scholarly journals Effect of monensin on secretory pathway in GH3 prolactin cells. A cytochemical study.

1983 ◽  
Vol 31 (6) ◽  
pp. 745-754 ◽  
Author(s):  
C Tougard ◽  
R Picart ◽  
A Morin ◽  
A Tixier-Vidal
Keyword(s):  
Endocrine Cells ◽  
Secretory Pathway ◽  
Culture Medium ◽  
Gh3 Cells ◽  
Secretory Product ◽  
Prolactin Cells ◽  
Cytochemical Study ◽  
Basal Release ◽  
Golgi Zone ◽  
Ionophore Monensin

The effects of the carboxylic ionophore monensin have been studied on a rat prolactin cell line (GH3 cells) under basal conditions or after acute stimulation by thyrotropin-releasing hormone (TRH). It was found that 1) monensin induces a rapid dilatation of Golgi elements in these endocrine cells; 2) secretory product, prolactin, is localized by electron microscope immunocytochemistry attached to the inner face of the membrane of these dilated vacuoles; 3) monensin induces preferentially a dilatation of the cis face of the Golgi zone, since the "GERL" complex identified by acid phosphate cytochemistry is disorganized or fragmented rather than vacuolized; and 4) monensin decreases strongly the basal release of prolactin in the culture medium but does not prevent the stimulating effect of TRH on this release. This suggests that monensin blocks preferentially the pathway of release of secretory product under basal conditions in GH3 cells but that another pathway less sensitive to monensin is involved under acute stimulation by TRH.

Immunolocalization of tissue inhibitor of metalloproteinases (TIMP) in human cells. Characterization and use of a specific antiserum

1985 ◽  
Vol 73 (1) ◽  
pp. 105-119
Author(s):  
R.M. Hembry ◽  
G. Murphy ◽  
J.J. Reynolds
Keyword(s):  
Smooth Muscle ◽  
Amniotic Fluid ◽  
Culture Medium ◽  
Lung Fibroblasts ◽  
Specific Antiserum ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Intracellular Accumulation ◽  
Human Amniotic Fluid ◽  
Foetal Lung ◽  
Ionophore Monensin

A specific antiserum to pure human amniotic fluid metalloproteinase inhibitor (TIMP) was raised in a sheep. This antiserum was used to demonstrate: firstly, the immunological identity of the TIMP activities from amniotic fluid and culture medium of human foetal lung fibroblasts; and secondly, by indirect immunofluorescence, the secretion of TIMP by human foetal lung fibroblasts, chondrocytes, epithelial cells and smooth muscle cells. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, was used to stimulate secretion of TIMP by human foetal lung fibroblasts and the ionophore monensin was used to demonstrate intracellular accumulation of TIMP in the Golgi apparatus of these cells. These results are discussed in relation to other inhibitors of collagenase reported in the literature, which are probably identical to TIMP.

scholarly journals Secretion of Cryparin, a Fungal Hydrophobin

1999 ◽  
Vol 65 (12) ◽  
pp. 5431-5435 ◽  
Author(s):  
Patricia M. McCabe ◽  
Neal K. Van Alfen
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Submerged Culture ◽  
Secretory Pathway ◽  
Culture Medium ◽  
Secretory Vesicle ◽  
Cryphonectria Parasitica ◽  
Secreted Protein ◽  
Filamentous Ascomycete ◽  
A Cell

ABSTRACT Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete Cryphonectria parasitica. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls ofC. parasitica, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly.

Movement of membrane tubules along microtubules in vitro: evidence for specialised sites of motor attachment

1994 ◽  
Vol 107 (7) ◽  
pp. 1885-1897 ◽  
Author(s):  
V. Allan ◽  
R. Vale
Keyword(s):  
Secretory Pathway ◽  
Motor Proteins ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Globular Domain ◽  
Secretory Product ◽  
Protein Activity ◽  
Golgi Stack ◽  
Membrane Tubules

We have studied the microtubule-dependent formation of tubular membrane networks in vitro, using a heterologous system composed of Xenopus egg cytosol combined with rat liver membrane fractions enriched in either Golgi stacks or rough endoplasmic reticulum. The first step in membrane network construction involves the extension of membrane tubules along microtubules by the action of microtubule-based motor proteins. We have observed for both membrane fractions that 80–95% of moving tubule tips possess a distinct globular domain. These structures do not form simply as a consequence of motor protein activity, but are stable domains that appear to be enriched in active microtubule motors. Negative stain electron microscopy reveals that the motile globular domains associated with the RER networks are generally smaller than those observed in networks derived from a crude Golgi stack fraction. The globular domains from the Golgi fraction are often packed with very low density lipoprotein particles (the major secretory product of hepatocytes) and albumin, which suggests that motor proteins may be specifically enriched in organelle regions where proteins for export are accumulated. These data raise the possibility that the concentration of active motor proteins into specialised membrane domains may be an important feature of the secretory pathway.

Release of gastric inhibitory polypeptide from cultured canine endocrine cells

1994 ◽  
Vol 267 (4) ◽  
pp. E489-E496 ◽  
Author(s):  
T. J. Kieffer ◽  
A. M. Buchan ◽  
H. Barker ◽  
J. C. Brown ◽  
R. A. Pederson
Keyword(s):  
Endocrine Cells ◽  
Gastric Inhibitory Polypeptide ◽  
Adherent Cells ◽  
Inhibitory Peptide ◽  
Cell Content ◽  
Gastrin Releasing Peptide ◽  
A 23187 ◽  
Basal Release ◽  
Cyclase Activator ◽  
Adenylate Cyclase Activator

Canine intestinal duodenal and jejunal epithelial cell preparations enriched for endocrine cells were obtained by sequential collagenase digestion and centrifugal elutriation and maintained in culture for a 40-h period. Adherent cells contained a total cell content (TCC) of 11.5 +/- 2.5 ng (mean +/- SE) immunoreactive gastric inhibitory peptide (IRGIP)/well and 1.4 +/- 0.2 ng immunoreactive somatostatin (IRS)/well. Release experiments were performed by incubation of the cells with various stimuli over a 2-h period. Basal release of IRGIP in 5 mM glucose-5 mM K+ was 2.7 +/- 0.4% TCC. Incubation with concentrations of K+ > 20 mM or glucose > 15 mM significantly increased IRGIP release, as did the addition of a somatostatin immunoneutralizing antibody to the basal media. The addition of the Ca2+ ionophore, A-23187 (10 microM), or the adenylate cyclase activator, forskolin (100 microM), resulted in an IRGIP output greater than four times basal. Porcine gastrin-releasing peptide (GRP), at 1-100 nM, significantly stimulated IRGIP release in a concentration-dependent fashion. IRS release was increased significantly by 55 mM K+, 20 mM glucose, 10 microM A-23187, 100 nM GRP, or 100 microM forskolin.

scholarly journals Pathways followed by membrane recovered from the surface of plasma cells and myeloma cells

1980 ◽  
Vol 152 (1) ◽  
pp. 1-19 ◽  
Author(s):  
PD Ottosen ◽  
PJ Courtoy ◽  
MG Farquhar
Keyword(s):  
Secretory Pathway ◽  
Plasma Cells ◽  
Surface Membrane ◽  
Secretory Cells ◽  
Secretory Product ◽  
Vesicle Membrane ◽  
Myeloma Cells ◽  
Glandular Cells ◽  
Lysosomal System ◽  
Endocytic Vesicles

Evidence for recovery of surface membrane and its fusion with Golgi cisternae has been obtained previously in several glandular cells. This study was conducted to determine whether or not membrane is similarly retrieved from the surfaces of plasma cells from lymph nodes (of rats immunized with horseradish peroxidase [HRP]) and mouse myeloma cells (RPC 5.4 and X63 Ag 8 cell lines). Electron-dense tracers (cationic and anionic ferritin, HRP) were used to trace the pathways followed by surface membrane recovered by endocytosis, and immunocytochemistry was used to identify the secretory compartments. When plasma cells or myeloma cells were incubated with cationized ferritin (CF), it bound to the cell surfaces and was taken up in endocytic vesicles, for the most part bound to the vesicle membrane. After 30-60 min, it was found increasingly within lysosomes and in several secretory compartments- notably in multiple stacked Golgi cisternae and secretory vacuoles. By immunocytochemistry the secretory product (immunoglobulins) and CF could be demonstrated in the same Golgi components. When myeloma cells were incubated with native (anionic) ferritin or in HRP, these tracers were taken up in much smaller amounts, primarily within the contents of endocytic vesicles. With continued incubation, they appeared only in lysosomes. When cells were doubly incubated, first in CF and then in HRP, both tracers were taken up (often within the same endocytic vesicle), but they maintained their same destinations as when incubated in a single tracer alone: the content marker, HRP, was localized exclusively within the lysosomal system, whereas the membrane marker, CF, was found within elements along the secretory pathway as well as within lysosomes. The findings demonstrate the existence of considerable membrane traffic between the cell membrane and the Golgi cisternae and lysosomes in both normal plasma cells and myeloma cells. Because myeloma cells behave like the glandular cells studied previously with regard to pathways of retrieved surface membrane, they represent a suitable and promising system for further studies of mechanisms and pathways of membrane retrieval and recycling in secretory cells.

scholarly journals Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

2001 ◽  
Vol 360 (3) ◽  
pp. 645-649 ◽  
Author(s):  
Renu K. JAIN ◽  
Paul B. M. JOYCE ◽  
Miguel MOLINETE ◽  
Philippe A. HALBAN ◽  
Sven-Ulrik GORR
Keyword(s):  
Green Fluorescent Protein ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Reporter Protein ◽  
Regulated Secretory Pathway ◽  
Green Fluorescent

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys49 and Cys71), which could play a role in this oligomerization. Site-directed mutagenesis of Cys49 and Cys71 showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

scholarly journals Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

2001 ◽  
Vol 360 (3) ◽  
pp. 645 ◽  
Author(s):  
Renu K. JAIN ◽  
Paul B.M. JOYCE ◽  
Miguel MOLINETE ◽  
Philippe A. HALBAN ◽  
Sven-Ulrik GORR
Keyword(s):  
Green Fluorescent Protein ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Ultrastructural and Immunofluorescence Studies of the Cells Associated With µ-Chain Disease

Blood ◽  
1971 ◽  
Vol 37 (3) ◽  
pp. 257-271 ◽  
Author(s):  
DOROTHEA ZUCKER-FRANKLIN ◽  
EDWARD C. FRANKLIN
Keyword(s):  
Electron Microscopy ◽  
Secretory Pathway ◽  
Plasma Cells ◽  
Bone Marrow Cells ◽  
Light Chains ◽  
Antigenic Determinants ◽  
Protein Fragments ◽  
Fluorescence And Electron Microscopy ◽  
Golgi Zone ◽  
Heavy Chain Disease

Abstract Fluorescence and electron microscopy studies were carried out on the blood and bone marrow cells of the first patient observed to have µ-chain disease. Since previously reported cases of γ and α heavy-chain disease did not elaborate any light chains, it was of interest to determine whether both the µ-chain fragments and the κ chains found in the serum of this patient originated in the same cell or whether mutations had affected two different clones. The use of rhodamine-conjugated and fluorescein-conjugated antisera to heavy and light chains respectively established that both antigenic determinants were present in the same cell. On electron microscopy, the plasma cells showed large vacuoles which appeared to form in the vicinity of the Golgi zone and frequently extended to the surface of the cell. It is postulated that the structural defect in the µ chain interferes with proper assembly of the gamma-globulin molecule. This in turn may preclude transport via the normal secretory pathway. The accumulated protein fragments may be released by a process of limited cytolysis.

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