scholarly journals Interleukin 1 beta is localized in the cytoplasmic ground substance but is largely absent from the Golgi apparatus and plasma membranes of stimulated human monocytes.

1988 ◽  
Vol 167 (2) ◽  
pp. 389-407 ◽  
Author(s):  
I I Singer ◽  
S Scott ◽  
G L Hall ◽  
G Limjuco ◽  
J Chin ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Interleukin 1 ◽  
Subcellular Location ◽  
Ground Substance ◽  
Outer Cell ◽  
Human Monocytes ◽  
Cytoplasmic Ground Substance

The subcellular location of IL-1 beta was determined using a postsectioning immunoelectron microscopic method on ultrathin frozen sections of human monocytes stimulated with LPS. This methodology permits access of antibody probes to all sectioned intracellular compartments, and their visualization at high resolution. Staining was performed with a rabbit antibody that specifically recognized amino acids 197-215 in the 33-kD IL-1 beta precursor molecule, followed by affinity-purified goat anti-rabbit IgG conjugated to 10 nm colloidal gold particles. Approximately 90% of the IL-1 beta antigens were localized in the ground substance of the cytoplasm at 4 or 20 h after activation, when both intracellular and extracellular accumulation of IL-1 beta was well underway. No significant IL-1 beta staining was observed on the outer cell membrane, nor within the lumens of the endoplasmic reticulum (ER), the Golgi apparatus, or secretory vesicles. In contrast, lysozyme was localized in the ER and dense secretory granules using these methods. Our results suggest that IL-1 beta is not anchored on the plasma membrane, and that its secretion occurs by a novel mechanism that does not use a secretory leader sequence, nor the classical secretory pathway involving the ER and Golgi apparatus.

scholarly journals The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

1995 ◽  
Vol 182 (5) ◽  
pp. 1447-1459 ◽  
Author(s):  
I I Singer ◽  
S Scott ◽  
J Chin ◽  
E K Bayne ◽  
G Limjuco ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Membrane ◽  
Interleukin 1 ◽  
Ground Substance ◽  
Human Monocytes ◽  
Interleukin 1 Beta ◽  
Converting Enzyme ◽  
Immuno Electron Microscopy ◽  
Ultrathin Cryosections ◽  
Cytoplasmic Ground Substance

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

scholarly journals Ferritin-Containing Bodies in Human Small Intestinal Epitheliuin

Blood ◽  
1963 ◽  
Vol 22 (4) ◽  
pp. 397-405 ◽  
Author(s):  
ROBERTA S. HARTMAN ◽  
MARCEL E. CONRAD ◽  
RICHARD E. HARTMAN ◽  
ROBERT J. T. JOY ◽  
WILLIAM H. CROSBY
Keyword(s):  
Golgi Apparatus ◽  
Inclusion Bodies ◽  
Iron Hydroxide ◽  
The Body ◽  
Ground Substance ◽  
Sharp Boundary ◽  
Apical Cytoplasm ◽  
Absorptive Cells ◽  
Small Intestinal ◽  
Cytoplasmic Ground Substance

Abstract Ferritin was identified in the absorptive cells of human jejunum from normal iron-replete subjects by the demonstration of the tetrad form of the iron hydroxide micelle of the ferritin molecule. In a few sections ferritin molecules were observed to be dispersed in the cytoplasm, but usually they were within inclusion bodies found in the apical cytoplasm. These ferritin-containing bodies were oval in profile and 0.5-1.5 µ long. They were not bounded by membrane but did have a moderately dense background substance which made a sharp boundary with the cytoplasmic ground substance. A few clues suggest that the body itself, but not necessarily its substance, is formed within the Golgi apparatus.

scholarly journals Production and characterization of monoclonal antibodies to insulin secretory granule membranes

1987 ◽  
Vol 245 (2) ◽  
pp. 557-566 ◽  
Author(s):  
K A Grimaldi ◽  
J C Hutton ◽  
K Siddle
Keyword(s):  
Monoclonal Antibodies ◽  
Secretory Granule ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Subcellular Location ◽  
Antigenic Determinants ◽  
Membrane Glycoproteins ◽  
Granule Membrane ◽  
Insulin Secretory Granule

Monoclonal antibodies to insulin secretory granule membranes were obtained following immunization of mice with granule membranes purified from a rat transplantable insulinoma. The specificities of the antibodies were investigated by using binding assays with different insulinoma subcellular fractions, by indirect immunofluorescence studies with intact and permeabilized cells, and by immunoblotting of granule membrane proteins fractionated by SDS/polyacrylamide-gel electrophoresis. Fifty-six antibodies were characterized initially, and 21 representative cell lines were cloned. The antibodies fell into four categories: (1) binding preferentially to secretory granules, and reacting with a component of approx. 80,000 Da on immunoblots (antigen designated SGM 80); (2) binding preferentially to secretory granules, and reacting with components of approx. 110,000 and 50,000 Da on immunoblots (antigen designated SGM 110); (3) binding preferentially to secretory granules but unreactive on immunoblots; (4) binding to membrane antigen(s) with a widespread intracellular distribution which included granules and plasma membranes. The antigens SGM 80 and SGM 110 were studied in more detail and both were shown to be integral membrane glycoproteins with antigenic determinants located on the internal face of the secretory granule membrane. These antigens were also present in normal rat islets of Langerhans and similar components were detected by immunoblotting in secretory granules from anterior pituitary and adrenal medulla. Proteins which were immunologically related to SGM 80 and SGM 110, but distinct in molecular size, were also identified in liver. It is concluded that secretory granules contain specific components which are restricted in subcellular location but widespread in tissue distribution. The antibodies obtained will be valuable reagents in the further investigation of the biogenesis and turnover of insulin secretory granules.

Three dimensional configuration of the secretory pathway and segregation of secretion granules in the yeast Saccharomyces cerevisiae

2001 ◽  
Vol 114 (12) ◽  
pp. 2231-2239 ◽  
Author(s):  
Alain Rambourg ◽  
Catherine L. Jackson ◽  
Yves Clermont
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Staining Intensity ◽  
Permissive Temperature ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Secretion Granules

The structural elements of the secretory pathway in the budding yeast Saccharomyces cerevisiae were analyzed by 3D stereo-electron microscopy using relatively thick sections in which membranes were selectively impregnated. In a wild-type strain, tubular networks of various sizes and staining properties were distributed throughout the cytoplasm. As a rule, wide-meshed, lightly stained polygonal networks were connected to more or less fenestrated sheets of endoplasmic reticulum (ER). Some of these networks were continuous with more intensely stained networks and narrower meshes that displayed at their intersections nodular dilations that progressively increased in size and staining properties to reach those of secretion granules. Such networks presumably corresponded to Golgi elements. Indeed, stacked cisternae typical of the mammalian Golgi apparatus are rarely found in wild-type cells. However, if it is assumed that the Golgi apparatus plays a key role in the segregation and maturation of secretion granules, then tubular networks with nodular dilations should be equivalent to parts of this organelle. In correlation with the increase in size and density of the nodules there was a decrease in diameter and staining intensity of the interconnecting tubules. These results parallel observations on the formation of secretory granules in mammalian cells and suggest that the segregation of secretory material is concomitant with the progressive perforation and tubulization of previously unperforated sheets. When the sec21-3 thermosensitive mutant was examined at the nonpermissive temperature (37°C), the secretory pathway was blocked at exit from the ER, which started to accumulate as clusters of narrow, anastomosed, unperforated ribbon-like elements. When the block was released by shifting down to permissive temperature (24°C), tubular networks of various sizes and caliber, presumably Golgi in nature, formed as soon as 5 minutes after release of the block. At later time intervals, granules of various sizes and densities appeared to be released by rupture of these tubular networks or even to form at the edges of ER fenestrae. These observations support a dynamic maturation process in which the formation of secretion granules occurs by means of an oriented series of membrane transformations starting at the ER and culminating with the liberation of secretion granules from Golgi networks.

Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells

1994 ◽  
Vol 107 (3) ◽  
pp. 539-549 ◽  
Author(s):  
C.S. Velez-Granell ◽  
A.E. Arias ◽  
J.A. Torres-Ruiz ◽  
M. Bendayan
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Tissue ◽  
Secretory Proteins ◽  
Trans Golgi Network ◽  
Hsp70 Protein ◽  
Golgi Network

Three chaperones, the chaperonins cpn10 and cpn60, and the hsp70 protein, were revealed by immunochemistry and cytochemistry in pancreatic rat acinar cells. Western immunoblotting analysis of rat pancreas homogenates has shown that antibodies against cpn10, cpn60 and hsp70 protein recognize single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpn10 and cpn60 were also detected in pancreatic juice. Immunofluorescence studies on rat pancreatic tissue revealed a strong positive signal in the apical region of the acinar cells for cpn10 and cpn60, while an immunoreaction was detected at the juxtanuclear Golgi region with the anti-hsp70 antibody. Immunocytochemical gold labeling confirmed the presence of these three chaperones in distinct cell compartments of pancreatic acinar cells. Chaperonin 10 and cpn60 were located in the endoplasmic reticulum, Golgi apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 followed the increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway. On the contrary, the labeling for hsp70 was mainly concentrated in the endoplasmic reticulum and the Golgi apparatus. In the latter, the hsp70 was found to be primary located in the trans-most cisternae and to colocalize with acid phosphatase in the trans-Golgi network. The three chaperones were also present in mitochondria. In view of the role played by the chaperones in the proper folding, sorting and aggregation of proteins, we postulate that hsp70 assists the adequate sorting and packaging of proteins from the ER to the trans-Golgi network while cpn10 and cpn60 play key roles in the proper packaging and aggregation of secretory proteins as well as, most probably, in the prevention of early enzyme activation in secretory granules.

Ultrastructure of midgut events in the pathogenesis of Bacillus sphaericus strain SSII-1 infections of Culex pipiens quinquefasciatus larvae

1979 ◽  
Vol 25 (2) ◽  
pp. 178-184 ◽  
Author(s):  
Elizabeth West Davidson
Keyword(s):  
Culex Pipiens ◽  
Bacillus Sphaericus ◽  
Wall Layer ◽  
Peritrophic Membrane ◽  
Ground Substance ◽  
Outer Cell ◽  
Bacterial Cells ◽  
Culex Pipiens Quinquefasciatus ◽  
Larval Host ◽  
Cytoplasmic Ground Substance

The fate of Bacillus sphaericus strain SSII-1 cells ingested by Culex pipiens quinquefasciatus (= C. pipiens fatigans, C. fatigans, C. quinquefasciatus of authors; Diptera: Culicidae) larvae and the cytological events preceding death of the host were observed using electron microscopy. Bacillus sphaericus cells were digested rapidly in the anterior and central midgut. The outer cell wall layer and cytoplasmic ground substance disappeared soon after ingestion. Cytolysosomes became prominent in midgut cells as these cells gradually separated from one another. All bacteria, including B. sphaericus, were confined within the peritrophic membrane until after death of the host. Digestion by the larval host is confirmed as a possible mechanism for release of B. sphaericus toxin from the bacterial cells.

scholarly journals The intracellular pathway of vitellogenin secretion in the frog hepatocyte as revealed by protein A-gold immunocytochemistry.

1984 ◽  
Vol 32 (7) ◽  
pp. 697-704 ◽  
Author(s):  
G H Herbener ◽  
M Bendayan ◽  
R C Feldhoff
Keyword(s):  
Golgi Apparatus ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Rana Catesbeiana ◽  
Secretory Granules ◽  
Plasma Concentrations ◽  
Protein A ◽  
Secretory Cells ◽  
Vitellogenin Synthesis ◽  
Cellular Compartments

The protein A-gold immunocytochemical technique was applied to the localization of vitellogenin in the hepatocyte of the bullfrog, Rana catesbeiana, eight days after treatment with estradiol-17 beta. Specific labeling was present in cellular compartments involved in protein secretion and was shown to progress in sequence through RER, Golgi apparatus, immature secretory granules, and mature secretory granules. Labeling intensities were quantitated and the values ranged from 34.6 to 172 gold particles/micron 2. In contrast, low background labeling was observed over mitochondria, nuclei, lipid droplets, and bile canaliculi. These observations support the hypothesis that vitellogenin synthesis and secretion in the frog hepatocyte lies exclusively along the RER-Golgi-granule secretory pathway. In addition to the cellular compartments involved in protein secretion, labeling was found over the majority of the lysosomes. The intensity of lysosomal labeling was intermediate between that of RER and Golgi apparatus. This labeling of lysosomes may be a consequence of the high blood plasma concentrations of vitellogenin that occur in the frog model, or to the well-known crinophagy phenomenon present in secretory cells.

scholarly journals High-resolution cytochemistry of neuraminic and hexuronic acid-containing macromolecules applying the enzyme-gold approach.

1988 ◽  
Vol 36 (8) ◽  
pp. 1005-1014 ◽  
Author(s):  
I Londoño ◽  
M Bendayan
Keyword(s):  
Golgi Apparatus ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Goblet Cells ◽  
Ultrastructural Level ◽  
Gold Complex ◽  
Thin Sections ◽  
Gold Particles ◽  
Tissue Sections ◽  
Apical Side

We localized acidic glycoconjugates at the ultrastructural level by applying the enzyme-gold approach. Neuraminidase and hyaluronidase were adsorbed to colloidal gold particles and applied to tissue sections under optimal conditions for their enzymatic activity. Neuraminidase-gold labeling was distributed over the Golgi apparatus and associated secretory granules in exocrine pancreatic cells and duodenal goblet cells. Mitochondria were labeled over inner membranes. Labeling was also found over the dispersed chromatin in the nucleus. Plasma membranes, particularly the apical side, were labeled by gold particles. On the other hand, incubation of tissue sections with the hyaluronidase-gold complex resulted in intense labeling of the rER membranes, the plasma membrane, and the dense chromatin in the nucleus. Labeling was also found over the Golgi apparatus and associated secretory granules, but only in duodenal goblet cells. Specificity of the results was confirmed by various control experiments performed, indicating that the enzyme-gold technique is useful for detecting linked-sugar residues on tissue thin sections. Labelings found over intra- and extracellular compartments in the present work are discussed in light of previous biochemical indications as well as of other histochemical detections of these glycoconjugates.

Organisation in the Structure of the Cytoplasmic Ground Substance

1978 ◽  
Vol 36 (3) ◽  
pp. 627-639
Author(s):  
K.R. Porter
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Random Distribution ◽  
Ground Substance ◽  
The Matrix ◽  
Cell Processes ◽  
Cytoplasmic Ground Substance

Most types of cells are known from their structure and overall form to possess a characteristic organization. In some instances this is evident in the non-random disposition of organelles and such system subunits as cisternae of the endoplasmic reticulum or the Golgi complex. In others it appears in the distribution and orientation of cytoplasmic fibrils. And in yet others the organization finds expression in the non-random distribution and orientation of microtubules, especially as found in highly anisometric cells and cell processes. The impression is unavoidable that in none of these cases is the organization achieved without the involvement of the cytoplasmic ground substance (CGS) or matrix. This impression is based on the fact that a matrix is present and that in all instances these formed structures, whether membranelimited or filamentous, are suspended in it. In some well-known instances, as in arrays of microtubules which make up axonemes and axostyles, the matrix resolves itself into bridges (and spokes) between the microtubules, bridges which are in some cases very regularly disposed and uniform in size (Mcintosh, 1973; Bloodgood and Miller, 1974; Warner and Satir, 1974).

scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

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