Showing papers on "Granule (cell biology) published in 1978"

Intracellular transport and packaging of prolactin: a quantitative electron microscope autoradiographic study of mammotrophs dissociated from rat pituitaries

Abstract: Dispersed pituitary cells prepared from estrogen-treated female rats were subjected to pulse labeling with [3H]leucine (5 min) followed by a chase incubation (up to 3 h) in order to study intracellular transport of PRL in mammotrophs. Sites of synthesis, rates of transport, and sites of packaging and storage of PRL were determined by quantitative electron microscopic autoradiography. Results of grain counts show that label is initially (end of pulse) distributed randomly over the rough endoplasmic reticulum (ER), but rapidly (5--15 min of chase) moves to the stacked Golgi cisternae where concentration into secretion granules takes place. The label moves successively from small (Type I) immature granules (15--55 min of chase) to large (Types II and III) polymorphic granules (55--115 min) in the Golgi region, to rounded or ovoid mature (Type IV) granules (55--185 min) usually found in the peripheral cytoplasm, indicating that these types of granules represent successive stages in granule concentration and assembly. Analysis of the relative grain density (percentage of total grains/percentage of total area) confirmed that there was progressive concentration (up to 20--150 times) along the transport route with the concentration lowest in the ER, higher in the Golgi, and highest in immature and mature secretion granules. These data indicate that synthesis of PRL occurs randomly in the ER, transport to the Golgi occurs rapidly (within 5--10 min), and is completed rapidly (90% within 15--20 min), and concentration into granules and aggregation of small granules into larger forms also occurs rapidly (by 15--20 min), but goes on over a prolonged period of time (up to 3 h). Use of dispersed cells has allowed a more precise determination of the location and kinetics of steps in the intracellular processing of PRL than has been possible previously using other systems.

Immunocytochemical identification of azurophilic and specific granule markers in the giant granules of Chediak-Higashi neutrophils.

Abstract: We used immunofluorescent microscopy to characterize the abnormal granules in neutrophils from five patients with Chediak-Higashi disease. Monospecific antiserums to the azurophilic markers myeloperoxidase, elastase, cathepsin G and lysozyme, and to the specific granule markers lactoferrin and lysozyme, were labeled with fluorescein and rhodamine and were used to demonstrate two antigens in the same cell simultaneously. The abnormal granules in Chediak-Higashi neutrophils contained both azurophilic and specific granule markers. Normal-appearing lactoferrin-positive granules were also present, but normal azurophilic granules were not seen. Analysis of bone-marrow samples from two of these patients suggested that the abnormal granules were formed during granulocyte maturation by the progressive aggregation and fusion of normally formed azurophilic and specific granules. These results are consistent with a membrane abnormality or a defect of microtubular function leading to inappropriate granule fusion, and suggest that the granular abnormality is more generalized than previously appreciated.

Neutrophil and eosinophil granulocytes in bacteral infection : Sequential studies of cellular and serum levels of granule proteins

Abstract: The intraneutrophilic concentrations of lactoferrin, myeloperoxidase, collagenase and chymotrypsin‐like cationic proteins were measured sequentially during acute bacterial infection. The serum levels of lactoferrin and myeloperoxidase were also followed as well as the ‘eosinophil’ cationic protein as a marker for eosinophil leucocytes. During the early course of infection there was a profound but reversible decrease of intraneutrophilic lactoferrin. The levels of cellular collagenase and chymotrypsin‐like cationic proteins also tended to decrease reversibly during day 2–8 in most cases; myeloperoxidase levels were normal except for two cases. Serum myeloperoxidase and lactoferrin correlated with blood neutrophil counts. In spite of the absence of peripheral eosinophils the ‘eosinophil’ cationic proteins of serum were increased on the first day of infection, which may reflect increased eosinophil turnover.

Identification of Human Anterior Pituitary Cells by Immunoelectron Microscopy

Abstract: An attempt was made to classify human pituitary cell types by electron microscopic immunohistochemistry. The immunoperoxidase technique involving the use of the peroxidase-antiperoxidase complex was applied to thin sections of human pituitaries removed surgically for breast cancer or diabetic retinopathy. Using specific antibodies against human PRL, GH, beta-FSH, beta-LH, beta-TSH, and porcine ACTH, the localization of each hormone was studied. Identification of 5 human pituitary cells was possible: 1) The PRL-secreting cell contains round or slightly ovoid secretory granules of a diameter of 275-350 nm. 2) The GH-secreting cell is densely granulated with granule diameters ranging from 350-500 nm. 3) The gonadotrophic cell, which stains for both beta-FSH and beta-LH, is characterized by the presence of a varying number of secretory granules ranging from 275-375 nm. 4) The cortico-lipotrophic cell has numerous granules of about 375-550 nm in diameter. 5) The TSH-secreting cell contains small secretory granules of about 125-200 nm in diameter. Another cell type of which the small secretory granules of about 100 nm in diameter could not be stained by any of the antisera was also observed. This ultrastructural identification of human pituitary cells should contribute to a better understanding of the pathophysiology of the human pituitary.

Bactericidal activity of specific and azurophil granules from human neutrophils: studies with outer-membrane mutants of Salmonella typhimurium LT-2.

Abstract: Extracts of specific granules and azurophil granules from human neutrophils were tested for their bactericidal activity against various lipopolysaccharide mutants of Salmonella typhimurium LT-2. Three purified granule populations, one specific and two azurophil, were obtained by isopycnic centrifugation of homogenized neutrophils. Each was extracted with 0.2 M acetate buffer (pH 4), and the extracts were dialyzed against phosphate-buffered saline (pH 7) to remove acetate. These extracts contained ≥84% of the lysozyme, lactoferrin, or myeloperoxidase initially present in the whole granules. The S. typhimurium mutants possessed Ra, Rc, Rd1, Rd2, or Re lipopolysaccharide. As the carbohydrate content of the lipopolysaccharide decreased, the bacteria became increasingly more susceptible to the bactericidal activity of all granule extracts. Bactericidal activity of the extracts was in the order: mixed (azurophil + specific) ≥ azurophil » specific. Specific granules were bacteriostatic for S through Rd2 bacteria. They were bactericidal only for the Re mutant. Both azurophil granule populations were equally bactericidal. Extracts boiled for 30 min retained none of their bactericidal activity for any of the bacteria; however, they remained bacteriostatic for the deep rough (Rd2, Re) mutants. Bactericidal activity was dependent upon pH, in that mixed and azurophil granule contents killed the smooth parent and Ra mutant best at pH 5, the Rc and Rd1 mutants to the same degree at pH 5 to 8, and the deep rough mutants (Rd2 and Re) best at pH 8. Specific granule contents were most bacteriostatic for S through Rd2 bacteria at pH 5 and killed the Re mutant only at pH 8. Thus, as the S. typhimurium lipopolysaccharide content decreased, the bactericidal pH optimum increased. Killing by all extracts was dependent upon incubation temperature, with almost no bactericidal or bacteriostatic activity observed when bacteria and granule fractions were incubated on ice (2°C) and plated immediately. Intermediate killing was observed at 22°C. If bacteria were incubated with granule extracts at 2°C, washed free of extract, suspended in medium without extract, and reincubated at 37°C, killing was observed. This suggested that a component(s) of the extracts was sticking to the bacteria at 2°C but killing only at 37°C.

Secretion granules of the rabbit parotid. Selective removal of secretory contaminants from granule membranes

Abstract: A membrane subfraction obtained from secretion granules isolated from rabbit parotid has been shown to be contaminated by residual secretory proteins to an estimated level of 25-30% of its total protein. In the present study an additional contaminant has been identified by improved mixing experiments and by comparative peptide mapping of specific polypeptides recovered from gels of membrane and content subfractions. This contaminant coelectrophoresis with (and probably comprises the bulk of) the majority component of the membrane subfraction (mol wt approximately 40,000). The contaminating polypeptides can be removed to a large extent by treating the membranes with low concentrations of saponin in the presence of 0.3 M Na2SO4. Although this treatment disrupts the typical bilayer structure of the granule membrane, it does not appear to cause dissociation of its phospholipids or bona fide membrane proteins.

Role of zinc and calcium in the formation and storage of insulin in the pancreatic β-cell

Abstract: 1. The effects of culture of isolated mouse islets of Langerhans for up to 9 days in media which had been depleted of zinc electrochemically or with the chelating agent Tris-(2-aminoethyl) amine, or of calcium, have been compared. 2. An 83% reduction of extracellular zinc concentration did not adversely affect proinsulin biosynthesis, conversion of proinsulin to insulin, or the ability of cells to store newly formed insulin in granules. When incubation media were depleted of both zinc and calcium the β-cells produced abnormally large electron-lucent granules, consistent with the failure of insulin to crystallise within the granule sac. 3. Very similar results, with formation of large electron lucent granules, were obtained after culture of islets in the absence of calcium but in the presence of normal concentrations of zinc. 4. It is suggested that zinc may play a less critical role in the biosynthesis of proinsulin and its conversion to insulin, while calcium may have a more important function in insulin storage, than has sometimes previously been supposed.

Ultrastructural localization of calcium in unfertilized sea-urchin eggs.

Abstract: The pyroantimonate technique was employed to identify the binding sites for calcium in unfertilized Arbacia punctulata and Strongylocentrotus purpuratus eggs. Since antimony is non-specific and binds with a variety of cations, the indentification of calcium was established by specific chelation with ethyleneglycol tetra-acetic acid (EGTA) and X-ray microprobe analysis. Antimony deposits were observed on the egg's membranes, i.e. plasma, cortical (secretory) granule, pigment granule, smooth-surfaced vesicle, and yolk platelet. Deposits were also observed in the mitochondria, rod-containing vesicles, and the vitelline layer. Two types of yolk platelets were observed: a more numerous electron-opaque platelet which had precipitate along its limiting membrane as well as within the stored-matrix substance, and a less-frequently seen platelet with lower electron opacity which contained precipitate only along its limiting membrane. Deposits were reduced at all sites following exposure of eggs to EGTA either prior to or after osmium-antimonate fixation. Initial fixation in glutaraldehyde followed by postfixation in osmium-antimonate solutions provided better preservation of structure but less precipitation than direct fixation in osmium-antimonate. The organelle sites of calcium binding identified within unfertilized sea-urchin eggs may participate in stimulus-secretion coupling (exocytosis of the cortical granules) and the activation of embryogenesis at fertilization.

Ultrastructural and cytochemical study of the gastric epithelium in a fresh water teleostean fish (Perca fluviatilis)

Abstract: A description of the cytology of the gastric mucosa of the perch has been made. Cytological and cytochemical features of the surface mucous cells, mucous neck cells, oxyntic cells and endocrine cells are described. The surface mucous cells are identified by their location and by the characteristics of their secretory granules. A proportion of these granules contain a core with electron density different from the rest of the granule. These granules, except for the electron-dense core, react cytochemically as glycoprotein. The mucous neck cells are distinguished by their shape, the appearance of their mucous granules and their location between surface mucous cells and glandular cells. The oxyntic cells contain a cytoplasmic “tubulovesicular system”, many large zymogen granules and a rich rough endoplasmic reticulum in the basal cytoplasm. Endocrine cells extending up to the gastric lumen were also observed.

Bactericidal capacity of phorbol myristate acetate-treated human polymorphonuclear leukocytes.

Abstract: Thus far, the functional capacity of phorbol myristate acetate- (PMA)-treated human polymorphonuclear leukocytes has been undefined. PMA induced exocytosis of lactoferrin, the specific granule marker, but not of myeloperoxidase, the azurophil granule marker. This phenomenon was demonstrated both biochemically and with fluorescent antibody conjugates. PMA-treated neutrophils contained virtually no specific granules when viewed by electron microscopy. Separation of the granule classes by linear sucrose density gradient centrifugation revealed the loss, from PMA-treated neutrophils, of lactoferrin and the specific granule (D20(20) = 1.89) band usually resolved from normal neutrophils. Cells treated with PMA appeared to retain those functions normally associated with intraleukocytic microbicidal action. The hexose monophosphate shunt activated by phagocytic challenge was present in PMA-treated neutrophils. As demonstrated by electron microscopy, the azurophil granules of these cells appeared intact, and they retained the capacity for degranulation with translocation of myeloperoxidase to the site of phagocytized Escherichia coli. The PMA-treated neutrophils also remained capable of degrading the ingested microorganisms. PMA-treated neutrophils exhibited a decrease in phagocytic ability at all levels of bacterial challenge. In the presence of a high multiplicity of bacteria they demonstrated an impairment in killing. These same cells were able to kill low multiplicities of E. coli as well as control cells. It thus appeared that the loss of the specific granules, plus other undefined PMA-induced alterations, impaired neither the viability of these neutrophils nor their killing ability in the presence of a modest phagocytic challenge.

SGC (small granule chromaffin) cells in the mouse adrenal medulla: light and electron microscopic identification using semi-thin and ultra-thin sections.

Abstract: Adrenal glands of the mouse, fixed either in glutaraldehyde followed by osmium tetroxide or in a mixture of potassium dichromate and glutaraldehyde, and embedded in Epon 812, were investigated by light and electron microscopy. An argentaffin reaction was applied to semi-thin sections for light microscopy and to ultra-thin sections for electron microscopy. Since the mature secretory granules in the Small Granule Chromaffin (SGC) cell were argentaffin and were mainly located along the cell membrane, this cell was clearly distinguishable under the light microscope both from the A (adrenaline) cell whose secretory granules were non-argentaffin and from the NA (noradrenaline) cell whose cytoplasm was rich and was filled with large, strongly argentaffin granules. Chromaffinity of the SGC cell was demonstrated under the light microscope. The SGC cell was intensively stained with toluidine blue without revealing metachromasia. It was demonstrated at the EM level that not only the secretory granules but also the synaptic-like vesicles in the SGC cell contained argentaffin substances. Possible functional relationship between the secretory granules and the synaptic-like vesicles was discussed.

Subcellular distribution of glycosidases in human polymorphonuclear leucocytes.

Abstract: The subcellular distribution of nine glycosidases were studied in fractions of homogenized human polymorphonuclear leucocytes (neutrophils) obtained by isopycnic centrifugation through linear sucrose density gradients. The substrates were 4-methylumbelliferyl glycosides. All nine glycosides were hydrolysed by enzymes in neutrophil cytosol fractions, and by enzymes in at least one granule population. alpha-Glucosidase activity sedimented in sucrose density gradients to a point (p = 1.180 g/ml) just above the specific granules, possibly the 'tertiary' granule population. The peak corresponding to alpha-glucosidase did not co-sediment with, but considerably overlapped, the peak corresponding to lactoferrin, a marker for specific granules (p = 1.187 g/ml). alpha-Galactosidase activity was found primarily in heavy azurophil granules (p = 1.222 g/ml). alpha-Mannosidase activity was found primarily in light azurophil granules (p = 1.206 g/ml), following the distribution of myeloperoxidase, the commonly used azurophil granule marker. beta-Glucosidase activity was concentrated in mitochondrial fractions (p = 1.160 g/ml). All other glycosidases presented complex distributions, with activities not restricted to one granule class. Granule-associated glycosidase activities were increased 2--38 times when measured in the presence of 0.05% Triton X-100, indicating latency of the enzymes within granules.

Freeze‐etch ultrastructure of waxy maize and acid hydrolyzed waxy maize starch granules

Abstract: Freeze-etching was used to study the ultrastructure of waxy maize starch and acid-treated (0.5M HCl, 25°C, 24 hr) waxy maize starch. The outer surfaces of the untreated granules were smooth with a faint fibrillar pattern. Granule cross-fractures had a fine particulate appearance with some radially-oriented ridges. Internal cracks were evident. Starch granules soaked in acid developed surface pits. Areas of hydrolysis had a distinctive structure and occurred in restricted zones. The shape of the acid-treated starch granules remained unchanged. However, inner regions showed extensive ultrastructural changes and water infiltration. Freeze-etching revealed bands of alternating high and low water content which formed lamellae in a few granules.

Postnatal quantitative changes in the cerebellar uvula of albino rats.

Abstract: The postnatal quantitative changes in cell diameter, cell density and total number of granule cells in the sublobule IXa of female rats from 6 to 760 days old were examined. There occurs and initial rapid increase in cell density from 1232.00±91.92 granule cells per 10-3 mm3 to 2995.50±322.07 granule cells per 10-3 mm3 and from 3 135 316±233 937 to more than 24 millions of granule cells between the 6th and 25th postnatal day. After the middle of the 3rd postnatal week, cell density and total number of granule cells decrease. The diameter of the granule cells reaches a maximum at the 6th day post partum and decreases continuously with progressing age. The possible mechanisms of these quantitative changes are discussed.

Morphology and cytochemistry of acinar secretory granules in normal and isoproterenol-treated rat submandibular glands.

Abstract: SUMMARY Several fixation procedures have been utilized in a fine-structural study of rat submandibular glands with the aim of correlating the morphology of granule substructure with its composition. Procedures included fixation with aldehyde in a variety of buffers, with or without fixation additives such as tannic acid or calcium chloride. Both immersion and perfusion fixation studies were performed. Osmium tetroxide postfixed tissue was compared with non-postfixed tissue. Thin sections were stained for carbohydrate-containing constituents by either periodic acid-thiocarbohydrazide-silver proteinate or tannic acid-ferric chloride sequences. The morphology and cytochemistry of acinar secretory granules were highly dependent upon the fixation procedure utilized. In postfixed tissue, fine filaments and vesicles were the major granule constituents. Filaments often aggregated into fibrils in fixatives containing phosphate buffer or calcium, whereas vesicles were prominent with fixatives containing collidine buffer. Tannic acid, as a fixation additive, imparted enhanced density to the peripheral rim of aggregated filaments. If post fixation was eliminated, an amorphous meshlike material was the major granule component. This stained readily with methods for carbohydrate whereas the granules of post-osmicated tissue did not. Following chronic isoproterenol treatment the degree of filament aggregation in postfixed tissue was augmented, and this corresponded to an increase in amount of material stainable for carbohydrate in non-post-osmicated tissue.

Mast cell granule formation in the beige mouse.

Abstract: The formation of mast cell granules was studied in the beige mouse utilizing histochemistry and electron microscopy. The time and sequence of appearance of heparin, histamine and the chymotrypsin-like protease were normal. By electron microscopy, the initial formation of progranules and subsequent aggregation was normal, but the granules from early stages were abnormally large. Reorganization of intermediate granule forms to homogeneous mature granules was delayed. Late fusions of intermediate and/or mature granules were not observed. Our findings indicate that the defect lies in the excessive initial fusion of progranules rather than in continued formation of new progranules or in fusion of mature granules with one another.

The ultrastructure of guinea pig heterophil granulocytes and the heterogeneity of the granules.

Abstract: The development of the heterophil granulocytes in the bone marrow of the guinea pig is described. During the maturation of these cells, three types of granule are formed, not only the azurophil and specific granules already described in other mammals but also a third type of granule referred to here as the nucleated granule. During the process of maturation of the cells, these three types of granule are formed successively. On this basis, two steps can be distinguished in the promyelocyte phase in which primary (nucleated and azurophil) granules are formed, i.e. an early and a late stage, nucleated granules being formed in early and azurophil granules in late promyelocytes. Secondary (specific) granules occur first in myelocytes. In mature heterophils of the guinea pig the granule population is composed of about 85% secondary granules, about 10% azurophil granules, and about 5% nucleated granules. The changes in the granule population during the maturation process were quantified. The observations and calculations point to the occurrence of three mitoses: one in the early and one in the late promyelocyte and the third in the myelocyte.

Impregnated porous granules with slow release pore membranes and process therefor

Abstract: Open-ended pores of a porous granule are filled with a liquid material which is at least partially immiscible with water, and sealed with a porous polyurea membrane which limits the release rate of the material into the surrounding medium. The membrane is formed by (a) applying to the bare granule an organic solution comprising the liquid material and an organic polyisocyanate, followed by (b) applying to the granule an aqueous solution comprising water and a catalytic amount of a catalyst selected from the group consisting of a basic organic tertiary amine and an alkyl tin carboxylate.

Ultrastructural identification of acid complex carbohydrate in cytoplasmic granules of normal and leukaemic human monocytes.

Abstract: Summary. Complex carbohydrate in granules of monocytes was compared with that in granules of neutrophils by ultrastructural cytochemical methods. The acid mucosubstance in granules of both cell types stained with dialysed iron after brief fixation with dilute glutaraldehyde, but that in monocyte granules differed in failing to stain after stronger fixation. Approximately 10% of granules in normal blood monocytes stained with this method, whereas more than 90% of granules in leukaemic monocytes from two of seven patients with acute myelomonocytic leukaemia stained intensely. This difference presumably results from unmasking of acid groups in immature granules or increased synthesis of granule mucosubstance in some leukaemic monocytes. Granules of monocytes differed further from those of neutrophils in failing after either type of fixation to stain with a high iron diamine technique for demonstration of sulphated mucosubstance. The absence of high iron diamine staining could reflect a lack of sulphate esters in monocyte granule mucosubstance, masking of the sulphate groups by other components, or extraction of the sulphated mucosubstance during specimen processing.

The fine structure of granules in eosinophil leucocytes from aquatic and terrestrial birds.

Abstract: The ultrastructure of eosinophil granules from various aquatic and terrestrial birds has been described. Granules of three basic types were found. The first had a crystalline internum and was found only in the order Anseriformes , which included the black-necked screamer, ducks, geese and swans. The crystals occurred in three morphological forms. The second and least common granule examined contained a non-crystalline internum which was either homogeneous or composed of microfilaments or microtubules. The largest and most common group of birds had a homogeneous granule with no internum shown. Homogeneous granules occurred less frequently than did those with interna.