scholarly journals Development of GP-2 and five zymogens in the fetal and young pig: biochemical and immunocytochemical evidence of an atypical zymogen granule composition in the fetus.

1996 ◽  
Vol 44 (5) ◽  
pp. 481-499 ◽  
Author(s):  
J Lainé ◽  
G Pelletier ◽  
G Grondin ◽  
M Peng ◽  
D LeBel
Keyword(s):  
Exocrine Pancreas ◽  
Secretory Granules ◽  
Fetal Tissue ◽  
Irregular Shape ◽  
Major Protein ◽  
Zymogen Granule ◽  
Mature Animal ◽  
Dense Granules ◽  
The Matrix ◽  
Light Material

To uncover the mechanisms involved in the biogenesis of secretory granules, we studied development of the exocrine pancreas in the pig from the fetus up to the mature animal by following the enzyme activities and expression (Northern blot) of five zymogens and GP-2, the major protein of the granule membrane. Fetal pancreas mainly contained chymotrypsinogen and barely detectable amounts of amylase, trypsin, lipase, and elastase. GP-2 was not notably expressed before the Day 21 of life. Ultrastructural examination of the fetal tissue embedded in Epon with osmium postfixation or in Lowicryl at -20 degrees C without postfixation showed dense granules with an irregular shape but also showed that most granules had uncondensed contents, with the aspect of immature granules, or had a dense core surrounded by light material. With immunogold cytochemistry, the concentration of chymotrypsinogen was directly associated with the acquisition of electron density by the granule matrix. These observations suggest that fetal granules have a slower rhythm of zymogen condensation and an irregular shape that could be due to the particular composition of the matrix and the absence of GP-2. We conclude that, in the exocrine pancreas, secretory granules can be formed under various conditions, even with a matrix containing a ratio of components very different from that of the normal mature animal.

scholarly journals Localization of bone sialoprotein (BSP) to Golgi and post-Golgi secretory structures in osteoblasts and to discrete sites in early bone matrix.

1993 ◽  
Vol 41 (2) ◽  
pp. 193-203 ◽  
Author(s):  
P Bianco ◽  
M Riminucci ◽  
G Silvestrini ◽  
E Bonucci ◽  
J D Termine ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Bone Matrix ◽  
Ultrastructural Level ◽  
Bone Sialoprotein ◽  
Protein Glycosylation ◽  
Secretory Structures ◽  
Mineral Deposition ◽  
The Matrix ◽  
Random Manner ◽  
Intracellular Labelling

Bone sialoprotein (BSP), a bone matrix-enriched glycoprotein containing the Arg-Gly-Asp (RGD) motif and endowed with cell binding properties, was localized in osteoblasts and early bone matrix of developing rat bone at the ultrastructural level. Preliminary light microscopic observations indicated that intracellular labelling was restricted to a paranuclear dot corresponding to the "negative Golgi image" of classical histology. The same pattern was observed whether antisera against the fully glycosylated protein or a peptide antiserum to a stretch of amino acids in human BSP sequence were employed. At the EM level, we obtained labeling over the Golgi area of osteoblasts but not over the rER. The labeling was concentrated over distensions of the trans Golgi and over pro-secretory granules. In the matrix, BSP was distributed in a non-random manner. The label was concentrated over spherical aggregates of finely fibrillar material corresponding to the sites of early mineral deposition (so-called "mineralization nodules"). Such BSP-positive foci were seen both close to and away from the cell surface. The predominant association of BSP with Golgi and post-Golgi secretory structures and its absence from rER, as well as the reproducibility of the same pattern of localization with different antisera, might indicate a slow transit of the protein through the Golgi, not necessarily associated with protein glycosylation.

Localization, turnover and conservation of gp15/400 in different stages ofBrugia malayi

Parasitology ◽  
1993 ◽  
Vol 107 (4) ◽  
pp. 449-457 ◽  
Author(s):  
M. E. Selkirk ◽  
W. F. Gregory ◽  
R. E. Jenkins ◽  
R. M. Maizels
Keyword(s):  
Protein Complex ◽  
Cdna Probe ◽  
The Body ◽  
Mammalian Host ◽  
Major Protein ◽  
Homologous Genes ◽  
Acanthocheilonema Viteae ◽  
Filarial Nematodes ◽  
The Matrix

SUMMARYThe expression of a protein complex designated gp15/400, previously identified via extrinsic iodination of adultBrugia malayi, was examined by labelling all stages found in the mammalian host and immunoprecipitation with a specific antibody raised to a recombinant protein. In this way, gp15/400 could be detected in L3, L4, adult worms and microfilariae recovered from jirds and labelled with Bolton–Hunter reagent. Metabolic labelling indicated that gp15/400 was released into culture medium when adult worms were maintainedin vitro, but at a rate slower than that of gp29, the major soluble cuticular glycoprotein. Immuno-electron microscopy showed that the protein complex was broadly distributed in different tissues, although it was not detectable in the cuticle of adult worms. Dense labelling was observed in the matrix of the basal laminae bordering the hypodermis, somatic musculature and oesophagus, and lower but significant labelling was seen in the cells overlying these extracellular matrices. Hybridization of genomic DNA with a cDNA probe encoding gp15/400 indicated that homologous genes were present inDirofilaria immitisandAcanthocheilonema viteae. The failure to detect related genes in non-filarial nematodes was presumed to be due to divergence beyond the practical limits of detection by nucleic acid probes, as antibody reagents showed that the protein cross-reacted immunologically with ABA-1, a major protein allergen from the body fluid ofAscaris.

Subcellular localization of oxytocin in the ovine corpus luteum

1985 ◽  
Vol 63 (4) ◽  
pp. 309-314 ◽  
Author(s):  
G. E. Rice ◽  
G. D. Thorburn
Keyword(s):  
Subcellular Localization ◽  
Physicochemical Properties ◽  
Corpus Luteum ◽  
Density Gradient Centrifugation ◽  
Secretory Granules ◽  
Gradient Centrifugation ◽  
Particulate Fraction ◽  
Dense Granules ◽  
Oxytocin Release ◽  
Midluteal Phase

The subcellular localization of oxytocin within the corpus luteum of sheep was investigated using differential and density gradient centrifugation. Oxytocin was associated with a particulate fraction which sedimented to a density of 1.054 – 1.061 g/mL. The exclusion of [3H]oxytocin from this particulate fraction is indicative that particulate oxytocin represents endogenous compartmentalization. Particulate oxytocin, incubated in buffered medium at 37 °C, was stable for up to 1 h and the release of oxytocin was not affected by the pH of the incubation medium, over the range 5.5 – 8.5. Oxytocin release, however, was stimulated by incubating particle-bound oxytocin in buffered medium of low osmolality (<200 mosmol). These data are similar to the physicochemical properties reported for peptide-containing neurohypophysial secretory granules. Ultrastructural analysis of oxytocin-containing fractions revealed the presence of electron-dense granules (diameter, 200–250 nm). These data are suggestive that oxytocin, in the corpus luteum of sheep, is contained within a population of secretory granules which occur in high numbers during the midluteal phase of the oestrous cycle.

scholarly journals Immunocytochemical assays of amylase and chymotrypsinogen in rat pancreas secretory granules. Efficacy of using immunogold-labeled ultrathin cryosections to estimate relative protein concentrations.

1984 ◽  
Vol 32 (10) ◽  
pp. 1028-1034 ◽  
Author(s):  
G Posthuma ◽  
J W Slot ◽  
H J Geuze
Keyword(s):  
Secretory Granules ◽  
Relative Concentration ◽  
Data Sets ◽  
Zymogen Granule ◽  
Protein Levels ◽  
Pancreatic Cells ◽  
Intracellular Proteins ◽  
Ultrathin Cryosections ◽  
Respective Protein ◽  
Normal Laboratory

An exploration was conducted as to whether the relative concentration of two intracellular proteins could be evaluated quantitatively from their labeling densities in ultrathin cryosections labeled with the immunogold technique. As a model rat pancreatic cells were used in which the content of amylase (Am) and chymotrypsinogen (Ch) was experimentally altered. Rats were fed either normal laboratory chow or food containing soybean trypsin inhibitor (STI), which affects the Am/Ch ratio in the tissues. The changes in Am and Ch protein levels and enzyme activities were measured biochemically in cell suspension homogenates or in zymogen granule fractions. Within 5 days a maximal change in the Am/Ch was observed as a result of adaptation to the STI diet. The Am/Ch ratio determined biochemically was compared with that from counts of gold particles bound to the respective protein in immunogold-labeled cryosections. The two data sets matched fairly well, indicating that the intensity of the immunoreaction is a reliable reflection of antigen concentration in this system.

Role of sulfated O-linked glycoproteins in zymogen granule formation

2002 ◽  
Vol 115 (14) ◽  
pp. 2941-2952 ◽  
Author(s):  
Robert C. De Lisle
Keyword(s):  
Acinar Cell ◽  
Secretory Granules ◽  
Sodium Chlorate ◽  
Pancreatic Acinar Cell ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Acidic Ph ◽  
Zymogen Granules ◽  
Granule Formation

Packaging of proteins into regulated secretory granules is mediated by the mildly acidic pH of the trans Golgi network and immature secretory granules. This need for an acidic pH indicates that ionic interactions are important. The mouse pancreatic acinar cell contains four major sulfated glycoproteins,including the zymogen granule structural component Muclin. I tested the hypothesis that sulfation and the O-linked glycosylation to which the sulfates are attached are required for normal formation of zymogen granules in the exocrine pancreas. Post-translational processing was perturbed with two chemicals: sodium chlorate was used to inhibit sulfation and benzyl-N-acetyl-α-galactosaminide was used to inhibit O-linked oligosaccharide elongation. Both chemicals resulted in the accumulation in the Golgi region of the cell of large vacuoles that appear to be immature secretory granules, and the effect was much more extensive with benzyl-N-acetyl-α-galactosaminide than chlorate. Both chemical treatments inhibited basal secretion at prolonged chase times, and again benzyl-N-acetyl-α-galactosaminide had a greater effect than chlorate. In addition, benzyl-N-acetyl-α-galactosaminide, but not chlorate, totally inhibited stimulated secretion of newly synthesized proteins. These data provide evidence for a role of sulfated O-linked glycoproteins in protein condensation and maturation of zymogen granules. Under maximal inhibition of O-linked oligosaccharide biosynthesis, anterograde post-Golgi traffic in the regulated pathway is almost totally shut down, demonstrating the importance of these post-translational modifications in progression of secretory proteins through the regulated pathway and normal granule formation in the pancreatic acinar cell.

scholarly journals Heterogeneity in the pattern of distribution of the specific hormonal product and secretogranins within the secretory granules of rat prolactin cells.

1994 ◽  
Vol 42 (8) ◽  
pp. 1097-1107 ◽  
Author(s):  
H Ozawa ◽  
R Picart ◽  
A Barret ◽  
C Tougard
Keyword(s):  
Subcellular Distribution ◽  
Secretory Granules ◽  
Culture Conditions ◽  
Male Rat ◽  
Immunogold Electron Microscopy ◽  
The Matrix ◽  
Granule Membrane ◽  
Adult Male Rat ◽  
Secretory Products ◽  
Rat Pituitary Tumor Cells

We investigated the subcellular distribution of secretogranins I, II (Sg I, Sg II), and prolactin (PRL) by double immunogold electron microscopy in GH3B6 rat pituitary tumor cells grown in different culture conditions and in normal PRL cells in adult male rat anterior pituitary. Co-localization of Sg I or Sg II with PRL was observed in most secretory granules in GH3B6 cells and normal PRL cells, except for some secretory granules containing only Sgs in GH3B6 cells and containing only PRL in normal PRL cells. In GH3B6 cells treated with thyrotropin-releasing hormone (TRH) for 2 hr, the newly formed small secretory granules within the Golgi zone contained preferentially immunoreactive PRL. Interestingly, when co-localized with PRL, Sgs (particularly Sg I) were observed at the periphery of the matrix of secretory granules in GH3B6 cells as well as in normal PRL cells, suggesting their possible interaction with the secretory granule membrane. The present study indicates a heterogeneous subcellular distribution of PRL, Sg I, and Sg II in individual secretory granules of GH3B6 cells and of normal PRL cells, pointing out the formation of different types of aggregates during the condensation of secretory products in these two PRL cell models.

scholarly journals Immunohistochemical localization of procollagens. III. Type I procollagen antigenicity in osteoblasts and prebone (osteoid).

1981 ◽  
Vol 29 (7) ◽  
pp. 791-804 ◽  
Author(s):  
G M Wright ◽  
C P Leblond
Keyword(s):  
Bone Growth ◽  
Secretory Granules ◽  
Immunohistochemical Localization ◽  
Multivesicular Bodies ◽  
Type I ◽  
Frozen Sections ◽  
Type I Procollagen ◽  
The Matrix ◽  
Old Rats ◽  
Procollagen Iii

Frozen sections of unfixed tibia and mandibles from day-old rats were immunostained by the peroxidase-antiperoxidase technique after exposure to antisera against procollagen I or other collagenous materials. Light microscopic study of bone growth areas showed that procollagen I antigenicity was present in osteoblasts and prebone (osteoid), but not bone tissue; neither procollagen III nor collagen IV antigenicity were detected. The localization of procollagen I antigenicity within osteoblasts was then attempted in the electron microscope. Chopper slices of formaldehyde-fixed tibia from day-old rats were incubated with affinity-purified anti-procollagen I antibodies linked to peroxidase and were treated with hydrogen peroxide in the presence of 3,3'-diaminobenzidine. The dot-like reaction indicative of procollagen I antigenicity was found to be moderate in rough endoplasmic reticulum (rER) cisternae, strong in spherical and cylindrical Golgi distensions, and intense in prosecretory and secretory granules. Reactivity was also observed within the matrix of multivesicular bodies Thus an increasing gradient of procollagen I antigenicity occurs from rER cisternae through Golgi distensions to secretory granules, that is, along the presumed pathway of procollagen synthesis. The antigenicity present within rER cisternae is attributed to the precursor pro alpha (I) chains, while that in the cylindrical Golgi distensions, secretory granules, and prebone is attributed to procollagen itself. The antigenicity of multivesicular bodies suggests some degradation of pro alpha chains or procollagen.

scholarly journals The localization of phospholipase A2 in the secretory granule

1994 ◽  
Vol 300 (3) ◽  
pp. 619-622 ◽  
Author(s):  
S P Chock ◽  
E A Schmauder-Chock ◽  
E Cordella-Miele ◽  
L Miele ◽  
A B Mukherjee
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Lipid Mediator ◽  
Arachidonic Acid Cascade ◽  
Surface Receptors ◽  
The Matrix ◽  
Immunocytochemical Techniques ◽  
Granule Matrix

A heat-resistant phospholipase A2 has been detected in the secretory granules of the mast cell [Chock, Rhee, Tang and Schmauder-Chock (1991) Eur. J. Biochem. 195, 707-713]. By using ultrastructural immunocytochemical techniques, we have now localized this enzyme to the matrix of the secretory granule. Like the cyclo-oxygenase [Schmauder-Chock and Chock (1989) J. Histochem. Cytochem. 37, 1319-1328], this enzyme also adheres tightly to the ribbon-like granule matrix components. The results from Western-blot analysis suggest that it has a molecular mass of about 14 kDa. The localization of the phospholipase A2, the presence of a phospholipid store with millimolar concentrations of calcium and the localization of the enzymes of the arachidonic acid cascade make the secretory granule a natural site for lipid-mediator synthesis. The packaging of phospholipase A2, together with its substrate and the components of the arachidonic acid cascade, in the secretory granule represents a physical arrangement by which the initiation of the cascade and the release of mediators can be directly linked to the stimulation of cell-surface receptors.

scholarly journals ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS

1969 ◽  
Vol 41 (1) ◽  
pp. 167-176 ◽  
Author(s):  
S. L. Howell ◽  
D. A. Young ◽  
P. E. Lacy
Keyword(s):  
Islets Of Langerhans ◽  
Secretory Granules ◽  
Sodium Deoxycholate ◽  
Rat Islets ◽  
The Matrix ◽  
Effects Of Ph ◽  
The Stability ◽  
Rat Islets Of Langerhans ◽  
Specific Insulin

A partially purified secretory granule fraction, isolated from rat islets of Langerhans by differential centrifugation, was used for investigating the stability of the beta granules during incubation in various conditions. Effects of pH, temperature, and time were studied; the granules possessed optimal stability at 4° and pH 6.0, and could be solubilized at pH 4.0 or 8.5, or in the presence of sodium deoxycholate, but not by phospholipase c, ouabain, or alloxan. Incubation with glucose or some of its metabolites, or with tolbutamide, ATP, or cyclic 3',5'-AMP did not alter the stability of the beta granules Exogenous insulin-131I was not bound by the isolated granules under the conditions used; no specific insulin-degrading activity could be detected in subcellular fractions of the islets. These findings indicate that intracellular solubilization of the granules with subsequent diffusion of the insulin into the extracellular space is not a likely mode of insulin secretion in vivo, and suggest that a crystalline zinc-insulin complex may exist in the matrix of the beta granules.

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