scholarly journals Differentiated microdomains on the luminal surface of the capillary endothelium. II. Partial characterization of their anionic sites.

1981 ◽  
Vol 90 (3) ◽  
pp. 614-621 ◽  
Author(s):  
M Simionescu ◽  
N Simionescu ◽  
J E Silbert ◽  
G E Palade
Keyword(s):  
Binding Sites ◽  
Chemical Nature ◽  
Capillary Endothelium ◽  
Anionic Sites ◽  
Coated Pits ◽  
Chondroitinase Abc ◽  
Mouse Pancreas ◽  
Broad Specificity

To investigate the chemical nature of the cationic ferritin (CF)-binding sites of the differentiated microdomains of the capillary endothelium, the vasculature of the mouse pancreas and intestinal mucosa was perfused in situ with neuraminidase, hyaluronidase, chondroitinase ABC, heparinase, and three proteases: trypsin, papain, and pronase. Proteases of broad specificity removed all anionic sites, suggesting that the latter are contributed by acid glycoproteins or proteoglycans. Neuraminidase, hyaluronidase, and chondroitinase ABC reduced the density of CF-binding sites on the plasmalemma proper, but had no effect on either coated pits or fenestral diaphragms. Heparinase removed CF-binding sites from fenestral diaphragms and had no effect on coated pits. Taken together, these results indicate that the anionic sites of the fenestral diaphragms are contributed primarily by heparan sulfate and/or heparin, whereas those of the plasmalemma proper are of mixed chemical nature. The membranes and diaphragms of plasmalemmal vesicles and transendothelial channels do not bind CF in control specimens; this condition is not affected by the enzymic treatments mentioned above.

scholarly journals Differentiated microdomains on the luminal surface of capillary endothelium: distribution of lectin receptors.

1982 ◽  
Vol 94 (2) ◽  
pp. 406-413 ◽  
Author(s):  
M Simionescu ◽  
N Simionescu ◽  
G E Palade
Keyword(s):  
Binding Sites ◽  
Ricinus Communis ◽  
Capillary Endothelium ◽  
Anionic Sites ◽  
Lectin Receptors ◽  
Fenestrated Endothelium ◽  
Phosphate Buffered Saline ◽  
Soybean Lectin ◽  
Galactosyl Residues

Lectins conjugated with either peroxidase or ferritin were used to detect specific monosaccharide residues on the luminal front of he fenestrated endothelium in the capillaries of murine pancreas and intestinal mucosa. The lectins tested recognize, if accessible, the following residues: alpha-N-acetylgalactosaminyl (soybean lectin), beta-D-galactosyl (peanut agglutinin [PA] and Ricinus communis agglutinin-120 [RCA]), beta-N-acetylglucosaminyl and sialyl residues (wheat germ agglutinin [WGA]), alpha-L-fucosyl (lotus tetragonolobus lectin), and alpha-D-glucosyl and beta-D-mannosyl (concanavalin A [ConA]). Thi labeled lectins were introduced by perfusion in situ after thoroughly flushing with phosphate-buffered saline the microvascular beds under investigation. Specimens were fixed by perfusion, and subsequently processed for peroxidase detection and electron microscopy. Control experiments included perfusion with: (a) unlabeled lectin before lectin conjugate; (b) labeled lectin together with the cognate hapten sugar, and (c) horseradish peroxidase or ferritin alone. Binding sites were found to be relatively homogeneously distributed on the plasmalemma proper, except for Lotus tetragonolobus lectin and Con A, which frequently bound in patches. Plasmalemmal vesicles, transendothelial channels, and their associated diaphragms were particularly rich in residues recognized by RCA and PA (beta-D-galactosyl residues) and by WGA (beta-N-acetylglucosaminyl residues). Receptors for all lectins tested appeared to be absent or considerably less concentrated on fenestral diaphragms. The results reported here extend and complement previous findings on the existence of microdomains generated by the preferential distribution of chemically different anionic sites (Simionescu et al., 1981, J. Cell Biol., 9:605-613 and 614-621).

scholarly journals Preferential distribution of anionic sites on the basement membrane and the abluminal aspect of the endothelium in fenestrated capillaries.

1982 ◽  
Vol 95 (2) ◽  
pp. 425-434 ◽  
Author(s):  
M Simionescu ◽  
N Simionescu ◽  
G E Palade
Keyword(s):  
Basement Membrane ◽  
Cationized Ferritin ◽  
Anionic Sites ◽  
Planar Lattice ◽  
Coated Pits ◽  
Mouse Pancreas ◽  
Collagenase Treatment ◽  
Fenestrated Capillaries ◽  
Collagenase Perfusion ◽  
Pronase Treatment

Cationized ferritin (CF) was injected interstitially to study the distribution of anionic sites on the basement membrane and abluminal aspect of the endothelium in the fenestrated capillaries of the mouse pancreas and intestinal mucosa. Extensive, but uneven removal of the basement membrane was obtained by collagenase perfusion of the vasculature before CF labeling. In the absence of collagenase treatment, CF label was essentially restricted to the lamina rara externa of the basement membrane and occurred in clusters distributed in a relatively ordered planar lattice. After collagenase digestion, labeling of the lamina rara interna and of the abluminal aspect of the endothelium became possible. In the lamina rara interna, the CF label occurred in clusters with a distribution comparable to that found in the lamina rara externa. On the abluminal aspect of the endothelium, the plasmalemma proper was extensively, though variably, labeled. Coated pits were heavily labeled, whereas the membranes and stomatal diaphragms of plasmalemmal vesicles and transendothelial channels remained free of CF decoration. In contradistinction with the heavy labeling of their luminal aspects, the abluminal surface of the fenestral diaphragms were free of any CF decoration. Pronase treatment removed all anionic sites detectable by CF binding. The findings establish the existence of differentiated microdomains on the abluminal aspect of the endothelial plasmalemma and suggest that the capillary wall selects permeant macromolecules according to charge, in addition to size.

scholarly journals Rings of membrane sterols surround the openings of vesicles and fenestrae, in capillary endothelium.

1983 ◽  
Vol 97 (5) ◽  
pp. 1592-1600 ◽  
Author(s):  
N Simionescu ◽  
F Lupu ◽  
M Simionescu
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Cell Surface ◽  
Freeze Fracture ◽  
Short Exposure ◽  
Capillary Endothelium ◽  
Anionic Sites ◽  
Microvascular Endothelium ◽  
Coated Pits ◽  
Plasmalemmal Vesicles

We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta-hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin-sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage of their fusion/fission process.

Cloning and characterization of a human leptin receptor using a biologically active leptin immunoadhesin

1997 ◽  
Vol 18 (1) ◽  
pp. 77-85 ◽  
Author(s):  
S-M Luoh ◽  
F Di Marco ◽  
N Levin ◽  
M Armanini ◽  
M-H Xie ◽  
...  
Keyword(s):  
Body Weight ◽  
Binding Sites ◽  
Leptin Receptor ◽  
Short Form ◽  
Biologically Active ◽  
Expression Cloning ◽  
Kidney Cells ◽  
Long Form

ABSTRACT Leptin, the product of the ob gene, is a hormone secreted by fat cells which is primarily involved in the regulation of body weight. We have generated a leptin immunoadhesin (leptin-IgG) which was more potent than leptin alone at reducing body weight and food intake when injected into ob/ob mice. This molecule was used to identify high affinity binding sites on human embryonic 293 kidney cells and subsequently to isolate a cDNA encoding the leptin receptor from this cell line by expression cloning. This receptor corresponds to the short form of the recently isolated leptin receptor. Analysis of the expression pattern of the two forms of receptor by Northern blot, in situ hybridization and quantitative PCR showed that the receptor is expressed in most tissues but that the long form is prevalent in the hypothalamus.

scholarly journals Differentiated microdomains on the luminal surface of the capillary endothelium. I. Preferential distribution of anionic sites.

1981 ◽  
Vol 90 (3) ◽  
pp. 605-613 ◽  
Author(s):  
N Simionescu ◽  
M Simionescu ◽  
G E Palade
Keyword(s):  
Distribution Pattern ◽  
Capillary Endothelium ◽  
Similar Distribution ◽  
Coated Pits ◽  
Free Aldehyde ◽  
Functional Implications ◽  
Aldehyde Groups ◽  
Similar Distribution Pattern

Cationized ferritin (CF), introduced systemically in vivo or by perfusion in situ, binds preferentially to certain microdomains of the luminal plasmalemma of fenestrated capillaries (mouse pancreas and jejunum). The density and affinity of binding decrease in the following order: fenestral diaphragms greater than coated pits greater than plasmalemma proper. CF binds neither to the membrane of plasmalemmal vesicles and transendothelial channels nor to the corresponding stomatal diaphragms. The distribution pattern is the same when glutaraldehyde fixation precedes the administration of the tracer by perfusion, provided fixation is followed by quenching of residual free aldehyde groups. A much smaller cationic probe (alcian blue) perfused together with the fixative reveals a similar distribution pattern. The functional implications of the association of these microdomains with structures involved in capillary permeability are discussed.

scholarly journals Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis.

1986 ◽  
Vol 102 (4) ◽  
pp. 1304-1311 ◽  
Author(s):  
L Ghitescu ◽  
A Fixman ◽  
M Simionescu ◽  
N Simionescu
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Specific Binding ◽  
Short Exposure ◽  
Gold Complex ◽  
Membrane Microdomains ◽  
Capillary Endothelium ◽  
Plasmalemmal Vesicles ◽  
Specific Binding Sites

The interaction of homologous and heterologous albumin-gold complex (Alb-Au) with capillary endothelium was investigated in the mouse lung, heart, and diaphragm. Perfusion of the tracer in situ for from 3 to 35 min was followed by washing with phosphate-buffered saline, fixation by perfusion, and processing for electron microscopy. From the earliest time examined, one and sometimes two rows of densely packed particles bound to some restricted plasma membrane microdomains that appeared as uncoated pits, and to plasmalemmal vesicles open on the luminal front. Morphometric analysis, using various albumin-gold concentrations, showed that the binding is saturable at a very low concentration of the ligand and short exposure. After 5 min, tracer-carrying vesicles appeared on the abluminal front, discharging their content into the subendothelial space. As a function of tracer concentration 1-10% of plasmalemmal vesicles contained Alb-Au particles in fluid phase; from 5 min on, multivesicular bodies were labeled by the tracer. Plasma membrane, coated pits, and coated vesicles were not significantly marked at any time interval. Heparin or high ionic strength did not displace the bound Alb-Au from vesicle membrane. No binding was obtained when Alb-Au was competed in situ with albumin or was injected in vivo. Gold complexes with fibrinogen, fibronectin, glucose oxidase, or polyethyleneglycol did not give a labeling comparable to that of albumin. These results suggest that on the capillary endothelia examined, the Alb-Au is adsorbed on specific binding sites restricted to uncoated pits and plasmalemmal vesicles. The tracer is transported in transcytotic vesicles across endothelium by receptor-mediated transcytosis, and to a lesser extent is taken up by pinocytotic vesicles. The existence of albumin receptors on these continuous capillary endothelia may provide a specific mechanism for the transport of albumin and other molecules carried by this protein.

Characterization of plasminogen binding to human capillary and arterial endothelial cells

1991 ◽  
Vol 69 (7) ◽  
pp. 442-448 ◽  
Author(s):  
Peter R. Ganz ◽  
Denis Dupuis ◽  
Anil K. Dudani ◽  
Sofia Hashemi
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Binding Sites ◽  
Phenotypic Diversity ◽  
Capillary Endothelium ◽  
Functional Differences ◽  
Equilibrium Binding ◽  
Capillary Endothelial Cells ◽  
Arterial Endothelial Cells

Phenotypic diversity of endothelial cells that line the various vascular spaces has been well established. However, it is not known if biochemical differences also exist, particularly in the numbers of receptors for plasma proteins. Equilibrium binding techniques were used to assess potential differences in the binding of 125I-labelled plasminogen to cultured human umbilical arterial endothelial cells and capillary endothelium, as compared with umbilical venous cells. The kinetic behaviour of plasminogen binding to all three types of cells was similar, with optimal binding occurring between 20 and 30 min of incubation. Binding of plasminogen to arterial, capillary, and venous cells was concentration dependent and reversible upon addition to excess unlabelled plasminogen. Scatchard analyses showed that artery, capillary, and venous endothelial cells all possess low affinity sites for plasminogen with Kd values of 0.30 ± 0.07, 0.40 ± 0.06, and 0.40 ± 0.08 μM, respectively. Vein cells also possess an additional higher affinity binding site with a Kd of 0.07 ± 0.01 μM, exhibiting a 6-fold greater affinity for plasminogen than the lower affinity sites on capillary and arterial endothelial cells. Assuming a stoichiometry of 1:1 for binding, the data indicate that arterial and capillary endothelial cells contain approximately 4.2 (± 0.9) × 106 and 4.1 (± 0.6) × 106 plasminogen receptors per cell. Venous cells contain both low and high density binding sites with 6.2 (± 0.8) × 106 and 12.4 (± 2.4) × 106 sites per endothelial cell. The presence of a higher affinity site on vein cells, but not on artery or capillary cells, may signal functional differences relating to fibrinolytic activity on the surface of these cells. Ligand blotting experiments, in which labelled plasminogen was adsorbed to polypeptides recovered from endothelial cell lysates, identified polypeptides of 46, 45, and 37 kDa, which may constitute the plasminogen-binding sites–receptors on endothelial cells.Key words: plasminogen, endothelial cells, receptors, fibrinolysis.

The Use of Advanced Analytical Techniques for Characterization of the Chemical, Physical, and Crystallographic Nature of a Surface

1973 ◽  
Vol 31 ◽  
pp. 132-133 ◽  
Author(s):  
R. E. Herfert
Keyword(s):  
Surface Characterization ◽  
Analytical Techniques ◽  
X Ray Diffraction ◽  
Depth Of Penetration ◽  
X Ray ◽  
The Past ◽  
Transmission Electron ◽  
Scanning Electron

Studies of the nature of a surface, either metallic or nonmetallic, in the past, have been limited to the instrumentation available for these measurements. In the past, optical microscopy, replica transmission electron microscopy, electron or X-ray diffraction and optical or X-ray spectroscopy have provided the means of surface characterization. Actually, some of these techniques are not purely surface; the depth of penetration may be a few thousands of an inch. Within the last five years, instrumentation has been made available which now makes it practical for use to study the outer few 100A of layers and characterize it completely from a chemical, physical, and crystallographic standpoint. The scanning electron microscope (SEM) provides a means of viewing the surface of a material in situ to magnifications as high as 250,000X.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

A very rapid in situ Kikuchi technique to determine relative crystal misorientation

1988 ◽  
Vol 46 ◽  
pp. 830-831
Author(s):  
J. I. Bennetch
Keyword(s):  
Electron Beam ◽  
Analytical Techniques ◽  
Superplastic Forming ◽  
The Other ◽  
Kikuchi Pattern ◽  
Kikuchi Line ◽  
Line Analysis

In a recent study of the superplastic forming (SPF) behavior of certain Al-Li-X alloys, the relative misorientation between adjacent (sub)grains proved to be an important parameter. It is well established that the most accurate way to determine misorientation across boundaries is by Kikuchi line analysis. However, the SPF study required the characterization of a large number of (sub)grains in each sample to be statistically meaningful, a very time-consuming task even for comparatively rapid Kikuchi analytical techniques.In order to circumvent this problem, an alternate, even more rapid in-situ Kikuchi technique was devised, eliminating the need for the developing of negatives and any subsequent measurements on photographic plates. All that is required is a double tilt low backlash goniometer capable of tilting ± 45° in one axis and ± 30° in the other axis. The procedure is as follows. While viewing the microscope screen, one merely tilts the specimen until a standard recognizable reference Kikuchi pattern is centered, making sure, at the same time, that the focused electron beam remains on the (sub)grain in question.

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