Transport of insulin and albumin by the microvascular endothelium of the rete mirabile

1996 ◽  
Vol 109 (7) ◽  
pp. 1857-1864 ◽  
Author(s):  
M. Bendayan ◽  
E.A. Rasio
Keyword(s):  
Serum Proteins ◽  
Protein A ◽  
Simultaneous Detection ◽  
Intercellular Junctions ◽  
Buffer Solution ◽  
Microvascular Endothelium ◽  
Double Labeling ◽  
Plasmalemmal Vesicles ◽  
Rete Mirabile ◽  
Paracellular Diffusion

Vascular permeability for albumin and insulin in the continuous capillary network of the rete mirabile of the eel swimbladder was evaluated by ultrastructural immunocytochemistry and countercurrent perfusion experiments. Upon perfusion of the rete capillaries with a buffer solution containing albumin and insulin, these serum proteins were revealed at the electron microscope level, by the Protein A-gold immunocytochemical technique on a post-embedding step. For the simultaneous detection of both proteins, the double labeling technique with different sized gold particles was used. Furthermore, labeling was performed with the mixture of anti-albumin and anti-insulin anti-bodies. The labelings obtained were morphometrically evaluated and demonstrate that: (1) serum proteins such as albumin and insulin are transported by the endothelial cells through their plasmalemmal vesicular system; (2) insulin is transported preferentially to albumin; and (3) this transport involves different populations of plasmalemmal vesicles. Measurements of diffusion permeability coefficients have confirmed the preferential transport of insulin, its coefficient being higher than that of albumin. Conversely, when compared to that of insulin or sucrose, which are assumed to be markers of the paracellular diffusion, it was found to be much lower, indicating that transcytosis through the vesicular system is less efficient than diffusion along the intercellular junctions. These results indicate that transcytosis of insulin and albumin occurs via different sets of plasmalemmal vesicles, probably through receptor-mediated mechanisms, and that the overall rate of transport across the rete capillaries, with respect to paracellular diffusion, is higher for insulin than for albumin.

scholarly journals Binding and transcytosis of glycoalbumin by the microvascular endothelium of the murine myocardium: evidence that glycoalbumin behaves as a bifunctional ligand.

1988 ◽  
Vol 107 (5) ◽  
pp. 1729-1738 ◽  
Author(s):  
D Predescu ◽  
M Simionescu ◽  
N Simionescu ◽  
G E Palade
Keyword(s):  
Intercellular Junctions ◽  
Concomitant Administration ◽  
Multivesicular Bodies ◽  
Microvascular Endothelium ◽  
Coated Pits ◽  
Gold Complexes ◽  
Plasmalemmal Vesicles ◽  
Bifunctional Ligand ◽  
Albumin Molecule

The binding and transport of glycoalbumin (gA) by the endothelium of murine myocardial microvessels were studied by perfusing in situ 125I-gA or gA-gold complexes (gA-Au) and examining the specimens by radioassays and EM, respectively. After a 3-min perfusion, the uptake of radioiodinated gA is 2.2-fold higher than that of native albumin; it is partially (approximately 55%) competed by either albumin or D-glucose, and almost completely abolished by the concomitant administration of both competitors or by gA. D-mannose and D-galactose are not effective competitors. Unlike albumin-gold complexes that bind restrictively to plasmalemmal vesicles, gA-Au labels the plasma-lemma proper, plasmalemmal vesicles open on the lumen, and most coated pits. Competing albumin prevents gA-Au binding to the membrane of plasmalemmal vesicles, while glucose significantly reduces the ligand binding to plasmalemma proper. Competition with albumin and glucose gives additive effects. Transcytosis of gA-Au, already detected at 3 min, becomes substantial by 30 min. No tracer exit via intercellular junctions was detected. gA-Au progressively accumulates in multivesicular bodies. The results of the binding and competition experiments indicate that the gA behaves as a bifunctional ligand which is recognized by two distinct binding sites: one, located on the plasma membrane, binds as a lectin the glucose residues of gA; whereas the other, confined to plasmalemmal vesicles, recognizes presumably specific domains of the albumin molecule.

Plasmalemmal vesicles represent the large pore system of continuous microvascular endothelium

1993 ◽  
Vol 265 (2) ◽  
pp. H725-H733 ◽  
Author(s):  
D. Predescu ◽  
G. E. Palade
Keyword(s):  
Intercellular Junctions ◽  
Pore System ◽  
Microvascular Endothelium ◽  
Plasmalemmal Vesicles ◽  
Morphological Studies ◽  
Large Pore ◽  
In Transit

In the capillary physiology literature, molecules and particles larger than 10 nm are assumed to leave the plasma mostly through large pores located at the level of intercellular junctions in microvessels lined with a continuous endothelium. In morphological studies of similar microvessels, outgoing particles > 10 nm were detected in endothelial plasmalemmal vesicles not in intercellular junctions. Because the probes may not be found in transit through the junctions because they may be swept away by strong currents generated by Starling forces, we have examined a large number of junctions in arteriolar, capillary, and venular segments of bipolar vascular fields of mouse diaphragms collected after perfusion with large pore probes. The results presented in this study indicate that 1) the perfused probes accumulate in the luminal introits of the junctions as filtration residues that decrease in size and frequency from arterioles to venules, and 2) large pore probes move across the endothelium exclusively through plasmalemmal vesicles.

scholarly journals Evidence of a Tubular System for Transendothelial Transport in Arterial Capillaries of the Rete Mirabile

1997 ◽  
Vol 45 (10) ◽  
pp. 1365-1378 ◽  
Author(s):  
Moöse Bendayan ◽  
Eugenio A. Rasio
Keyword(s):  
Electron Microscopy ◽  
Serum Proteins ◽  
Protein A ◽  
Freeze Fracture ◽  
Thin Sections ◽  
Functional Studies ◽  
Transendothelial Transport ◽  
Rete Mirabile ◽  
Osmium Impregnation ◽  
Membrane Bound

The arterial endothelial cells of the rete capillaries of the eel were examined by transmission electron microscopy on thin sections, on freeze-fracture replicas, by scanning electron microscopy, after cytochemical osmium impregnation and perfusion with peroxidase. The study revealed the existence of membrane-bound tubules and vesicles that open at both the luminal and abluminal poles of the cell and at the level of the intercellular space. The tubules are straight or present successive dilations and constrictions. They branch in various directions and intrude deeply into the cell cytoplasm, forming a complex tubular network within the cell. Immunocytochemical techniques were applied on immersion-fixed tissues and on perfusion of the capillaries with albumin and insulin. These demonstrated that the tubular–vesicular system is involved in the transport of circulating proteins. Furthermore, protein A–gold immunocytochemistry has revealed the association of actin with the membranes of this system. On the basis of these results, we suggest that the transendothelial transport of serum proteins takes place by a transcytotic process through a membrane-bound tubular–vesicular system and is equivalent to the large pore system presumed from functional studies.

scholarly journals A double-immunostaining procedure using colloidal gold, ferritin, and peroxidase as markers for simultaneous detection of two hypophysial hormones with the electron microscope.

1984 ◽  
Vol 32 (11) ◽  
pp. 1159-1166 ◽  
Author(s):  
J Doerr-Schott ◽  
C M Lichte
Keyword(s):  
Electron Microscope ◽  
Protein A ◽  
Simultaneous Detection ◽  
Ultrastructural Localization ◽  
Double Immunostaining ◽  
Double Labeling ◽  
Gold Particles ◽  
Ultrathin Sections ◽  
Species Specific ◽  
Simultaneous Visualization

For simultaneous ultrastructural localization of intracellular peptides, protein A-gold techniques or immuno-gold techniques have generally been applied. The present study reports a double immunostaining procedure for simultaneous visualization of two hypophysial hormones (prolactin and corticotropin) on a single ultrathin section of the pars distalis of an amphibian. Prolactin and corticotropin antisera were respectively raised in guinea pigs and rabbits and were applied simultaneously to ultrathin sections. Antigenic binding sites were detected under the electron microscope using differently labeled species-specific secondary antisera raised in goats or sheep. Three labels (gold particles, ferritin, peroxidase) were checked for double labeling. The combinations investigated were: 1) two gold preparations or IgG-gold labeled with different-sized gold particles; 2)IgG-gold and IgG-ferritin; 3) IgG-gold and IgG-PAP (peroxidase-antiperoxidase). The double-immunostaining procedures described here have proved useful in the simultaneous ultrastructural localizations of two intracellular antigens on a single tissue section. These procedures constitute a basis for the development of triple immunostaining methods.

scholarly journals Surfactant protein D binding to alveolar macrophages

1994 ◽  
Vol 300 (1) ◽  
pp. 237-242 ◽  
Author(s):  
K Miyamura ◽  
L E A Leigh ◽  
J Lu ◽  
J Hopkin ◽  
A López Bernal ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Serum Proteins ◽  
Specific Binding ◽  
Lectin Binding ◽  
Protein A ◽  
Direct Interaction ◽  
Surfactant Protein ◽  
Lung Epithelial Cells ◽  
Surfactant Protein D ◽  
Apparent Dissociation Constant

Surfactant protein D (SP-D) is a lung-specific protein, synthesized and secreted by lung epithelial cells. It belongs to group III of the family of C-type lectins; each member of this group has an unusual overall structure consisting of multiple globular ‘head’ regions (which contain the C-type lectin domains) linked by triple-helical, collagen-like, strands. This group includes the surfactant protein A (SP-A) and the serum proteins mannan-binding protein, conglutinin and collectin-43, all of which have been shown to bind to the C1q receptor found on a wide variety of cells, including macrophages. Both SP-D and SP-A have been shown to enhance oxygen radical production by alveolar macrophages. Although this strongly suggests a direct interaction between SP-D and a specific receptor on alveolar macrophages, it is still unclear whether SP-D binds to the same receptor used by SP-A and/or C1q. Human SP-D was isolated from amniotic fluid and was radiolabelled using 125I. Alveolar macrophages were isolated from human bronchioalveolar lavage fluid, and also from bovine lung washings, by differential adhesion to 24-well tissue-culture plates. The study was carried out using EDTA-containing buffers, to eliminate Ca(2+)-dependent C-type lectin binding, and was also carried out at 4 degrees C to eliminate possible internalization by the cells. 125I-SP-D showed specific binding to alveolar macrophages in both a time- and concentration-saturable manner. The binding was inhibited, by approx. 90%, on addition of a 200-fold excess of unlabelled SP-D. The apparent dissociation constant (Kd) was (3.6 +/- 1.3) x 10(-11) M, based on the assumption that native SP-D is assembled as a dodecamer of 12 identical polypeptides of 43 kDa to yield a protein of 516 kDa. C1q was also shown to bind alveolar macrophages (Kd 3 x 10(-6) M), but addition of C1q did not show inhibition of the binding of 125I-SP-D to the macrophages. We conclude that SP-D binds specifically to alveolar macrophages and the receptor involved is different from that utilized by C1q.

scholarly journals Development of QCM Biosensor with Specific Cow Milk Protein Antibody for Candidate Milk Adulteration Detection

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Setyawan P. Sakti ◽  
Nur Chabibah ◽  
Senja P. Ayu ◽  
Masdiana C. Padaga ◽  
Aulanni’am Aulanni’am
Keyword(s):  
Milk Protein ◽  
Protein A ◽  
Goat Milk ◽  
Specific Protein ◽  
Cow Milk ◽  
Frequency Change ◽  
Milk Product ◽  
Reaction Cell ◽  
Buffer Solution ◽  
Milk Adulteration

Adulteration of goat milk is usually done using cow’s milk product. Cow milk is used as it is widely available and its price is cheaper compared to goat milk. This paper shows a development of candidate tools for milk adulteration using cow milk. A quartz crystal microbalance immunosensor was developed using commercial crystal resonator and polyclonal antibody specific to cow milk protein. A specific protein at 208 KDa is found only in cow milk and does not exist in goat milk. The existence of this protein can be used as an indicator of cow milk content in a target solution. To detect the PSS 208 kDa protein, antibody specific to the PSS 208 was developed. The purified antibody was immobilized on top of the sensor surface on a polystyrene layer. The fraction of the immobilized antibody on the sensor was found at 1.5% of the given antibody. Using a static reaction cell, the developed immunosensor could detect the specific cow milk protein in buffer solution. The detection limit is 1 ppm. A linear relationship between frequency change and specific protein of cow milk concentration is found from a concentration of 1 ppm to 120 ppm.

scholarly journals THE OCCURRENCE DURING ACUTE INFECTIONS OF A PROTEIN NOT NORMALLY PRESENT IN THE BLOOD

1947 ◽  
Vol 85 (5) ◽  
pp. 491-498 ◽  
Author(s):  
Maclyn McCarty
Keyword(s):  
Infectious Diseases ◽  
Early Phase ◽  
Serum Proteins ◽  
Protein A ◽  
Normal Serum ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Acute Infections

A procedure is described for the isolation and crystallization from human serous fluids of the C-reactive protein, a substance which appears in the blood especially in the early phase of certain acute infectious diseases. Immunological studies confirm earlier work in showing that the protein is highly antigenic and serologically specific, and demonstrate that crystallization of the protein effectively separates it from normal serum proteins.

scholarly journals Double immunocytochemical labeling applying the protein A-gold technique.

1982 ◽  
Vol 30 (1) ◽  
pp. 81-85 ◽  
Author(s):  
M Bendayan
Keyword(s):  
Tissue Section ◽  
Protein A ◽  
Antigenic Site ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Double Labeling ◽  
Gold Particles ◽  
Antigenic Sites

In the present study we report the modifications and the different steps of the protein A-gold (pAg) technique that allow the simultaneous demonstration of two antigenic sites on the same tissue section. The labeling is carried out in the following manner: face A of the tissue section is incubated with an antiserum followed by a pAg complex prepared with large gold particles; face B of the same tissue section is then incubated with a second antiserum followed by a pAg complex prepared with small gold particles. Each of the pAg complexes reveals a different antigenic site on opposite faces of the tissue section. The transparency of the section in the electron beam allows the visualization of the gold particles present on both faces. The double labeling pAg technique was applied for the simultaneous demonstration of two secretory proteins in the same Golgi, condensing vacuoles, and zymogen granules of the rat pancreatic acinar cells.

scholarly journals Double labeling of SIgG+ cells harboring a lymphotropic herpesvirus by avidin-biotin complex immunoperoxidase and immunogold-silver staining techniques.

1988 ◽  
Vol 36 (9) ◽  
pp. 1187-1189 ◽  
Author(s):  
Z Nagy-Oltvai ◽  
T Brady ◽  
G D Hsiung
Keyword(s):  
Cell Surface ◽  
Silver Staining ◽  
Simultaneous Detection ◽  
Double Labeling ◽  
Tissue Sections ◽  
Surface Immunoglobulin ◽  
Staining Techniques ◽  
Double Immunolabeling ◽  
Usual Order ◽  
Avidin Biotin Complex

By reversing the usual order of double-staining procedures, we obtained optimal conditions for simultaneous detection of guinea pig lymphotropic herpesvirus (GPHLV) and surface immunoglobulin G-positive (SIgG+) cells in paraffin-embedded tissue sections. Of the different fixatives tested, Bouin's solution gave the best preservation of morphology and cell surface IgG. Double immunolabeling was best achieved when avidin-biotin complex immunoperoxidase (ABC-IP) staining was performed first for detection of intracellular viral antigen, followed by immunogold-silver staining (IGSS) for localization of cell surface IgG.

Pattern Analysis Meets Cell Biology

1999 ◽  
Vol 5 (S2) ◽  
pp. 510-511
Author(s):  
Robert F. Murphy ◽  
Michael V. Boland
Keyword(s):  
Cell Biology ◽  
Fluorescent Dyes ◽  
Protein A ◽  
Chinese Hamster ◽  
Subcellular Location ◽  
Computer Applications ◽  
Double Labeling ◽  
A Cell ◽  
Widespread Availability

The widespread availability of automated fluorescence microscope systems has led to an explosion in the acquisition of digital images by biologists. This has created a need for computer applications that automate the analysis of these images and an opportunity to develop new approaches to classical problems. An example is the determination of the subcellular location of a protein from immunofluorescence images (or, more recently, images of GFP fluorescence). Current practice is to compare such images to mental images that a cell biologist has developed over time, and to reach a tentative conclusion about the structure (i.e., organelle) that a protein is found in. Since this determination is subjective, it often must be followed up by double labeling with a marker protein from the suspected structure.As an initial exploration of the feasibility of automating the determination of subcellular location, we developed a system that is able to classify the localization patterns characteristic of five cellular molecules (proteins and DNA) in Chinese Hamster Ovary (CHO) cells. Images were acquired on an epifluorescence microscope after the cells had been fixed, permeabilized, and labeled with appropriate fluorescent reagents (usually antibodies conjugated to fluorescent dyes). The labels used were directed against a Golgi protein, a lysosomal protein, a nuclear protein, a cytoskeletal protein, and DNA.

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