Biomedical subjects

M Bendayan

Publications and source records attributed to M Bendayan.

At least 109 records · Page 6Linked to original sources

PubMed1991

Ontogenesis of glomerular basement membrane: structural and functional properties.

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.

Animals↗

PubMed1991

Protein AG-gold complex: an alternative probe in immunocytochemistry.

The purpose of this study was to evaluate the use of protein AG tagged with colloidal gold as a reliable immunocytochemical reagent. Protein AG is a recombinant of 47.3 KD molecular weight and pI = 4.3, which displays immunoglobulin Fc binding sites for both staphylococcal protein A and streptococcal protein G. It adsorbs to 10-nm colloidal gold particles with a lower affinity than does protein A, and is saturable. A maximal number of 12 protein AG molecules could be accommodated on the gold particle surface. Protein AG-gold conjugates yielded positive signals in post-embedding immunocytochemical assays when used as a secondary reagent in conjunction with several species and classes of polyclonal (rabbit, goat, sheep, guinea pig) and mouse monoclonal immunoglobulins (IgG1, IgG2, and IgG3). In addition, protein AG-gold was found to be a useful reagent in immunoblot analysis because of its ability to bind and identify nitrocellulose-immobilized IgGs (rabbit, mouse, goat, sheep, rat, and cow). Its spectrum of specificity towards various types of antibodies combines those of the parental protein A and protein G molecules. The protein AG-gold complex therefore appears to be a highly versatile and convenient alternative probe for immunochemical and immunocytochemical studies.

Bacterial Proteins↗

PubMed1990

Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria.

In avian smooth muscle cells, desmin-containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, including the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti-desmin and protein-A-colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.

Animals↗

PubMed1990

Immunogold studies of monomeric elements from the globular domain (NC1) of type IV collagen in renal basement membranes during experimental diabetes in the rat.

The protein A-gold immunocytochemical technique was applied to reveal the monomeric elements M1, M2* and M3 from the non-collagenous globular domain (NC1) of type IV collagen over various renal basement membranes from control and long-term streptozotocin-induced diabetic rats. This study includes the basement membranes of the proximal tubule, the Bowman's capsule and the glomerulus as well as the extracellular matrix of the mesangium. The labellings obtained were confined to basement membrane material. The quantitative analysis demonstrated changes in labelling intensities and distribution between tissues from normal and diabetic animals. Increased labelling intensities were observed for M1 and M2* monomers in all the basement membranes studied except for the mesangial matrix which remained unchanged. In addition, the labelling for M1 monomers, present on the endothelial side of the glomerular basement membrane of control animals, was found to be distributed throughout the entire thickness of the basement membrane of diabetic animals. In contrast, neither the intensity of the labelling, nor the distribution of M3 monomers were altered in diabetic animals. Since M1 monomers are markers of the alpha 1(IV) and alpha 2(IV) chains of type IV collagen while M2* and M3 mark alpha 3(IV) and alpha 4(IV) chains respectively, the present results demonstrate changes in the nature of the collagenous elements of basement membranes during diabetes. Furthermore, the results indicate that the alpha 3(IV) and the alpha 4(IV) chains are not necessarily present in the same molecule. The modifications of the collagenous elements of the basement membranes during diabetes must alter the structural characteristics of these matrices which in turn might influence their functional properties.

Animals↗

PubMed1990

Morpho-cytochemical and biochemical evidence for insulin absorption by the rat ileal epithelium.

In order to investigate the mechanism through which insulin is absorbed by the intestinal epithelium and transferred to the circulation where it exercises its biological activity of lowering blood glucose levels, a combined biochemical morpho-cytochemical study was undertaken on rat ileal tissue, in vivo. Insulin was introduced into the lumen of the ileum in combination with sodium cholate and aprotinin and allowed to be absorbed for various periods of time. Analysis of blood samples from the inferior vena cava, at different time points has demonstrated an increase in plasma insulin followed by a decrease in blood glucose levels. The ileal tissues were studied at different time points after the introduction of the insulin, by applying the protein A-gold immunocytochemical technique. Insulin antigenic sites were detected with high resolution, at various levels of the enterocytes but were absent from goblet cells. At 2 to 5 min, the labelling was mainly associated with the microvilli and endocytotic vesicles in the apical portion of the epithelial cells. Some gold particles were in contact with the lateral membranes. At 10 min, the labelling was found at the level of the trans-side of the Golgi apparatus and mainly along the baso-lateral membranes of the epithelial cells. Labelling was also detected in the interstitial space. The control experiments have demonstrated the specificity of the labelling and confirmed the nature of the insulin molecules detected. Furthermore, the morphological study has confirmed that exposure of the tissue to the insulin-cholate-aprotinin solution does not affect the integrity of the epithelium while promoting insulin absorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells.

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.

Albumins↗

PubMed1990

Immunolabeling efficiency of protein A-gold complexes.

A systematic study of the adsorption of protein A on colloidal gold particles varying in size from 5-16 nm was performed at different protein concentrations. The number of protein A molecules bound per colloidal particle was evaluated and the Scatchard analysis of the adsorption parameters was applied for each size of the colloid. The binding of protein A to the colloidal gold surface exhibited the same affinity pattern for all of the particle sizes. At low concentrations of stabilizing protein, adsorption took place with high affinity (Kd 1.96-3.3 nM) and the maximum number of protein A molecules attached with this affinity correlated well with the surface of the particle. At higher concentrations of protein A, adsorption exhibited a significantly lower affinity (Kd 530-800 nM), and no saturation was recorded. Competition by albumin did not reveal a preferential removal of the "low-affinity" bound protein A molecules, contradicting the model of successive shells of stabilizing protein around the colloidal particle. The immunolabeling efficiency of conjugates having the same size of gold nucleus but carrying different numbers of protein A molecules was comparatively investigated by quantitative post-embedding immunocytochemistry. Protein A-gold formed with 5-10-nm colloids gave the highest intensity of labeling when carrying the maximum number of protein A molecules that could be adsorbed with high affinity. Overloading as well as underloading these complexes resulted in a significant decrease of their immunoreactivity. The most efficient conjugates were obtained when stabilization was performed with 6 micrograms protein A/ml gold sol of 5 and 10 nm particle diameter, and 15 micrograms protein/ml of 15-nm colloid.

Animals↗

PubMed1990

Comparative pyrimidine- and purine-specific RNAse-gold labeling on pancreatic acinar cells and isolated hepatocytes.

We applied the enzyme-gold approach to investigate the potential of various ribonucleases displaying different affinities for ultrastructural localization of particular RNA molecules. Five specific ribonucleases were used: three from a pancreatic source, RNAses A, B, and S with affinities for pyrimidine bases; and two from Aspergillus oryzae, RNAses T1 and T2 specific for purine bases. Conditions required for preparing each RNAse-gold complex, as well as for obtaining specific labelings, were determined. Application of the probes on thin sections of pancreatic acinar cells yielded labeling patterns that differed according to the enzyme used. Pancreatic RNAses labeled mostly the rough endoplasmic reticulum and the nucleolus, whereas fungal RNAses labeled more intensely the interchromatin space and the nucleolus, the rough endoplasmic reticulum being labeled to a lesser extent. Areas rich in interchromatin granules were intensely labeled by the RNAses T1 and T2. This was confirmed on DRB-treated hepatocytes, which displayed large clusters of interchromatin granules. Perichromatin granules were labeled by the RNAse A- and T1-gold complexes. These results provide a strong indication for the presence of RNA molecules in both types of granules. Nuclear pores were labeled, particularly by the RNAses T1 and T2, thus supporting the hypothesis for the site of RNA transit between nucleus and cytoplasm. The differences in patterns of labeling among the various enzyme-gold complexes could be related to differences in affinities. The use of a panel of specific RNAses, displaying different affinities, could thus allow for the topographical distribution of particular RNA molecules according to their relative content of specific bases.

Animals↗

PubMed1990

Ultrastructural distribution of lectin-binding sites in the glomerular wall of streptozotocin-induced diabetic rats.

Since carbohydrates-containing molecules are known to be preferentially altered in diabetes mellitus and that major functional and morphological alterations do occur during diabetes in the renal tissue, we revealed in the present study various lectin-binding sites in the glomerular wall of control and long-term diabetic animals. Lectin-binding sites specific to N-acetyl-galactosamine, N-acetyl-glucosamine, sialic acid, galactose and fucose were revealed using the appropriate lectin and the lectin-gold complex at the electron microscope level. Differences in intensity of labeling as well as in distribution were detected for several lectin-binding sites particularly in the glomerular basement membrane, reflecting the presence of additional glycoconjugates and changes in the molecular organization of the basement membrane components during diabetes. Alterations in the glycocomponents and the glycoproteins of the glomerular basement membrane as well as non-enzymatic glycosylation of the basement membrane components have been described in diabetes, going along with our present results. The alteration in the distribution of some lectin-binding sites gives support to modifications in the three dimensional organization of some glycoproteins which could occur in diabetes. Since the glomerular wall is actively involved in blood filtration, these changes may either induce, or result from, the loss in selective permeability and the massive proteinuria occurring during diabetes.

Animals↗

PubMed1990

Heterogeneous distribution of monomeric elements from the globular domain (NC1) of type IV collagen in renal basement membranes as revealed by high resolution quantitative immunocytochemistry.

Heterogeneity in the distribution of major components, namely laminin, entactin/nidogen, heparan sulfate proteoglycan and type IV collagen among renal basement membranes, was previously demonstrated (Desjardins and Bendayan, J Histochem Cytochem 37:885-897, 1989). To further investigate the nature of basement membranes, we applied high resolution immunocytochemistry to reveal monomers M1, M2* and M3 composing the NC1 domain of type IV collagen of various rat renal basement membranes. Labeling for the three monomers was confined to basement membranes. Quantitative morphometrical analysis demonstrated that the labeling for each monomer was not evenly distributed among the various basement membranes. Labeling for M1 was intense over the proximal tubule and the Bowman's capsule basement membranes. In the glomerulus, the mesangial matrix was highly labeled, whereas the glomerular basement membrane was labeled with a lower intensity. In contrast, labeling for M2* and M3 were high in the glomerular basement membrane, while very low in the mesangial matrix. Labeling intensities were intermediate in the other renal basement membranes. The ultrastructural distribution analysis made over the glomerular basement membrane showed a preferential subendothelial localization of M1 monomers. M2 and M3 on the other hand, were found throughout the entire thickness of the basement membrane. M1 monomers being associated to alpha 1(IV) and alpha 2(IV) chains of type IV collagen, and M2* and M3 to alpha 3(IV) and alpha 4(IV) chains respectively, our results thus demonstrate the existence of an heterogeneity in the collagenous nature of renal basement membranes, particularly the glomerular one. This heterogeneity must reflect variations in the structural arrangement of basement membranes and therefore in their functional properties.

Animals↗

PubMed1990

Apolipoprotein B-100: immunolocalization and synthesis in human intestinal mucosa.

Despite the evidence that the human small intestine produces two separate species of apoB mRNA encoding for B-100 and B-48, there is a paucity of data concerning the expression of the latter form in this organ. Using a high resolution immunogold approach, with specific polyclonal antibodies and a panel of monoclonal antibodies (2D8, 3A10, 4G3), both forms of apoB (B-48 and B-100) were revealed over enterocytes of pediatric intestinal samples. Intense labeling was observed over microvilli, apical smooth membrane vesicles, multivesicular bodies, the basolateral membrane, as well as the trans Golgi region. Only low labeling was found over the rough endoplasmic reticulum (rER). Similar patterns of apoB distribution characterized both duodenal and jejunal regions. The presence of labeling over the Golgi apparatus and rER suggests a synthetic activity of both forms of apoB by the epithelial cells. To test this hypothesis, human intestine was incubated with [3H]leucine, homogenized, and subjected to immunoprecipitation for apoB. Immunoprecipitates contained radioactivity mainly in apoB-48 with relatively small amounts in apoB-100 when examined by NaDodSO4-polyacrylamide gel electrophoresis. These findings were further supported by the biochemical determination of apoB-100 and apoB-48 in chylomicron particles isolated from thoracic duct lymph of a human donor. Taken together, our data suggest that the human intestine is able to synthesize and to express the apoB-100.

Apolipoprotein B-100↗

PubMed1990

Immunocytochemical distribution of endogenous albumin and immunoglobulins in the glomerular wall of the sucrose-fed "Cohen" diabetic rat.

Endogenous circulating albumin and immunoglobulin G (IgG) molecules were revealed with high resolution over the glomerular wall of renal tissue from normoglycemic and sucrose-fed hyperglycemic Cohen rats applying the protein A-gold immunocytochemical approach. Due to the length of the experiment, the hyperglycemic animals demonstrated only minor renal morphological alterations. In spite of this, labelling for albumin and immunoglobulins in the glomerular wall showed significant differences between the normoglycemic and hyperglycemic animals. In the former, the labelings were restricted to the endothelial side of the glomerular basement membrane, while in the latter, the labelings were distributed throughout the entire thickness of the basement membrane. In addition, the hyperglycemic animals display some labelling associated with the slit diaphragms between podocytes and in the urinary space. Numerous lysosomal structures present in podocytes and mesangial cells of the hyperglycemic animals were intensely labeled for albumin and IgGs, reflecting a reabsorption activity. Dense deposits labeled for IgGs were found in the glomerular wall and mesangial regions of hyperglycemic animals corresponding to depositions of immune-complexes. These results demonstrated that in hyperglycemic conditions induced by environmental factors such as the diet, the permselectivity of the glomerular basement membrane is lost; proteins such as albumin and IgGs are not restricted by the endothelial side of the basement membrane and cross the glomerular wall leading to proteinuria. The loss of selective permeability properties of the glomerular basement membrane was found to precede the morphological changes occurring in the glomerular wall during diabetes.

Animals↗

PubMed1990

Ultrastructural distribution of glomerular basement membrane components in experimental diabetes.

To investigate possible changes in the organization of the constitutive elements of the glomerular basement membrane during diabetes we have localized type IV collagen, entactin, laminin and heparan sulfate proteoglycan on the glomerular wall of control and long-term streptozotocin-induced diabetic animals. To do so, we made use of specific polyclonal antibodies and the high resolution protein A-gold immunocytochemical technique. The morphometrical analysis of the labelling has demonstrated that each antigen displays a specific distribution in the glomerular basement membrane of normal animals. In diabetes, changes in type IV collagen were detected with an accentuation of the labelling on the endothelial side and the presence of a wider region weakly labelled on the epithelial side of the basement membrane. No differences were found in the distribution of other components in normal and diabetic conditions, beside a decrease in the labelling for heparan sulfate proteoglycan in the laminae rarae. These modifications may influence the interactions among the various basement membrane components which in turn, will affect the structural organization of the polymeric matrix of the basement membrane, leading to a loss in selective permeability.

Animals↗

PubMed1990

Distribution of endogenous albumin in the glomerular wall of proteinuric patients.

Glomerular proteinuria seems to be related, in part, to loss or impairment of the normal barrier function of the glomerular capillary wall. To investigate the functional properties of this barrier, endogenous albumin was revealed in the glomerular wall of proteinuric patients and compared with a nonproteinuric control by immunoelectron microscopy using the protein A-gold method. In the control biopsy, peaks of albumin accumulation were noted in the subendothelial area and in the inner portion of the lamina densa, with gradual tapering of the distribution toward the epithelial side of the basement membrane. The urinary space and epithelial cells were weakly labeled. In tissues from proteinuric patients, albumin was distributed throughout the entire width of the glomerular basement membrane, although the pattern of accumulation varied between patients. The urinary space showed significant labeling associated with some flocculent material. Mesangial areas were heavily labeled in tissues from both control and proteinuric patients. In the latter, lysozomes in glomerular and tubular epithelial cells also accumulated albumin, which is evidence of reabsorption. These results reveal the existence, in normal conditions, of a barrier located in the subendothelial area of the glomerular basement membrane, the loss of which, as in the idiopathic nephrotic syndrome, leads to diffuse distribution of albumin in the glomerular capillary wall.

Basement Membrane↗

PubMed1989

Distribution of endogenous albumin across the rat aortic wall as revealed by quantitative immunocytochemistry.

Endogenous albumin was revealed over thin sections of rat aortic wall, with high resolution and specificity, by applying the protein A-gold immunocytochemical technique. Gold particles, revealing albumin antigenic sites, were observed over plasmalemmal vesicles in endothelial cells and over the interstitial space throughout the thickness of the aortic wall. The distribution of the labeling in the interstitial space varied from region to region and was associated with the collagen fibers, following the orientation of the bundles. The morphometric evaluation of this labeling demonstrated a first peak in labeling intensity in the intima followed by a steep decrease with low levels in the media, and an increasing gradient towards the adventitia. In the subendothelium, a moderate labeling was observed at the base of the endothelial cells of both aortic and capillary endothelia, followed by a decreasing gradient. Ratios between the labeling density in the intima as well as in the adventitia and that in the capillary lumen (plasma albumin) revealed different concentrations of albumin in these compartments. Endogenous albumin, under steady-state conditions, is thus unevenly distributed over the interstitial spaces across the rat aortic wall, and appears associated along the collagen fibers.

Albumins↗

PubMed1989

Effect of vanadate administration on blood glucose and insulin levels as well as on the exocrine pancreatic function in streptozotocin-diabetic rats.

In the present study, streptozotocin-induced diabetic rats with their corresponding controls, were treated orally with sodium metavanadate. A gradual increase of the vanadate concentration up to 0.8 mg/ml in the drinking water, lowered the blood glucose levels of the diabetic animals to normal values without changing the insulin levels. On the other hand, vanadate did not affect the blood glucose levels of the non-diabetic animals; it did however induce lower levels of circulating insulin in these animals. The lowering of the glycaemic values of the diabetic animals was closely related to the consumption of vanadate. When the treatment was ceased, the blood glucose levels rose rapidly. The diabetic animals responded to the vanadate treatment with two sensitivities; while the large majority of the diabetic animals displayed stable normoglycaemic values, others had fluctuating values. Amylase content in the exocrine pancreas of these two subgroups of animals was studied separately and compared to that from the non-treated control and diabetic animals. The presence of amylase in the pancreatic acinar cells was assessed by the protein A-gold immunocytochemical approach and biochemical determinations. Amylase was found to be very low in the non-treated diabetic animals. Lowering of the blood glucose levels induced by the vanadate treatment restored the amylase to levels similar to those of the controls. However, vanadate-treated diabetic animals with fluctuating levels of blood glucose, demonstrated only a partial recovery of amylase. Thus, vanadate treatment was found to have a normalizing effect on blood glucose levels in diabetic animals as well as restoring amylase content in the pancreas of diabetic animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗

PubMed1989

Prolactin crinophagy is induced in the estrogen-stimulated male rat pituitary.

The phenomenon of crinophagy in rat pituitary mammotrophs, or lysosomal uptake of prolactin secretory granules, was confirmed by means of double-label immunogold electron microscopy, and shown to be induced in estrogen-stimulated male rats. Rabbit antibodies to rat cathepsin D were used to label lysosomes, and to rat prolactin to label secretory granules. The pituitaries were fixed in 4% formaldehyde and 1% glutaraldehyde, embedded in Lowicryl K4M, and thin sections were exposed successively to primary antibodies, biotin-labelled second antibodies, and streptavidin-gold, with an amplification procedure for cathepsin D. Cathepsin D and prolactin were detected separately on opposite sides of the sections, using 5-nm and 15-nm gold particles. Lysosomal uptake of prolactin secretory granules was not observed in untreated control rats. It was detected in about 26% of lysosome-containing mammotroph cell sections in estrogen-stimulated rats and at 7 h after estrogen withdrawal, but fell to 14% at 24 h and to 2% at 72 h after estrogen withdrawal.

Animals↗

PubMed1989

Ultrastructural distribution of endogenous IgGs in the glomerular wall of control and diabetic rats.

Endogenous IgG molecules were revealed with high resolution EM over the glomerular wall in renal tissues sampled from short and longterm control and streptozotocin induced diabetic rats by applying the protein A-gold immunocytochemical approach. In tissues from control animals, IgG antigenic sites were revealed on the subendothelial side of the basement membrane, the epithelial side being only weakly labelled. In contrast, in longterm diabetic animals IgG antigenic sites were present throughout the entire thickness of the basement membrane, and in patches closely associated with the plasma membrane of the epithelial cells. Deposits of basement membrane-like material present in the mesangial area were also highly labelled for IgG. Numerous intensely labelled lysosome-like structures were present in the epithelial cells. Morphometrical evaluation of the distribution of the labelling over the basement membrane confirmed these observations. In control animals a peak of labelling was found at 30 nm from the endothelial cell region corresponding to the subendothelial side of the lamina densa. In longterm diabetic animals the labelling was more uniformly distributed throughout the entire thickness of the basement membrane. These data were correlated to biochemical determinations of proteinuria and IgG excretion in urine samples from the control and the diabetic animals. These results suggest that in normal conditions the lamina densa may represent the main barrier for the restriction of the passage of IgGs through the glomerular wall. Modifications at that level occur during diabetes leading to or participating in the loss of the selective permeability of the basement membrane.

Animals↗