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 Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes.

1992 ◽  
Vol 40 (1) ◽  
pp. 73-82 ◽  
Author(s):  
Y Fukui ◽  
A Yamamoto ◽  
R Masaki ◽  
K Miyauchi ◽  
Y Tashiro
Keyword(s):  
Electron Microscopy ◽  
Particle Density ◽  
Protein A ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Thin Sections ◽  
Gold Particles ◽  
Rough Er

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.

The Effect of Electron Microscopy on Pathological Diagnosis of Neoplasm

1990 ◽  
Vol 48 (3) ◽  
pp. 248-249
Author(s):  
Xiaojun Zhou ◽  
Taihe Zhang
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Frozen Section ◽  
Protein A ◽  
Functional Classification ◽  
Lead Citrate ◽  
Thin Sections ◽  
Tumor Tissues ◽  
Frozen Section Diagnosis

Although electron microscopy (EM) has contributed enormously to an understanding of the structural intricacies of tumor cells, the usefulness of EM in pathological diagnoses of neoplasms has not been readily appreciated by general pathologists. In the present study, 223 tumors submitted for EM diagnosis were analyzed in an attempt to gain further information concerning the contribution of EM to tumor diagnosis.223 neoplasms were submitted to EM for their final diagnoses when histopathological diagnoses were obscure, which represented about of the total number of surgical tumor specimens. Most specimens were taken at the time of frozen section diagnosis and a small number of tissues were originally fixed informaldehyde. All of tissues were fixed with buffered glutaradehyde, postfixed with osmium tetroxide and embedded in Epon 812. Ultrasections were made after semith in sections were examined to verify that representative tumor tissues were present. Thin sections were stained with uranium acetate and lead citrate, and examined under JEM-1200 EX electron microscope. In selected cases, mainly with neuroendocrine tumors, nickel grid-mounted sections were subjected to post embedding immunoelectron microscopy (IEM) using protein A-gold for more detailed functional classification. Protein A-gold probes were prepared as Wang and co-workers described.

Aggregation of band 3 in hereditary ovalocytic red blood cell membranes. Electron microscopy and protein rotational diffusion studies

1993 ◽  
Vol 105 (3) ◽  
pp. 655-660
Author(s):  
A. Che ◽  
R.J. Cherry ◽  
L.H. Bannister ◽  
A.R. Dluzewski
Keyword(s):  
Electron Microscopy ◽  
Rotational Diffusion ◽  
Freeze Fracture ◽  
Band 3 ◽  
Normal Band ◽  
Cytoskeletal Proteins ◽  
Rotational Mobility ◽  
Normal Cells ◽  
Membrane Bound ◽  
Red Blood Cell Membranes

Microaggregation of band 3 proteins in hereditary ovalocytic membranes was investigated by rotational diffusion measurements and by electron microscopy. It was previously shown that band 3 in ovalocytic membranes has decreased rotational mobility compared with band 3 in normal cells (Tilley, L., Nash, G.B., Jones, G.L. and Sawyer, W.L. (1991) J. Membr. Biol. 121, 59–66). This result could arise from either altered interactions with cytoskeletal proteins or from band 3 microaggregation. In the present study it was found that removal of spectrin and actin from the membrane had no effect on the rotational mobility of ovalocytic band 3. Additional removal of ankyrin and band 4.1, as well as cleavage of the cytoplasmic domain of band 3 with trypsin, did enhance band 3 mobility, as is the case in the membranes from normal cells. However, the rotational mobility of ovalocytic band 3 was always considerably less than that of normal band 3 under the same conditions. Scanning electron microscopy and low power electron micrographs of freeze-fracture replicas revealed that the surfaces of ovalocytes were more irregular than those of normal erythrocytes. At higher magnification, numerous linearly arranged intramembranous particles were observed on the P-faces of freeze-fractured ovalocytes but not on normal cells. These clusters consist of straight or slightly curved lines of 10–15 particles in single rows. From these results it is deduced that the reduced rotational mobility of band 3 in ovalocytes is a consequence of the formation of microaggregates, which are very probably induced by the mutation in the membrane-bound domain of ovalocytic band 3.

Differentiation in Trypanosoma brucei: host-parasite cell junctions and their persistence during acquisition of the variable antigen coat

1985 ◽  
Vol 74 (1) ◽  
pp. 1-19 ◽  
Author(s):  
L. Tetley ◽  
K. Vickerman
Keyword(s):  
Electron Microscopy ◽  
Trypanosoma Brucei ◽  
Morphological Changes ◽  
Freeze Fracture ◽  
Tsetse Fly ◽  
Cell Junctions ◽  
Surface Coat ◽  
Thin Sections ◽  
Variable Antigen ◽  
Host Parasite

Acquisition of the variable antigen-containing surface coat of Trypanosoma brucei occurs at the metacyclic stage in the salivary glands of the tsetse fly vector. The differentiation of the metacyclic trypanosome in the gland has been studied by scanning electron microscopy and by transmission electron microscopy of thin sections and freeze-fracture replicas. The uncoated epimastigote trypanosomes (with a prenuclear kinetoplast) divide while attached to the salivary gland epithelium brush border by elaborate branched flagellar outgrowths, which ramify between the host cell microvilli and form punctate hemidesmosome-like attachment plaques where they are indented by the microvilli. These outgrowths become reduced as the epimastigotes transform to uncoated trypomastigotes (with postnuclear kinetoplast), which remain attached and capable of binary fission. The flagellar outgrowths disappear but the attachment plaques persist as the uncoated trypomastigotes (premetacyclics) stop dividing and acquire the surface coat to become ‘nascent metacyclics’. Coat acquisition therefore occurs in the attached trypanosome and not, as previously believed, after detachment. Coating is accompanied by morphological changes in the glycosomes and mitochondrion of the parasite. Freeze-fracture replicas of the host-parasite junctional complexes show membrane particle aggregates on the host membrane but not on the parasite membrane. It is suggested that disruption of the complex occurs when maximum packing of the glycoprotein molecules has been achieved in the trypanosome surface coat, releasing the metacyclic trypanosome into the lumen of the gland.

scholarly journals Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles.

1989 ◽  
Vol 37 (11) ◽  
pp. 1635-1643 ◽  
Author(s):  
A H Schrijvers ◽  
P M Frederik ◽  
M C Stuart ◽  
K N Burger ◽  
V V Heijnen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Tannic Acid ◽  
Freeze Fracture ◽  
Tissue Preparation ◽  
Unilamellar Vesicles ◽  
Thin Sections ◽  
Multilamellar Vesicles ◽  
Morphological Studies ◽  
Rat Hearts ◽  
Small Unilamellar Vesicles

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.

The extrarhabdomeral cytoskeleton in photoreceptors of Diptera. II. Plasmalemmal undercoats

1984 ◽  
Vol 220 (1220) ◽  
pp. 353-359 ◽  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Genetic Analysis ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Basal Region ◽  
Thin Sections ◽  
En Face ◽  
Thiol Proteases ◽  
Planar Membranes

The plasmalemmal undercoats of those regions of the photoreceptors of the blowfly Lucilia that flank the central extracellular space of each ommatidium are described from en face and transverse thin sections. Labile structures were stabilized before fixation for electron microscopy by using an inhibitor of thiol proteases, Ep-475, as described in the previous paper (Blest et al., Proc. R. Soc. Lond . B 220, 339-352, 1984). Membranes of R 1-6 are underlain by a closely associated, randomly organized filamentous meshwork. That of the basal region of R 7 is highly organized, and consists of very long, about 8 nm filaments running parallel to each other and to the longitudinal ommatidial axis; these ‘backbone’ filaments are tightly adherent to the plasma membrane, and are spaced some 190-200 nm apart. They are linked by abundant transverse filaments that form a reticulum between them. The degree of ordering of the reticulum in life is not clear, but some well-preserved profiles suggest that it may be high. Replicas obtained by the freeze-fracture technique show that extrarhabdomeral membranes have dense populations of intramembrane particles, just as they do in Drosophila where a genetic analysis has shown them to consist largely of rhodopsin. It is proposed as a working hypothesis that these planar membranes can be regarded as flat equivalents of the microvillar membranes, that some fraction of the integral membrane proteins may be immobilized by bonding to the plasmalemmal undercoat, and that the latter may help to constrain both the translational and rotational movements of rhodopsin molecules.

scholarly journals A novel methodology for double protein A-gold immunolabeling utilizing the monovalent fragment of protein A.

1992 ◽  
Vol 40 (3) ◽  
pp. 435-441 ◽  
Author(s):  
J R Thorpe
Keyword(s):  
Electron Microscopy ◽  
Binding Sites ◽  
Secretory Granules ◽  
Protein A ◽  
Model System ◽  
Normal Serum ◽  
Gold Complex ◽  
Thin Sections ◽  
Igg Binding ◽  
Gold Probe

A method for sequential protein A-gold immunolabeling is described whereby the binding of second gold probe to the first antibody-protein A-gold complex is reduced to acceptably minimal levels. Immunolabeling of thin sections of embedded pituitary tissue was used as a model system. After an initial immunolabeling for prolactin, sections were incubated in normal serum (rabbit) followed by a monovalent fragment of protein A. These latter two incubations reduced artifactual second gold probe label over prolactin-labeled secretory granules to minimal levels (much less than 1 particle per granule) when sections were subsequently immunolabeled with normal serum. The combination of normal serum and protein A fragment incubations saturates IgG and protein A binding sites on the first antibody-gold probe complex. The latter is thereafter unable to bind further IgG (and thus gold probe) because of the monovalent nature of the protein A fragment. It is suggested that this methodology may be extended to multiple immunolabeling procedures for electron microscopy. In addition, when used before single labeling this method may be an effective way to minimize nonspecific IgG binding in cases where the tissue or antibody under study may be a problem.

scholarly journals Ultrastructure of Na,K-transport vesicles reconstituted with purified renal Na,K-ATPase.

1980 ◽  
Vol 86 (3) ◽  
pp. 746-754 ◽  
Author(s):  
E Skriver ◽  
A B Maunsbach ◽  
P L Jørgensen
Keyword(s):  
Electron Microscopy ◽  
Freeze Fracture ◽  
Cation Transport ◽  
Coupled Transport ◽  
Vesicle Membrane ◽  
Thin Sections ◽  
Intramembrane Particles ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
K Transport

To study the size and structure of the Na,K-pump molecule, the ultrastructure of phospholipid vesicles was examined after incorporation of purified Na,K-ATPase which catalyzes active coupled transport of Na+ and K+ in a ratio close to 3Na/2K. The vesicles were analyzed by thin sectioning and freeze-fracture electron microscopy after reconstitution with different ratios of Na,K-ATPase protein to lipid, and the ultrastructural observations were correlated to the cation transport capacity. The purified Na,K-ATPase reconstituted with phospholipids to form a very uniform population of vesicles. Thin sections of preparations fixed with glutaraldehyde and osmium tetroxide showed vesicles limited by a single membrane which in samples stained with tannic acid appeared triple-layered with a thickness of 70 A. Also, freeze-fracture electron microscopy demonstrated uniform vesicles with diameters in the range of 700-1,100 A and an average value close to 900 A. The vesicle diameter was independent of the amount of protein used for reconstitution. Intramembrane particles appeared only in the vesicle membrane after introduction of Na,K-ATPase and the frequency of intramembrane particles was proportional to the amount of Na,K-ATPase protein used in the reconstitution. The particles were evenly distributed on the inner and the outer leaflet of the vesicle membrane. The diameter of the particles was 90 A and similar to our previous values for the diameter of intramembrane particles in the purified Na,K-ATPase. The capacity for active cation transport in the reconstituted vesicles was proportional to the frequency of intramembrane particles over a range of 0.2-16 particles per vesicle. The data therefore show that active coupled Na,K transport can be carried out by units of Na,K-ATPase which appear as single intramembrane particles with diameters close fo 90 A in the freeze-fracture micrographs.

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.

Sign in / Sign up

Export Citation Format

Share Document