule matrix of guinea pig basophils, unlike the cytoplasmic granules of other granulocytes or mast cells, was little altered by high or low salt concentration but was disrupted into insoluble fragments by 0.01 N HCl and by Triton X-100. Granules were solubilized by papain and by urea-SDS but enzyme activity was destroyed. Triton X-100 incubation with freeze-thawing proved to be the optimal method for extracting esterase activities. Esterase activities were not released from basophils under conditions of anaphylactic degranulation that liberated the great majority of basophil granule histamine.

Animals↗

PubMed2002

The precrystalline cytoplasmic granules of alveolar soft part sarcoma contain monocarboxylate transporter 1 and CD147.

Alveolar soft part sarcoma (ASPS) is an unusual tumor of young adults with the characteristic presence on ultrastructural analysis of rhomboid or rectangular cytoplasmic crystals. These membrane-bound crystals are known to form within specific PAS-diastase-resistant electron-dense cytoplasmic granules. The composition of these crystals and the dense granules from which they are derived has remained elusive. After the detection of strong discrete granular cytoplasmic immunoreactivity in ASPS for monocarboxylate transporter 1 (MCT1) in the course of a broad immunohistochemical characterization of an MCT1 antibody, we studied the expression of MCT1 and its interacting partner, CD147, in a panel of 10 ASPS cases using appropriate antibodies. MCT1 is one of a family of widely expressed proton-linked transporters for monocarboxylates such as lactate and pyruvate. In all normal and neoplastic tissues studied to date, MCT1 immunoreactivity is limited to the cell surface. We find that the periodic acid-Schiff-diastase-resistant cytoplasmic granules of ASPS are strongly immunoreactive for MCT1 and CD147. Specifically, intense cytoplasmic granular positivity for MCT1 and CD147 was found in 7 of 10 and 8 of 10 ASPSs, respectively. Ultrastructural immunohistochemistry with immunogold labeling confirmed that the MCT1 immunoreactivity localized to the cytoplasmic electron-dense granules in ASPS. Western blot analysis of several ASPS cases confirmed that the protein reactive with the MCT1 antibody and that reactive with the CD147 antibody both migrated at the size expected for MCT1 and CD147, respectively. Thus, ASPS cells seem to accumulate MCT1-CD147 complexes in the specific cytoplasmic granules known to undergo crystallization. The possible basis for the overproduction or impaired surface localization of these proteins in ASPS remains unclear.

Adult↗

PubMed1987

Defective cytoplasmic granule formation. I. Abnormalities affecting tissue mast cells and pancreatic acinar cells of beige mice.

Beige mice (C57BL/6-bgJ/bgJ) express the Chediak-Higashi syndrome, a genetically determined constellation of morphologic and functional abnormalities affecting cells that synthesize cytoplasmic granules; a similar disorder also occurs in humans and several other mammalian species. We used a computer-assisted morphometric approach to identify and quantitate the effect of the beige mutation on the structure of mast cell or pancreatic acinar cell cytoplasmic granules. Beige and control mouse mast cell or pancreatic acinar cell granules exhibited periodic, multimodal distributions of equivalent volumes in which the modes fell at volumes that were integral multiples of the volume of the "unit granule," whose volume (the "unit volume" or v1) was defined by the first mode in the granule equivalent volume distribution. But the modal frequency of the C57BL/6-bgJ/bgJ mast cell granule equivalent volume distribution fell at v1, a pattern consistent with a haphazard pattern of "unit granule" fusion, whereas the corresponding modal frequency for the control mast cell granules fell at v3, a pattern consistent with a "unit addition" model of granule fusion. In addition, the unit volume of beige mouse mast cell granules was 18 times that of control mouse mast cell granules. By contrast, the unit volume of beige mouse pancreatic acinar cell granules was only slightly (23%) greater than that of control cells. C57BL/6-bgJ/bgJ and control cells did not differ significantly in total cell or nuclear volume, or in the aggregate volume of their cytoplasmic granules. However, C57BL/6-bgJ/bgJ mast cells or pancreatic acinar cells contained significantly fewer granules than did their normal counterparts. These findings are consistent with the hypothesis that the beige mutation affects the formation of unit granules and also alters the pattern of aggregation and fusion of unit granules. The data also identify quantitative differences in the expression of the beige mutation in mast cells and pancreatic acinar cells.

Animals↗

PubMed1988

Immunohistochemical identification of plasma proteins in cytoplasmic granules of peripolar cells of the sheep.

The cytoplasmic granules of glomerular periopolar cells of the newborn lamb and sheep have been shown to contain immunoreactive albumin and immunoglobulin using immunohistochemistry and ultrastructural immunocytochemistry. It is most likely that the granule contents include material derived from filtered plasma proteins, although the basis for such uptake, localised to glomerular epithelial cells in the peripolar region, remains unexplained.

Animals↗

PubMedDate not supplied

An ultrastructural study of cutaneous panniculitis-like T-cell lymphoma: cytoplasmic granules and active cellular and cell-to-matrix interaction mimic cytotoxic T-cells.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare type of peripheral T-cell-derived lymphoma. A 30-year-old Japanese woman presented, complaining of skin lesions on her left hip. The cellular constituents in this SPTCL were of a mixed population. Not only tumor lymphoid cells, but also many macrophages, endothelial, fibroblasts, and fat cells were seen. The tumor cells immunostained positive for CD3 and CD8, but negative for CD4. Cytotoxic injury granule-related antigens of TIA-1 and granzyme B were positive in tumor cells. CD30. CD56, EBNA-2, LMP-1, CD20cy, and CD68 were all negative in the tumor cells. An ultrastructural study revealed that the lymphoma cells showed primitive cellular contacts with the neighboring tumor cells, interacted with the short villous dendrites of the opposing macrophage and fibroblast cellular membranes, and were associated with the vascular constituents, fat cells, and the extracellular matrix. Small aggregations of the granules were frequently seen in the cytoplasm. It was speculated that the tumor cells to some extent preserve the cytotoxic T-cell structure and function, have active cellular and cell-to-matrix interaction, contain characteristic cytoplasmic granules, and reveal unique histology like panniculities.

Adaptor Proteins, Signal Transducing↗

PubMed2005

A comparative study of cytoplasmic granules imaged by the real-time microscope, Nile Red and Filipin in fibroblasts from patients with lipid storage diseases.

Cytoplasmic granules in fibroblasts, visualized without stains, or labelled with Nile red, Filipin, or anti-LAMP-1 (lysosome-associated membrane protein 1), were imaged using the real-time microscope (RTM). New advances in light microscope technology were applied to detect cytoplasmic granules (RTM-visible granules) and characterize them by imaging contrast, size, shape, cellular distribution, composition, motion dynamics and quantity. Appearing as solid spheroids or ring structures, the majority of the RTM-visible granules contained Nile-red labelled neutral lipids. A smaller subpopulation, appearing dimmer, with less imaging contrast, contained Filipin-labelled free cholesterol. Most lipid storage granules have a diameter ranging from 0.3 mum to 0.6 mum, with a small population measuring up to 1 mum. They typically clustered in the perinuclear region and displayed relatively small oscillatory motion. Immunofluorescence based on LAMP-1 labelling highlighted granular structures that were distinct and separate from RTM-visible granules and other structures in the light modality of the microscope. RTM-visible granules were associated with disease phenotypes that have increased cellular neutral lipid stores corresponding to the Nile red-labelled droplets (e.g. triacylglycerides, cholesterol esters). As predicted, the fibroblast strains with a defect resulting in Wolman disease, when compared to control samples, consistently had RTM-visible granules, higher in imaging contrast and with larger diameters, that were labelled with Nile red, and also an increased frequency of Filipin-cholesterol complexes. By comparison, in fibroblasts where the lipid storage is less evident (Gaucher and Farber diseases) or from GM(1) gangliosidosis, where the primary storage substances are oligosaccharides, fewer and smaller RTM-visible granules were observed. In some cases, changes in contrast and morphology in the unstained cytoplasmic compartments were more evident than in the labelled structures. In summary, applying the RTM imaging system to fibroblasts enables differences between the various disease types to be seen and, in specific examples, a unique phenotype can be readily discerned.

Antifungal Agents↗

PubMed2004

Intratracheal instillation of cytoplasmic granules from Phleum pratense pollen induces IgE- and cell-mediated responses in the Brown Norway rat.

BACKGROUND: Release of cytoplasmic granules from grass pollen upon contact with water is thought to be an important source of airborne allergens. OBJECTIVES: To investigate the humoral and cellular responses to intratracheal instillation of Phleum pratense (timothy grass) pollen cytoplasmic granules (PCG) in the Brown Norway rat. METHODS: PCG were purified from timothy grass pollen by filtration through 5-microm-mesh filters. Rats were sensitized (day 0) and challenged (day 21) intratracheally with purified PCG suspended in saline (6 x 10(6) PCG/rat). Rats were then challenged 4 weeks later (1.5 x 10(6) PCG/rat). Blood samples, bronchial lymph nodes and lungs were collected from the rats 4 days after the second challenge. PCG-specific IgE and IgG1 levels and specificity were determined by ELISA and Western blotting. Pollen, pollen extract and PCG-induced proliferation of lymph node cells were monitored by [(3)H]-thymidine incorporation in a lymph node assay. Histopathological examination was carried out on the lungs. RESULTS: Specific IgE and IgG1 were present in the sera. Cultured lymph node cells proliferated in the presence of pollen, pollen extract and PCG. Western blots showed that all major pollen allergens are recognized by IgE and IgG1 from PCG-treated rats. Histopathological examination revealed features of a mild allergic reaction. CONCLUSIONS: In our rat model of allergy, purified timothy grass PCG instillation induced specific antibodies and lymph node cell responses, comparable to those obtained with intact pollen.

Administration, Inhalation↗

PubMed1987

Simultaneous in situ detection of IgE receptors and cytoplasmic granules in murine cutaneous mast cells.

A method is described for the simultaneous in situ detection of surface receptors and cytoplasmic granules in mast cells of frozen sections of mouse skin. Surface IgE receptors are detected after saturation of the receptors with a murine monoclonal antibody of IgE isotype. The latter is subsequently detected by monospecific rabbit anti-mouse IgE (purified on protein A-Sepharose) followed by FITC-conjugated goat anti-rabbit IgG. Cytoplasmic granules are localized by staining with TRITC-avidin conjugate. Normal cutaneous mast cells show green surface fluorescence and red intracellular granules. The method is specific for mast cells; other cells with Fc receptors for IgE are not seen. This method should be useful in the study of situations in which mast cells may have become degranulated.

Animals↗

PubMed1994

Effects of interleukin-3 with or without the c-kit ligand, stem cell factor, on the survival and cytoplasmic granule formation of mouse basophils and mast cells in vitro.

We assessed the ultrastructure and the cell-surface expression of receptors for immunoglobulin E (Fc epsilon R), and c-kit, the receptor for stem cell factor (SCF), in mouse basophils and mast cells present in short-term cultures of mouse bone marrow cells in interleukin-3 (IL-3) with or without SCF. Basophils did not develop increased numbers of cytoplasmic granules and underwent apoptosis in cultures containing IL-3 and SCF, whereas mast cells thrived and developed increased numbers of granules. Basophils were nearly all Fc epsilon R+ c-kit- when sorted after culture in IL-3 and SCF; most mast cells were Fc epsilon R+ c-kit+. However, a second population of Fc epsilon R+ c-kit- mast cells was present after culture in IL-3 and SCF. These c-kit receptor-negative mast cells were less mature than c-kit+ mast cells and contained significantly fewer cytoplasmic granules than the c-kit+ mast cells present in the same cultures (P < 0.001). Thus, mouse basophils express little or no c-kit receptor on their surface, nor can they survive for long periods in SCF-supplemented cultures. By contrast, mouse mast cells seem to express the Fc epsilon R early in their development, even before they express detectable c-kit receptors on their surface. IL-3 promotes cytoplasmic granule formation in immature mast cells, but even more granules are formed when c-kit receptor-positive immature mast cells are cultured in both SCF and IL-3.

Animals↗

PubMed1987

Cytochemical characterization of leukemic cells with numerous cytoplasmic granules.

Ten cases in which leukemic cells contained numerous cytoplasmic granules were examined by using a panel of cytochemical reactions. The diagnoses in the 10 cases were mast cell leukemia, chronic basophilic leukemia, and acute myeloid leukemia with basophilic differentiation in one case each, acute promyelocytic leukemia in two cases, acute megakaryoblastic leukemia in two cases, and blastic hairy cell leukemia in three cases. The cytochemical panel consisted of peroxidase, toluidine blue, chloroacetate esterase, aminocaproate esterase, tartrate-resistant acid phosphatase, and immunoalkaline phosphatase for platelet/megakaryocyte-specific antigen. The unusual cytologic features of leukemic cells in cases similar to our 10 cases have caused considerable diagnostic difficulties. In our 10 cases, however, the effective use of cytochemical studies helped to achieve accurate identification of the various types of leukemic cells. We conclude that the intelligent application of cytochemical techniques continues to be useful for the accurate cytodiagnosis of hematopoietic neoplasms.

Adult↗

PubMed1987

Histopathologic alterations in the decidua in human spontaneous abortion: loss of cells with large cytoplasmic granules.

The histopathology of decidua obtained from the placental bed was evaluated by phloxine-tartrazine staining, which allows clear definition of cells with cytoplasmic granules. Mononuclear cells with large granules were seen in biopsy specimens taken from women at 8-31 weeks of normal pregnancy. In contrast, cells with large granules were missing in sections taken from the decidua of five women who were aborting or were destined to abort. Since the presence of suppressor cell activity in murine decidua correlates with the success of pregnancy and since this suppression is associated with small lymphocytes with cytoplasmic granules, the observations made using human placental bed biopsy material suggest that a possible suppressor cell deficiency might occur in the early stages of spontaneous abortion in human females.

Abortion, Spontaneous↗

PubMed1980

Kinetics of fusion of the cytoplasmic granules with phagocytic vacuoles in human polymorphonuclear leukocytes. Biochemical and morphological studies.

This study on human neutrophils was conducted to measure the kinetics of degranulation of the different cytoplasmic granules into phagocytic vacuoles, and to relate the timing of these events to the burst of respiration that accompanies phagocytosis by these cells. Purified neutrophils were incubated with latex particles opsonized with human immunoglobulin (Ig)G, and phagocytosis was stopped at timed intervals. The cells were examined by electron microscopy to document the sequence of degranulation of the cytoplasmic granules. The azurophil granules and lyosomes were identified by histochemical staining for peroxidase and acid phosphatase, respectively. Phagocytic vacuoles were separated from cell homogenates by floatation on sucrose gradients and assayed for contained lactoferrin, myeloperoxidase, and acid hydrolases. The conclusions drawn from the biochemical and morphological studies were in agreement and indicated: particle uptake and vacuole closure can be completed within 20 s; both the specific and azurophil granules fuse with the phagocytic vacuole much earlier than is generally appreciated, with half-saturation times of 39 s (99% confidence limits, 15-72); oxygen consumption has kinetics similar to those of the fusion of these granules with the phagosome; degranulation of the acid hydrolases beta-glucuronidase, N-acetyl-beta-glucosaminidase (biochemical assays), and acid phosphatase (biochemical assay and electron microscopic cytochemistry) have kinetics of degranulation that are similar to each other but totally different from and much slower than that of myeloperoxidase with half-saturation times of between 354 and 682 s (99% confidence limits, 246-883). This suggests that the acid hydrolases are not co-located with myeloperoxidase in the azurophil granule but are contained in distinct lysosomes, or "tertiary granules".

Cytoplasmic Granules↗

PubMed1990

Morphological and quantitative aspects of the development of cytoplasmic granules in concanavalin A-stimulated normal human lymphocytes.

We have studied normal human lymphocytes stimulated by concanavalin A to determine the rate and extent of development of cytoplasmic granules. Morphological studies on stimulated lymphocytes were undertaken at the light and electron microscopic level. The percentage of T lymphocytes bearing different CD antigens (CD3, CD4, and CD8) that also contained cytoplasmic granules was also assessed. Granules were identified in a high percentage of lymphocytes bearing each of the three CD markers studied.

Cell Survival↗

PubMed1995

[Saltatory movements of the cytoplasmic granules in the cells of an ESK culture].

Saltatory movements of large (0.3-0.8 micron) granules in the cytoplasm of PK cells were described in norm and after nocodazole and sodium azide treatments. In untreated cells the length of single movements was up to 2 micron and even more (sometimes up to 5 micron). Nocodazole at a concentration of 0.2 micron and sodium azide at a concentration of 20 micron inhibited all rapid movements longer than 1.2 micron, but did not affect the frequency of movements shorter than 1 micron. The effect of nocodazole was reversible: the long rapid movements were seen resumed after its removal. A comparison of histograms (length versus frequency) of the rapid movements of granules in norm and after nocodazole and sodium azide treatments made it possible to conclude that real saltations (ATP and microtubule-dependent motions) are rapid translocations which exceed 1 micron.

Animals↗

PubMed1989

Regulation of superoxide responses of human neutrophils by adenine compounds. Independence of requirement for cytoplasmic granules.

Recent evidence suggests that the adenine compounds ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), and adenosine have important regulatory effects on O2- responses of human neutrophils stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP). Because of evidence that receptors on neutrophils may be modified by granule fusion events, we assessed the extent to which these adenine compounds affect fMLP and CR3 (C3bi) receptors on neutrophils and whether cytoplasmic granules are required for the ability of the adenine compounds to modify O2- responses in neutrophils stimulated with fMLP. Incubation of neutrophils with ATP gamma S or adenosine led to a decrease in numbers of fMLP receptors (17 and 9.2%, respectively) but no change in receptor affinity (Kd). Paradoxically, ATP gamma S caused an increase in CR3 receptors (Mo1, CD-11b antigen), suggesting that fMLP and CR3 receptors may be under separate control. The ability of the adenine compounds to modify O2- responses in fMLP-stimulated cells was equivalent in both neutrophils and cytoplasts, suggesting that the regulatory effect of ATP, ATP gamma S, and adenosine do not require the presence of cytoplasmic granules. ATP gamma S caused enhancement of O2- responses of neutrophils to phorbol 12-myristate 13-acetate, raising the possibility that ATP gamma S may be affecting late events in the signal transduction pathway.

Adenosine↗

PubMed1985

A C9 related channel forming protein in the cytoplasmic granules of human large granular lymphocytes.

Large granular lymphocytes (LGL) from human blood maintained in culture for 2 to 6 weeks with IL-2 were found positive in the K562 cell killing assay. The cytoplasmic granules of the LGL were isolated, lysed and the soluble proteins were passed over a Sepharose-anti-C9 column. The retained protein was eluted with NaCl and found to consist by SDS polyacrylamide gel electrophoresis of essentially one component with a molecular weight of approximately 70 000. The protein did not give a positive precipitation test with anti-human C9 by Ouchterlony analysis, but it reacted reproducibly with anti-human C9 by Western blot analysis. By ELISA the cross reaction with human C9 was less than 1%. The C9 related lymphocyte protein lacked C9 hemolytic activity, but it formed functional pores in liposomes in presence of Ca++. These results suggest that the cytoplasmic granules of human LGL that are capable of killing NK target cells contain C9 related protein which is involved in the cellular cytotoxicity reaction.

Cells, Cultured↗