Biomedical subjects

M Bendayan

Publications and source records attributed to M Bendayan.

At least 127 records · Page 7Linked to original sources

PubMed1989

Effect of reduced energy metabolism and reperfusion on the permeability and morphology of the capillaries of an isolated rete mirabile.

The effects of reduction in energy metabolism were explored in the eel rete mirabile, an organ composed predominantly of capillaries. In vitro experiments showed that glycolysis is the major pathway of energy production in this capillary tissue, and that iodoacetate, KCN, and low PO2 in combination markedly reduce its ATP generation. When in situ energy generation was inhibited by this combination during countercurrent perfusion of the arterial and venous capillaries of the rete, an approximate doubling of the intercapillary barrier permeability for human [125I]albumin, [14C]sucrose, and 22Na was found. Structural damage was evident, but the intercellular junctions remained intact. The effect of cessation of flow for 30 minutes, followed by reperfusion, was then explored. Stasis alone altered the structure, chiefly of the venous capillary endothelium, but not the permeability of the intercapillary barrier. Stasis with a hypoxic medium containing the inhibitors of energy generation, followed by reperfusion with oxygenated control medium, resulted in a progressive breakdown of the intercapillary barrier, with a threefold to fourfold increase in solute (labeled albumin, sucrose, and sodium) permeability, evolving during early reperfusion, but no change for labeled water permeability. Morphologically, the endothelial cells, especially those in venous capillaries, showed substantial damage; they appeared vacuolated, their cytoplasm was extracted, and cytoplasmic and membrane debris were found in the lumen; intercellular junctions remained intact. Local pericyte detachment with interstitial edema also appeared. Thus, stasis and reperfusion amplified the effects of reduction in energy generation and hypoxia on both permeability and morphological change.

Anguilla↗

PubMed1989

Effect of phalloidin on structure and permeability of rete capillaries in the normal and hypoxic state.

The effects of 10(-6) M phalloidin on reperfusion-injured blood capillary structure and permeability were studied in the countercurrent perfused rete mirabile of the eel swim bladder. In the normal rete, the addition of phalloidin to the perfusion medium did not induce morphological or functional changes. When flow was arrested for 30 minutes, during which time the capillaries were exposed to inhibitors of ATP generation, and flow was then resumed with an oxygenated medium, cell membrane blebs and vacuolization, mitochondrial swelling, pericyte shrinkage, and interstitial space edema were observed. The permeability coefficients for labeled albumin, sucrose, and sodium increased to three to four times baseline values, whereas the permeability to water was not significantly modified. When the same protocol was repeated with phalloidin present in the medium throughout the experiment, the structural integrity of the endothelial cells was completely preserved and pericyte shrinkage was abolished, but interstitial space edema still occurred. The permeability to albumin, sucrose, and sodium increased only to 1.5 times baseline values, a significantly decreased increment in comparison with the experiments performed without phalloidin. We concluded that although phalloidin does not improve the capillary barrier of the normal rete, it provides protection against the structural and functional damage induced by hypoxia and reperfusion.

Air Sacs↗

PubMed1989

Lung surfactant-associated proteins and type IV collagen share common epitopes. An immunocytochemical demonstration.

Among the surfactant-associated proteins (SP-A) characterized so far, there is a group of glycoproteins 26 to 34 kDa that features collagenlike sequences near their N-terminal end. We herein report the cross-reactivity of a rabbit polyclonal antibody to EHS tumor-derived type IV collagen towards rat SP-A. Rat lung tissues were processed for the localization of both type IV collagen and SP-A by high-resolution immunocytochemistry, applying the protein A-gold technique with specific antibodies. In addition to the various basal laminae of the pulmonary tissue, the antitype IV collagen antibody labeled the surfactant material found in alveolar spaces and macrophages, as well as in type II pneumocytes. The surfactant nature of the alveolar material labeled by the antiserum to type IV collagen was confirmed by the positive labeling obtained using an antibody to SP-A. This antibody labeled specifically the alveolar surfactant material, without binding any basal laminae. Several control experiments demonstrated the specificity of each labeling. These results were further supported by immunoblot experiments on nitrocellulose membrane. These findings thus provide further support to the existence of collagenlike sequences on SP-A, and further demonstrate that this structural similarity with collagens can lead to some cross-antigenicity.

Animals↗

PubMed1989

Cytochemical demonstration of increased phospholipid content in cell membranes in chlorphentermine-induced phospholipidosis.

We recently introduced a novel cytochemical approach to high-resolution cytochemistry of phospholipids in biological tissues. The technique consists of adsorption of bee venom phospholipase A2 to colloidal gold particles (PLA2-gold complex) and subsequent application of this complex for localization of the enzyme substrate, i.e., glycerophospholipids. In the present study, this technique was applied at the post-embedding level, in both light (LM) and transmission electron microscopy (TEM), to investigate drug-induced phospholipidosis, an experimental disorder in which the lysosomal catabolism of phospholipids is inhibited. Rats received one week of daily treatment (40 mg IP/kg) with chlorphentermine (CP), a cationic amphiphilic drug known to induce phospholipidosis in several tissues. Glutaraldehyde- and osmium-fixed lung and kidney tissues from both treated and control animals, were embedded in Epon and sections processed for labeling by PLA2-gold. In CP-treated specimens the presence of large osmiophilic inclusions in several cell types of lung parenchyma and kidney cortex confirmed the onset of phospholipidosis. These inclusions were densely labeled by PLA2-gold at both LM and TEM levels. Two general types of abnormal inclusions were distinguished on the basis of their ultrastructure and labeling pattern by PLA2-gold, suggesting different content or configuration of phospholipids. Moreover, quantitative evaluation of labeling density over various membrane compartments in lung alveolar cells evidenced significantly increased phospholipid content after CP treatment. In type II pneumocytes, such increases were measured in membranes of the RER, Golgi complex, outer and inner nuclear envelope, and the basolateral and apical domains of the plasma membrane. In capillary endothelial cells, the basal and luminal domains of the plasma membrane also showed an increase in labeling density. These results further demonstrate the potential usefulness of the PLA2-gold technique for in situ ultrastructural localization of phospholipids in normal and pathological tissues.

Animals↗

PubMed1989

Electron spectroscopic imaging for high-resolution immunocytochemistry: use of boronated protein A.

In the present study we adapted electron spectroscopic imaging (ESI) for high-resolution immunocytochemistry. To accomplish this, we applied boronated protein A (B-pA) for indirect detection of specific antigenic sites using pre-embedding and post-embedding protocols. Isolated acinar cells were exposed to wheat germ agglutinin (WGA) and anti-WGA, followed by B-pA, to reveal WGA binding sites at the level of the plasma membrane. The cells were then embedded in Epon and unstained ultra-thin sections were examined by electron microscopy using the ESI mode. For post-embedding, ultra-thin sections of glutaraldehyde-fixed, Lowicryl-embedded pancreatic tissue were exposed to specific antibodies (anti-insulin or anti-amylase), followed by B-pA. The unstained sections were examined using the ESI mode. In both cases, boron was detected with high resolution either at the level of the plasma membrane of acinar cells, demonstrating WGA binding sites, or over secretory granules in pancreatic insulin-secreting cells or acinar cells, demonstrating insulin and amylase, respectively. These findings were compared to those obtained with the protein A-gold technique, and have demonstrated the analogy of both types of labeling. In addition, several control experiments assessed this novel approach. They have demonstrated the specificity of labeling and the high reactivity of B-pA, as well as its antibody-binding properties. Finally, electron energy loss spectral analysis confirmed the presence of boron in the tissue sections at sites where immunolabeling was detected. These results demonstrate that ESI is an appropriate approach for cytochemistry. Since the technique is based on detection of elements, spatial resolution is considered to be in the magnitude of 0.5 nm, which represents a major improvement in resolution over actual electron microscopic cytochemical techniques.

Animals↗

PubMed1989

Heterogenous distribution of type IV collagen, entactin, heparan sulfate proteoglycan, and laminin among renal basement membranes as demonstrated by quantitative immunocytochemistry.

Type IV collagen, entactin, heparan sulfate proteoglycan, and laminin antigenic sites were revealed on various rat renal basement membranes by use of protein A-gold immunocytochemistry. The basement membranes of the proximal and distal convoluted tubules, those of Bowman's capsule and glomerulus, and the mesangial matrix were labeled for all the antigens but to differing extents. Control experiments confirmed the specificity of these labelings. Quantitative evaluation revealed an important heterogeneity for each antigen among the various basement membranes. This heterogeneity suggests that the basement membrane components must arrange themselves in different ways, possibly to account for differences in functional properties of the various renal structures.

Animals↗

PubMed1989

Heterogeneous distribution of phospholipids in membranes along the secretory pathway in pancreatic B-cells.

The membrane content in phospholipids along the secretory pathway in rat pancreatic B-cells was studied in situ by high-resolution cytochemistry, applying the recently introduced phospholipase A2-gold technique. The gold particles were mostly associated with cell membranes, and the various types of membranes were labeled to a different extent. Quantitation of the labeling over these membranes revealed a heterogeneous distribution of the labeling across the secretory pathway. This heretogeneity occurred mainly as a progressive, decreasing gradient in the first half of this pathway, between the rough endoplasmic reticulum and the mi-cisternae of the Golgi apparatus. The labeling density remained at a lower level in the trans-most Golgi cisternae and immature secretory granule membranes, to increase in the mature secretory granule membrane, where it reached the value found in the plasma membrane. These results provide evidence that the functional heterogeneity existing across the membrane forming the secretory pathway is parallelled by substantial changes in their phospholipid content.

Animals↗

PubMed1989

Brief review on progresses in enzyme-gold cytochemistry.

Initially developed for the in situ localization of nucleic acids, the enzyme-gold approach has been extended to the detection of a large variety of biological molecules. The enzyme-gold approach, based on the highly specific interaction existing between an enzyme and its substrate, can be used both in pre-embedding and post-embedding labeling procedures. Fixation and embedding conditions for the best preservation of each particular substrate under study have to be defined. On the other hand, conditions required to adsorb purified enzymes on colloidal gold particles should be determined according to the biochemical properties of each protein. Labeling protocols must be performed taking into consideration the optimal conditions for the enzymatic activity. The enzyme-gold complexes have been shown to retain their biochemical properties and the specificity of each labeling obtained has been assessed through various control experiments. Initially applied for the demonstration of nucleic acids, the approach has been extended to the ultrastructural localization of various substrates, and in particular, more recently, glycoconjugates and phospholipids. Indeed, various glycosidase-gold complexes and a phospholipase-gold complex, applied in pre- and post-embedding labeling protocols, did specifically label plasma membranes as well as various defined subcellular compartments. In addition, the morphometrical evaluation of labeling intensities revealed differences in amounts of binding sites between compartments. Considering its versatility, simplicity and efficiency, the enzyme-gold technique provides an alternative, very valuable cytochemical tool for the localization of a variety of biological molecules at the cellular and subcellular level.

Animals↗

PubMed1989

Relationship of decreased hepatic lipase activity and lipoprotein abnormalities to essential fatty acid deficiency in cystic fibrosis patients.

Polyunsaturated fatty acids are known to affect plasma lipids and lipoproteins but there is no information on the effect of essential fatty acid (EFA) deficiency on lipoprotein composition. The purpose of this study was to characterize lipoproteins from 17 cystic fibrosis (CF) patients in relationship to their EFA status (eicosatrienoic/arachidonic acid ratio) and compare them with those of 10 healthy siblings (SIB) and of 10 unrelated controls. In 7 EFA-deficient (EFAD) and 10 EFA-sufficient (EFAS) patients, hypocholesterolemia was associated with a decrease of HDL-cholesterol and of LDL-cholesterol which was more marked in the EFAD group. Similarly, although triglyceride enrichment of VLDL, LDL, HDL2, and HDL3 with a concomitant reduction of cholesteryl esters from all particles except HDL2 was observed in both CF groups, it was more sizable in the EFAD patients. These changes led to an increase in the particle size of VLDL, LDL, and HDL2 whereas the distribution of HDL3 was skewed to smaller particles. Alterations in the apoprotein composition of particles were greater in EFAD than in EFAS. A decrease of total postheparin lipolytic activity was observed in the two groups of CF patients as well as in siblings. It was entirely accounted for by hepatic lipase (mumol FFA/ml per h) which was more severely diminished in EFAD (2.8 +/- 0.6) than in EFAS (4.4 +/- 0.7) and SIB (5.1 +/- 0.5). Although the two groups of CF children differed in terms of growth, severity of malabsorption, and vitamin E status, these data suggest that disturbance of lipoprotein concentration, composition, size, and metabolism (hepatic lipase) may be in part related to EFA deficiency. Further studies are necessary to explore the effect of EFA deficiency on hepatic lipase activity.

Adolescent↗

PubMed1988

Arrangement of desmin intermediate filaments in smooth muscle cells as shown by high-resolution immunocytochemistry.

To gain additional information about the arrangement of intermediate filaments (IF) in normal smooth muscle, fresh avian gizzard was processed for immunoelectron microscopy. The protein A-gold immunocytochemical technique was applied for the localization of desmin antigenic sites. Desmin-containing IFs were located in an axial bundle that partially surrounds the nucleus and were associated with numerous mitochondria near the poles of the nucleus. The bundle probably extends the length of the cell. Antibody labeling also showed concentrations of IF around and between cytoplasmic dense bodies (CDB) and also between CDB and membrane-associated dense bodies (MADB). The relationship between the axial bundle and the nucleus and associated mitochondria suggests that the bundle may support and define the position of these organelles in the cell. A fraying or branching of the bundle may integrate the bundle into the remaining cytoskeletal network of the cell.

Animals↗

PubMed1988

A correlated morphometric and cytochemical study on hepatocyte nucleolar size and RNA distribution during vitellogenesis.

The enzyme-gold cytochemical technique was used to label RNA in the nucleolus and rough endoplasmic reticulum (RER) of the hepatocytes of normal, male American bullfrogs (Rana catesbeiana) and in bullfrogs eight days following treatment with estradiol-17 beta. Concurrently, stereology was applied to quantitate: (1) the density of RNA labelling, and (2) changes in the size of the nucleus and nucleolus in response to estrogen treatment. In the hepatocytes from untreated frogs, specific labelling for RNA was present over the fibrillar and, to a greater extent, the granular portions of the nucleolus, and, to the greatest extent, over the RER. Following estrogen treatment, the density of RNA labelling increased over both parts of the nucleolus but was unchanged over the RER. The size of the nucleolus enlarged in response to estrogen: in combination with the increase in its RNA labelling, this suggested an increase of about 80% in the total amount of RNA in the nucleolus. Previous data on enlargement of the RER compartment, along with the present data on RNA labelling of RER, suggested that the total amount of this nucleic acid increased about 430% in this entity, in response to estrogen. However, the density of RNA labelling over the RER appears to be constant in spite of changes in the amount of RER.

Animals↗

PubMed1988

Lipid abnormalities in pancreatic tissue of streptozotocin-induced diabetic rats.

Pancreatic lipid and fatty acid composition was determined in streptozotocin-induced diabetic rats and compared to control and insulin-treated diabetic rats. A pronounced decrease of total fatty acids was recorded and mainly accounted for by a drop of fatty acids in glycerides. Cholesterol, on the other hand, was significantly increased two- to three-fold, leading to an elevated cholesterol/phospholipid ratio. Morpho-cytochemical studies confirmed these findings because the multiple lipid droplets present in acinar cells of diabetic animals were found to be of cholesterol nature. The major alterations in phospholipid-fatty acid composition were characterized by an increase of linoleate coupled to a decrease of monounsaturates and arachidonate, suggesting defective metabolism of saturates and of linoleate. This was further supported by fatty acid ratios that suggested low delta 5 and delta 9 desaturation. Daily administration of insulin for 10 days restored and overcorrected the various lipid alterations. This study suggests that there are alterations in lipid composition and metabolism in the exocrine pancreas of chronic streptozotocin-induced diabetic rats. Their possible role in the mechanism regulating pancreatic function and secretion remains to be elucidated.

Animals↗

PubMed1988

Immunocytochemical and biochemical evaluation of pancreatic lipase in acinar cells of control and streptozotocin-induced diabetic rats.

Pancreatic lipase was revealed by immunocytochemistry and analyzed biochemically in pancreatic tissue from control, diabetic, and insulin-treated diabetic rats. In the three groups of animals, lipase antigenic sites were detected with high resolution in the acinar cells in the compartments involved in protein secretion: rough endoplasmic reticulum, Golgi apparatus, and secretory zymogen granules. The quantitative evaluation of the intensities of labeling has demonstrated that, in contrast to other pancreatic proteins, lipase is concentrated only at the transition between the Golgi apparatus and the condensing vacuoles. This indicates that, although sharing the same secretory pathway as amylase and chymotrypsinogen, lipase may in fact be processed differently. On the other hand, when compared with controls, lipase immunolabelings in tissues with diabetic condition were higher in all the cellular compartments. Treatment of diabetic animals with insulin was found to restore these levels to those obtained in control condition. The biochemical determination of lipase activities in pancreatic tissues confirmed the immunocytochemical data. These results, together with those obtained previously for amylase and chymotrypsinogen, indicate that in diabetic condition secretion from the acinar cells is significantly altered, which may influence intestinal digestion and absorption processes. These modifications, and the enhancement of lipase in particular, could play a role in the pathogenesis of the hyperlipidemic condition present in diabetes.

Animals↗

PubMed1988

Protein G-gold complex: comparative evaluation with protein A-gold for high-resolution immunocytochemistry.

We combined the protein G-gold complex with several polyclonal and monoclonal antibodies for localization of various antigenic sites. The labelings were compared with those obtained using the protein A-gold complex. The results from either the immunodot experiment or immunoelectron microscopy have demonstrated that, for rabbit and guinea pig antibodies, both protein G-gold and protein A-gold complexes label several different specific antibodies with similar efficiency. However, with antibodies raised in goats or in mice, and particularly with mouse monoclonal antibodies, protein G-gold yielded intense and specific labeling, whereas protein A-gold yielded intense and specific labeling, whereas protein A-gold was very variable; it either gave weaker signals or failed to reveal any specific site or, as with one monoclonal, both protein G and protein A gave similar results. The higher affinity and versatility of protein G over protein A, established by the immunochemical approach, was confirmed by immunocytochemistry. Because of its enhanced reactivity with monoclonal antibodies and its broader affinity for polyclonal antibodies, protein G-gold complex appears to be a better and more versatile probe for high-resolution immunocytochemistry.

Amylases↗

PubMed1988

High-resolution cytochemistry of neuraminic and hexuronic acid-containing macromolecules applying the enzyme-gold approach.

We localized acidic glycoconjugates at the ultrastructural level by applying the enzyme-gold approach. Neuraminidase and hyaluronidase were adsorbed to colloidal gold particles and applied to tissue sections under optimal conditions for their enzymatic activity. Neuraminidase-gold labeling was distributed over the Golgi apparatus and associated secretory granules in exocrine pancreatic cells and duodenal goblet cells. Mitochondria were labeled over inner membranes. Labeling was also found over the dispersed chromatin in the nucleus. Plasma membranes, particularly the apical side, were labeled by gold particles. On the other hand, incubation of tissue sections with the hyaluronidase-gold complex resulted in intense labeling of the rER membranes, the plasma membrane, and the dense chromatin in the nucleus. Labeling was also found over the Golgi apparatus and associated secretory granules, but only in duodenal goblet cells. Specificity of the results was confirmed by various control experiments performed, indicating that the enzyme-gold technique is useful for detecting linked-sugar residues on tissue thin sections. Labelings found over intra- and extracellular compartments in the present work are discussed in light of previous biochemical indications as well as of other histochemical detections of these glycoconjugates.

Animals↗

PubMed1988

Circulating lipids and lipoproteins in glycogen storage disease type I with nocturnal intragastric feeding.

With the advent of nocturnal intragastric feeding which protects against acute metabolic complications and promotes growth, patients with glycogen storage disease type I are attracting less attention. However, several biochemical alterations persist and suggest that the long-term risk of atherosclerotic heart disease remains high. Persisting hypertriglyceridemia and hypercholesterolemia were found in seven glycogen storage disease type I subjects, six of them following 5-6 yr of nocturnal intragastric feeding. When compared to ten age-matched controls, the patients showed significantly (P less than 0.001) higher low density lipoprotein cholesterol (LDL-C) (247.7 +/- 46.8 vs. 115.3 +/- 5.0 mg/dl) and lower high density lipoprotein cholesterol (HDL-C) (26.4 +/- 3.4 vs. 55.8 +/- 2.9 mg/dl). Triglyceride (TG) enrichment with cholesteryl ester depletion characterized the lipoprotein classes. The diameters of very low density lipoproteins (VLDL) and LDL were larger, while that of HDL was smaller and consistent with the predominance of the HDL3 subclass and a lower apoA-I/apoA-II ratio. The raised levels of TG appeared attributable not only to the well-described lipogenesis, but also to impaired catabolism of fat, as evidenced by the significantly (P less than 0.001) decreased activity of both peripheral lipoprotein lipase (3.17 +/- 0.43 vs. 14.15 +/- 0.50 mumol FFA.ml-1.hr-1) and hepatic lipase (1.88 +/- 0.30 vs. 4.83 +/- 0.90). This may well explain the high concentration of intermediate density lipoprotein (IDL) and the impaired conversion of HDL3 to HDL2. Low apoC-II/apoC-III1 could be related to defective lipoprotein lipase activity. These data suggest that glycogen storage disease type I patients on nocturnal intragastric feeding remain at risk for atherosclerosis and its complications.

Adolescent↗

PubMed1988

Introduction of a high-resolution cytochemical method for studying the distribution of phospholipids in biological tissues.

A novel cytochemical method for the in situ, ultrastructural localization of phospholipids in biological tissues is reported. The method is based on the enzyme-gold approach (M. Bendayan: J. Histochem. Cytochem. 29, 531, 1981). Phospholipase A2 from bee venom was adsorbed on colloidal gold particles (PLA2-gold) and applied for the specific labeling of its substrate, sn3-glycerophospholipids. The binding and enzymic competence of the PLA2-gold complex were confirmed by in vitro, preembedding experiments with erythrocytes and a crude lung surfactant preparation. The substrate specificity of the probe was assessed by labeling Epon thin sections of pure phospholipids. To test the potential applications of the PLA2-gold complex, lung and pancreatic tissues were fixed with glutaraldehyde-osmium and embedded in Epon for transmission electron microscopy (TEM). They were also prepared for critical-point-drying fracture-label (CPD-FL) replicas and thin-section fracture-label (TS-FL) specimens. On TEM thin sections incubated with PLA2-gold, all cellular membranes were labeled. The labeling density over each membrane compartment, as quantitated in lung type II pneumocytes, was classified in order of magnitude as follows: a) nuclear membranes; b) outer mitochondrial membrane and rough endoplasmic reticulm (RER); and c) Golgi complex, mitochondrial cristae and plasma membranes. In lung alveoli, the phospholipid-rich surfactant material was intensely labeled. Labeling of lung thin sections from chlorphentermine-treated rats (phospholipidosis-inducing drug) further demonstrates the reliability of PLA2-gold to label phospholipids. CPD-FL replicas and TS-FL specimens further extended the TEM observations: nuclear membranes and RER were more intensely labeled than plasma membranes. In exocrine pancreatic cells, two distinct labeling patterns were found for secretory granule membranes: sparse and dense. The specificity and reliability of the labeling were confirmed through several control experiments. The studies performed thus demonstrate the great potential of the PLA2-gold technique as a new approach to the high-resolution study of phospholipid distribution and density among biological structures.

Animals↗

PubMed1987

The interaction of rat liver carbamoyl phosphate synthetase and ornithine transcarbamoylase with inner mitochondrial membranes.

The intramitochondrial localization of the urea cycle enzymes, carbamoyl phosphate synthetase and ornithine transcarbamoylase, has been examined by both in vitro and in situ studies. The following three lines of evidence are presented to establish that significant fractions of the rat liver enzymes are loosely associated with the inner mitochondrial membrane: 1) when the mitochondrion is fractionated, the enzymes partition between the matrix and membrane fractions in the absence of detergent and partition solely to the matrix in the presence of detergent; 2) the purified enzymes associate with purified inner membrane preparations; and, 3) protein A-gold electron microscopic immunocytochemical analysis of rat liver sections reveals a nonrandom arrangement of the enzyme, with the maximal enzyme density adjacent to the inner mitochondrial membrane. These findings serve as the basis for novel potential mechanisms for regulation of the activity of the enzymes and provide additional evidence for the extensive organization of the mitochondrial matrix. The membrane interaction might also serve as the organizing factor for a carbamoyl phosphate synthetase-ornithine transcarbamoylase or other multienzyme complex.

Animals↗