Biomedical subjects

M Bendayan

Publications and source records attributed to M Bendayan.

At least 145 records · Page 8Linked to original sources

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↗

PubMed1987

Immunocytochemical studies of pancreatic acinar cells from spontaneously diabetic BB Wistar rats.

Amylase in pancreatic tissue from normal and spontaneously diabetic BB Wistar rats was assessed by immunocytochemistry and analyzed by biochemical approach. Amylase immunofluorescence, in pancreatic tissues from control "non-BB" Wistar rats, gave a positive reaction. By electron microscopy, it was detected in the rough endoplasmic reticulum, Golgi apparatus, immature, and mature secretory zymogen granules of the acinar cells. Quantitative evaluations of the intensity of labeling have demonstrated the presence of an increasing gradient which followed precisely the secretory pathway. In the spontaneously diabetic BB Wistar rats, concomitant with the disappearance of insulin containing cells in the islets of Langerhans, no reaction for amylase was found in the acinar cells. Labeling for amylase was markedly reduced in the cellular organelles and the gradient along the secretory pathway was altered. In insulin-treated diabetic rats, labeling for amylase was restored. These results were in agreement with those obtained by the biochemical approach and demonstrated that, in diabetic conditions, secretion of amylase by pancreatic acinar cells is selectively impaired. This alteration, found also in pancreatic tissue from streptozotocin-diabetic rats, demonstrates that the exocrine parenchyma is under the influence of islet hormones and that both the pancreatic exocrine and endocrine tissues are closely related forming an integrated organ.

Amylases↗

PubMed1987

Immunohisto- and cytochemical studies of pancreatic enzymes in peri-insular and tele-insular acinar cells of streptozotocin-induced diabetic rats.

Previous studies have demonstrated that the exocrine pancreatic tissue is influenced by islet hormones and that under diabetic condition, amylase secretion is altered. On the other hand according to the topographical location of the acini with reference to the islet of Langerhans, the exocrine parenchyma has been partitioned into peri- and tele-insular regions. In the present study, we have analyzed by immunofluorescence and protein A-gold immunocytochemistry the distribution of two enzymes, amylase and chymotrypsinogen, on pancreatic tissues of short- and long-term diabetic rats. The alteration in amylase secretion occurring under diabetic condition was found to follow the topographical partition of the pancreas. Acinar cells, located in the peri-insular regions of the pancreas (close to the islets of Langerhans), appeared totally devoid of amylase immunolabeling while positive and negative cells were found in the tele-insular regions (at a distance from the islets). In addition, quantitation of amylase immunolabeling present in positive cells has demonstrated that although amylase is still secreted by these cells, its processing is altered. Since the partition of the exocrine parenchyma is due to the paracrine influence of islet hormones, the data obtained demonstrate and reinforce the fact that the endocrine and exocrine components of the pancreas are functionally interrelated in normal as well as in pathological conditions.

Amylases↗

PubMed1987

Presence of endocrine cells in pancreatic ducts.

Pancreatic endocrine cells were found in close contact with epithelial cells (either the centro-acinar or those lining the ducts). Junctional specializations were present between both cell types, demonstrating that they are structurally associated. In some instances, the endocrine cells present in the wall of the ducts reached the luminal space having direct contact with the pancreatic juice. These cells may well be responsible for the secretion of islet hormones directly in the duct lumen. The islet hormones present in the pancreatic juice as reported previously, seems to play a significant role in the interactions between the gut and the pancreas for optimal regulation of digestive activity.

Animals↗

PubMed1987

Ultrastructural localization of glucoside residues on tissue sections by applying the enzyme-gold approach.

The enzyme-gold approach was applied for ultrastructural localization of glucoside residues in animal and plant tissues. A beta-glucosidase-gold complex was prepared and used on thin tissue sections to reveal the corresponding substrate molecules by electron microscopy. Conditions for preparation of the complex, as well as for its application, were determined. Once applied on thin tissue sections, the glucosidase-gold complex yielded labeling over the rough endoplasmic reticulum, mainly on the ribosomal side of the membranes, and over the dense chromatin in the nucleus. Mitochondria, Golgi apparatus, and secretory granules in liver and pancreatic cells were free of gold particles. In plant cells, the labeling pattern was similar. In addition, the stroma regions of chloroplasts were densely labeled. In the extracellular space, labeling was found over the basal laminae of cells in animal tissues and over the fibrillar wall material bordering the intercellular space in plant tissues. Fungal cell cytoplasm was also labeled, as well as the membrane delineating mycoplasma-like organisms. Control conditions confirmed these labelings, demonstrating the possibility of revealing glucoside residues on tissue sections with high resolution and specificity.

Animals↗

PubMed1987

Effect of tissue processing on colloidal gold cytochemistry.

The aim of cytochemical techniques is to localize specific biochemical components in particular tissue and cell compartments. However, since preparation of tissues for structural observation results in major alterations of the properties of their components, a major problem is to retain an adequate degree of their biochemical properties as well as adequate structural preservation. In the present study, we describe results obtained using various colloidal gold cytochemical techniques on tissues processed through different approaches. We found that any manipulation of the tissue during its processing can result in modifications of tissue components, leading to problems in cytochemistry. Indeed, washing of the tissue before fixation, the nature of the fixative solution, the chemical basis of the resins, and the physical conditions of embedding can all introduce changes in tissue components which can be cytochemically demonstrated. This has been illustrated with application of the protein A-gold, lectin-gold, and enzyme-gold cytochemical techniques on tissues submitted to different processings: fixation by perfusion or by immersion; glutaraldehyde vs paraformaldehyde fixative solutions; cryo-ultramicrotomy; embedding in epoxy, GMA, Lowicryl, or LR resins. The results obtained have demonstrated that conditions for optimal labeling must be worked out for each class of binding sites, and that no single procedure can be recommended as THE best approach in cytochemistry.

Acrylic Resins↗

PubMed1987

Intestinal apoB synthesis, lipids, and lipoproteins in chylomicron retention disease.

Chylomicron retention disease is characterized by fat malabsorption, hypocholesterolemia, normal fasting triglycerides, and marked intestinal steatosis despite the presence of both plasma and intestinal apoprotein B. The defect remains unknown but presumably involves the synthesis or secretion of chylomicrons. The present investigation examines this hypothesis by studying the biosynthesis of chylomicrons in cultured jejunal explants and by defining the quantitative and qualitative abnormalities of plasma lipids and of circulating lipoproteins. Following 2-3 years of a low fat diet supplemented with medium chain triglycerides, six patients with chylomicron retention disease had significantly higher triglyceride (TG) levels coupled with a decrease in both free (FC) and esterified cholesterol (EC) as well as in essential fatty acids and phospholipids (PL) when compared to healthy controls. The low total plasma cholesterol was largely accounted for by low levels of both low density (LDL) and high density lipoprotein (HDL) cholesterol. VLDL and LDL were characterized by a diminished percentage of CE with an increase of TG while HDL contained relatively more FC as well as PL and less CE. The diameter of VLDL was larger whereas those of LDL and HDL were smaller than in normal controls. Jejunal explants, when incubated with [14C]palmitate, were capable of normal biosynthesis of TG, diglycerides, PL, and CE. These lipids, however, except for PL, were retained in the tissue and could not be secreted into the culture medium. Incubation of intestinal biopsies with [3H]leucine and [14C]mannose resulted in normal protein synthesis and reduced glycosylation. The presence of intestinal apoB-48 was confirmed by immunoblot using 2D8 antibodies. These data suggest that the intestinal defect in this disease results from a disorder of the final assembly of chylomicrons or in the mechanism of their exocytosis.

Apolipoproteins B↗

PubMed1987

Ultrastructural localization of mannoside residues on tissue sections: comparative evaluation of the enzyme-gold and the lectin-gold approaches.

Mannoside residues were revealed at the ultrastructural level in different cellular and extracellular compartments by means of the enzyme-gold and the lectin-gold approaches. For the enzyme-gold technique, an alpha-mannosidase-gold complex was prepared and conditions for the preparation of this complex as well as for its application were determined. Labeling was found over the rough endoplasmic reticulum mainly at the level of the membranes, the lumen of the cisternae being devoid of labeling. In the nucleus, the dense chromatin and the edge of the fibrillar threads in the nucleolus were intensely labeled. Few gold particles were present over the Golgi apparatus and mitochondria. The secretory granules in pancreatic cells, the peroxisomes in liver and the mucin in duodenal goblet cells were devoid of labeling. In the extracellular space, the basal lamina was labeled. Over the glomerular basal lamina, the labeling was mainly towards the epithelial side, in close contact with the podocytes. The results with the concanavalin A horseradish peroxidase (Con A-HRP)-gold technique were similar to those found with the enzyme-gold approach. Some differences were, however, detected at the level of the rough endoplasmic reticulum and the nucleus. In the endoplasmic reticulum, Con A-HRP-gold labeling was present over both the membranes and the lumen of the cisternae. In the nucleus, the labeling was mainly over the dispersed chromatin. These differences may be due to the binding of Con A not only to mannoside but also to other sugar residues as well as to the affinity of HRP-gold for some nucleoplasmic components.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Immunocytochemical studies of pancreatic acinar cells in normal and streptozotocin-induced diabetic rats.

Amylase and chymotrypsinogen in pancreatic tissue from normal and diabetic rats were revealed by immunocytochemistry and analyzed biochemically. In acinar cells of control animals, both enzymes were localized with high resolution in the rough endoplasmic reticulum, Golgi apparatus, immature and mature secretory granules. Quantitative evaluations of the intensities of labelings have demonstrated, for both enzymes, the presence of an increasing gradient which followed precisely their secretory pathway. This gradient reflects the normal processing of both proteins through secretion. In streptozotocin-induced diabetic animals, labeling for amylase in acinar cells was markedly reduced (remaining about 11% of the normal values). The gradient along the secretory pathway was abolished, indicating an alteration in the processing and secretion of amylase. On the other hand, labeling for chymotrypsinogen was significantly increased (to 170% p less than 0.0005), and its processing remained normal. In insulin-treated diabetic animals, immunolabeling for amylase was restored and the gradient re-established, indicating a normalization of the secretion. Labeling for chymotrypsinogen was reduced towards normal values. These results were found to be in agreement with those obtained by biochemical approaches and demonstrate that, in the diabetic condition, secretion of amylase is selectively impaired.

Amylases↗

PubMed1986

Distribution of endogenous albumin in the glomerular wall of streptozotocin-induced diabetic rats as revealed by high-resolution immunocytochemistry.

Endogenous albumin was revealed with high resolution in the glomerular wall of renal tissue from normoglycaemic and long-term streptozotocin-induced hyperglycaemic rats applying the protein A-gold immunocytochemical approach. In tissues from normal animals, albumin antigenic sites were detected at the level of the endothelial cell basal plasma membrane and in the subendothelial side of the lamina densa of the glomerular basal laminae. The epithelial side of the laminae was weakly labelled, while the urinary space was devoid of labelling. In the podocytes, labelling for albumin was confined to few lysosomal structures. In diabetic animals, concomitant with hyperglycaemy, low insulin levels, significant glycosuria, proteinuria and albuminuria, the glomerular basal laminae displayed the characteristic increase in thickness found in diabetic microangiopathy (404 +/- 45 nm versus 190 +/- 10 nm). Major basal laminae deposits were also found in the mesangial regions. Albumin antigenic sites were detected throughout the entire thickness of the glomerular basal laminae without any preferential accumulation at any particular site. Labelling was also found over flocculent material present in the urinary space. Numerous densely labelled lysosomal structures were present in the podocytes. The basal laminae deposits in the mesangial regions were labelled for albumin. Morphometrical evaluations made on the distribution of the labelling confirmed the qualitative observations. Two sites for albumin retention were revealed in the glomerular wall of the normal animal: the endothelial cell basal membrane (less than 10 nm) and the subendothelial side of the lamina densa (50 nm).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Characterization of pancreatic-type tissue in the liver of rat induced by polychlorinated biphenyls.

Pancreatic-type tissue induced in the livers of rats treated with polychlorinated biphenyls was characterized by transmission electron microscopy and high-resolution immunocytochemistry. The cells of pancreatic-type tissue were arranged as acini and in small groups. By electron microscopy the pancreatic-type tissue showed features very similar to normal pancreatic acinar tissue, such as well developed rough endoplasmic reticulum (RER), large numbers of mature zymogen granules, and a basally located nucleus. Protein A-gold imunocytochemical technique showed localization of amylase and trypsinogen over the zymogen granules and RER. These findings confirm that this tissue in the liver is morphologically and functionally identical to pancreatic acinar tissue.

Amylases↗

PubMed1986

Modification of the protein A-gold immunocytochemical technique for the enhancement of its efficiency.

A modification in the protein A-gold immunocytochemical technique has been introduced for amplification of the labeling. This modification consists of performing additional incubation steps with an anti-protein A antibody and the protein A-gold complex. The original antigen-antibody-protein A-gold complex was further incubated with an antibody directed against protein A and then, in a fourth step, again with protein A-gold. This multiple-step protocol results in significant enhancement of the original signal. The modified technique can be applied to either light or electron microscopy protein A-gold immunocytochemistry. The advantage of such an approach is double: it allows for either amplification of the labeling when the original signal is of low intensity or use of highly diluted antibody solutions. The modification introduced was thus found to significantly enhance the efficiency of the technique.

Amylases↗

PubMed1986

Albumin localization in the frog hepatocyte during vitellogenesis as revealed by protein A-gold immunocytochemistry.

The protein A-gold immunocytochemical technique was used to localize albumin in the hepatocyte of the normal male American bullfrog, Rana catesbeiana, and also in the hepatocyte of this animal 8 days after treatment with estradiol-71 beta. Albumin concentration in plasma also was estimated biochemically. In the normal animal, specific immunolabeling for albumin was present in the intracellular compartments involved in protein secretion, i.e., rough endoplasmic reticulum (RER), Golgi apparatus and secretory granules, and also in lysosomes. Density of labeling increased as it progressed along the secretory pathway. In the hepatocyte of the estrogen-treated frog, specific immunolabeling for albumin was also present along the entire secretory pathway and in the lysosomes. Density of labeling over the RER was similar to that seen for this organelle in normal tissue; however, no progressive increase, but rather significant decreases, in labeling density occurred further along the secretory pathway. The biochemical data demonstrated no change in the concentration of plasma albumin in the treated frog, compared with the normal one. These observations localize albumin along its secretory pathway in frog hepatocyte and demonstrate a perturbation in its secretion in response to estrogen treatment.

Albumins↗

PubMed1986

Studies on pancreatic acinar cells in tissue culture: basal lamina (basement membrane matrix promotes three-dimensional reorganization.

Rat pancreatic acinar cells have been dissociated and maintained in culture under specific conditions which allow the retention of their differentiated state and three-dimensional organization. When cultured on a basal lamina (basement membrane) matrix, the cells first formed large monolayer patches and then reorganized themselves into acini-like structures. The cells regained their polarity around luminal spaces which appeared to be sealed off by well developed junctional complexes. Typical microvilli appeared at the "apical" plasma membrane projecting themselves into the luminal spaces. The intracellular organization resembled that of the cells in situ: a well developed rough endoplasmic reticulum located towards the "base" of the cell around a nucleus; a supranuclearly positioned Golgi apparatus and numerous secretory granules located in the "apical" region of the cell. Immunocytochemistry has revealed the presence of two pancreatic enzymes, amylase and chymotrypsinogen, in the various cellular compartments involved in secretion; the rough endoplasmic reticulum and Golgi cisternae as well as in the secretory granules. Biochemical evaluations have also shown the presence of amylase in the acinar cells and culture medium. These results thus demonstrate that dissociated pancreatic acinar cells maintained in culture under specific conditions reaggregate themselves into acini-like structures and retain their differentiated morphology as well as their ability to secrete.

Animals↗

PubMed1985

Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa.

RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.

Animals↗