Demonstration of fluid-phase endocytosis in epithelial cells of the male reproductive system by means of horseradish peroxidase-colloidal gold complex

1983 ◽  
Vol 230 (3) ◽  
pp. 503-510 ◽  
Author(s):  
C. Morales ◽  
L. Hermo
Keyword(s):  
Epithelial Cells ◽  
Horseradish Peroxidase ◽  
Reproductive System ◽  
Colloidal Gold ◽  
Fluid Phase ◽  
Male Reproductive System ◽  
Gold Complex ◽  
Fluid Phase Endocytosis

Hepatocyte horseradish peroxidase uptake is saturable and inhibited by mannose-terminal glycoproteins

1993 ◽  
Vol 264 (5) ◽  
pp. G880-G885 ◽  
Author(s):  
Y. Yamaguchi ◽  
E. Dalle-Molle ◽  
W. G. Hardison
Keyword(s):  
Horseradish Peroxidase ◽  
Parenchymal Cell ◽  
Fluid Phase ◽  
Mannose Receptor ◽  
Isolated Hepatocytes ◽  
Cell Counting ◽  
Cell Fractionation ◽  
Morphological Studies ◽  
Fluid Phase Endocytosis ◽  
Peroxidase Uptake

In the liver, horseradish peroxidase (HRP) is thought to be taken up via mannose receptor-mediated endocytosis by non-parenchymal cells (NPC) and via fluid-phase endocytosis by hepatocytes. When we attempted to inhibit NPC uptake of HRP with mannan in the whole perfused rat liver, > 80% of HRP uptake was eliminated. Liver cell fractionation revealed that mannan not only inhibited HRP uptake by NPC (91%) but also by hepatocytes (81%). In isolated hepatocytes, HRP uptake was linear over 60 min and saturable in the range of 0 to 200 mg/l (Vmax = 4.3 ng.mg protein-1.min-1; Km = 8.3 mg/l). Mannan inhibited uptake competitively (Ki = 2.0-2.5 mg/l). At high concentrations of HRP, a nonsaturable component of HRP uptake became evident (k = 2.8 pg.mg protein-1.min-1.mg HRP-1.l-1). Hepatocyte uptake of HRP was inhibited by other glycoproteins and glycopeptides with mannose-terminal groups, as well as by mannan, but not by asialofetuin (ASF) or bovine serum albumin. Hepatocyte uptake of 125I-labeled ASF, which is taken up via the asialoglycoprotein receptor, was saturable and not inhibited by mannan. HRP binding to hepatocytes, determined at 4 degrees C, was also inhibited by mannan. Quantification of contamination of the parenchymal cell fraction by NPC by cell counting and by pronase digestibility suggested our results could not be explained by contamination of hepatocytes by NPC. At concentrations used for most morphological studies (1,000-10,000 mg/l), fluid-phase endocytosis accounts for much of HRP uptake. However, at low concentrations, a saturable low-capacity mechanism is responsible for most HRP uptake by the hepatocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of colchicine on endocytosis of horseradish peroxidase by rat peritoneal macrophages

1980 ◽  
Vol 45 (1) ◽  
pp. 59-71 ◽  
Author(s):  
A. Piasek ◽  
J. Thyberg
Keyword(s):  
Horseradish Peroxidase ◽  
Digestive Enzymes ◽  
Cytochalasin B ◽  
Fluid Phase ◽  
Peritoneal Macrophages ◽  
The Other ◽  
Fluid Phase Endocytosis ◽  
Cytoplasmic Microtubules ◽  
Endocytic Vesicles ◽  
Cytoplasmic Complex

Horseradish peroxidase (HRP) was used as an exogenous marker to study the effects of microtubule-disruptive drugs on endocytosis in cultures of thioglycollate-elicited rat peritoneal macrophages. Colchicine and vinblastine, but not lumicolchicine or cytochalasin B, reduced HRP uptake by about 30–40%. However, as determined by stereological measurements, the size of the HRP-containing compartment within the cells remained unaltered. In both control cells and cells treated with colchicine or vinblastine the HRP-reactive vesicles were preferentially located close to the dictyosomes (stacks of cisternae) despite the fact that the Golgi complex was disorganized in the treated cells. These results suggest that intact cytoplasmic complex was disorganized in the treated cells. These results suggest that intact cytoplasmic microtubules are required to maintain a normal rate of fluid phase endocytosis in macrophages. On the other hand, it seems as if microtubules are not necessary for the translocation of newly formed endocytic vesicles/lysosomes to the dictyosomes, from which they probably are supplied with digestive enzymes.

scholarly journals Okadaic acid induces Golgi apparatus fragmentation and arrest of intracellular transport

1991 ◽  
Vol 100 (4) ◽  
pp. 753-759 ◽  
Author(s):  
J. Lucocq ◽  
G. Warren ◽  
J. Pryde
Keyword(s):  
Golgi Apparatus ◽  
Horseradish Peroxidase ◽  
Vesicular Stomatitis Virus ◽  
Okadaic Acid ◽  
Intracellular Transport ◽  
Fluid Phase ◽  
Fluid Phase Endocytosis ◽  
Vesicular Stomatitis ◽  
Dose Dependent ◽  
Mitotic Cells

The specific phosphatase inhibitor okadaic acid (OA) induced fragmentation of the Golgi apparatus in interphase HeLa cells. Immunoelectron microscopy for galactosyltransferase identified a major Golgi fragment composed of a cluster of vesicles and tubules that was morphologically indistinguishable from the ‘Golgi cluster’ previously described in mitotic cells. The presence of homogeneous immunofluorescence staining for galactosyltransferase in OA-treated cells also suggested that isolated Golgi vesicles, previously found in mitotic cells, existed along with the clusters. After removal of OA, both clusters and vesicles appeared to participate in a reassembly pathway that strongly resembled that occurring during telophase. OA also induced inhibition of intracellular transport, another feature of mitotic cells. OA treatment prevented newly synthesised G protein of vesicular stomatitis virus (VSV) from acquiring resistance to endoglycosidase H and from arriving at the cell surface. In addition, fluid phase endocytosis of horseradish peroxidase (HRP) was reduced to less than 10% of control values. All these effects were dose-dependent and reversible. OA should be a useful tool to study the Golgi division and membrane traffic.

Ultrastructure of the male reproductive system of the free-living marine nematode Paracyatholaimus pugettensis Wieser & Hopper, 1967 (Chromadorida: Cyatholaimidae)

Nematology ◽  
2010 ◽  
Vol 12 (2) ◽  
pp. 255-268 ◽  
Author(s):  
Julia K. Zograf
Keyword(s):  
Epithelial Cells ◽  
Seminal Vesicle ◽  
Reproductive System ◽  
Vas Deferens ◽  
Proximal Part ◽  
Male Reproductive System ◽  
Synthetic Activity ◽  
The Sea Of Japan ◽  
Marine Nematode ◽  
Free Living

AbstractAlthough nematodes are a well studied group of multicellular organisms, until now the only information on the cellular structure of the male reproductive system of marine nematodes is that on the histology of free-living marine nematode from the order Enoplida. The fine structure of the male reproductive system of the free-living marine nematode Paracyatholaimus pugettensis (Chromadorida: Cyatholaimidae) from the Sea of Japan has been studied using TEM. The testis epithelium has a large distal tip cell similar to that described for representatives of the subclass Rhabditia. The epithelial wall of the testis is differentiated along its length. The proximal part of the epithelial tube consists of relatively large cells bearing numerous surface outgrowths that permeate between the developing spermatocytes. The epithelium in the middle region of the testis is formed from extremely flattened cells. The distal part of the testis – the seminal vesicle – is filled with immature spermatozoa and consists of absorptive cells. The seminal vesicle is followed by the vas deferens. The gonoduct is also differentiated along its length, the first third being formed from synthetically active epithelial cells, the two layers of which form a tiled structure. There is no lumen in the gonoduct and it is probable that, due to the tiled structure, the epithelial cells move apart to create space for the spermatozoa during ejaculation. The posterior two-thirds of the duct is surrounded by muscle cells that create the necessary pressure during ejaculation. The enlarged epithelial cells of the vas deferens show vigorous synthetic activity, which is probably involved in the transformation of immature spermatozoa into mature gametes.

scholarly journals Fluid-phase endocytosis by intrahepatic bile duct epithelial cells isolated from normal rat liver.

1990 ◽  
Vol 38 (4) ◽  
pp. 515-524 ◽  
Author(s):  
M Ishii ◽  
B Vroman ◽  
N F LaRusso
Keyword(s):  
Acid Phosphatase ◽  
Bile Duct ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Rat Liver ◽  
Intrahepatic Bile Duct ◽  
Fluid Phase ◽  
Fluid Phase Endocytosis ◽  
Normal Rat ◽  
Secondary Lysosomes

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.

The Application of Protein A-Gold Immunocytochemistry to Studies of Protein Secretion

1985 ◽  
Vol 43 ◽  
pp. 426-429
Author(s):  
George H. Herbener ◽  
Antonio Nanci ◽  
Moise Bendayan
Keyword(s):  
Tissue Section ◽  
Colloidal Gold ◽  
Unit Area ◽  
Protein A ◽  
Extracellular Proteins ◽  
Gold Complex ◽  
Second Step ◽  
Igg Antibodies ◽  
Tissue Sections ◽  
Antigenic Sites

Protein A-gold immunocytochemistry is a two-step, post-embedding labeling procedure which may be applied to tissue sections to localize intra- and extracellular proteins. The key requisite for immunocytochemistry is the availability of the appropriate antibody to react in an immune response with the antigenic sites on the protein of interest. During the second step, protein A-gold complex is reacted with the antibody. This is a non- specific reaction in that protein A will combine with most IgG antibodies. The ‘label’ visualized in the electron microscope is colloidal gold. Since labeling is restricted to the surface of the tissue section and since colloidal gold is particulate, labeling density, i.e., the number of gold particles per unit area of tissue section, may be quantitated with ease and accuracy.

Comparative strengths and weaknesses of peroxidase and colloidal gold in pre- and post-embedding immunolabeling techniques

1987 ◽  
Vol 45 ◽  
pp. 1002-1005
Author(s):  
D. R. Abrahamson ◽  
P. L. St.John ◽  
E. W. Perry
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Light Microscopy ◽  
Colloidal Gold ◽  
Light Microscope ◽  
Ultrastructural Localization ◽  
The Other ◽  
Quantitative Studies ◽  
Dense Suspension ◽  
Antigen Localization

Antibodies coupled to tracers for electron microscopy have been instrumental in the ultrastructural localization of antigens within cells and tissues. Among the most popular tracers are horseradish peroxidase (HRP), an enzyme that yields an osmiophilic reaction product, and colloidal gold, an electron dense suspension of particles. Some advantages of IgG-HRP conjugates are that they are readily synthesized, relatively small, and the immunolabeling obtained in a given experiment can be evaluated in the light microscope. In contrast, colloidal gold conjugates are available in different size ranges and multiple labeling as well as quantitative studies can therefore be undertaken through particle counting. On the other hand, gold conjugates are generally larger than those of HRP but usually can not be visualized with light microscopy. Concern has been raised, however, that HRP reaction product, which is exquisitely sensitive when generated properly, may in some cases distribute to sites distant from the original binding of the conjugate and therefore result in spurious antigen localization.

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