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M Bendayan

Publications and source records attributed to M Bendayan.

At least 181 records · Page 10Linked to original sources
PubMed1983

Intracellular transport and storage of secretory proteins in relation to cytodifferentiation in neoplastic pancreatic acinar cells.

The pancreatic acinar carcinoma established in rat by Reddy and Rao (1977, Science 198:78-80) demonstrates heterogeneity of cytodifferentiation ranging from cells containing abundant well-developed secretory granules to those with virtually none. We examined the synthesis intracellular transport and storage of secretory proteins in secretory granule-enriched (GEF) and secretory granule-deficient (GDF) subpopulations of neoplastic acinar cells separable by Percoll gradient centrifugation, to determine the secretory process in cells with distinctly different cytodifferentiation. The cells pulse-labeled with [3H]leucine for 3 min and chase incubated for up to 4 h were analyzed by quantitative electron microscope autoradiography. In GEF neoplastic cells, the results of grain counts and relative grain density estimates establish that the label moves successively from rough endoplasmic reticulum (RER) leads to the Golgi apparatus leads to post-Golgi vesicles (vacuoles or immature granules) leads to mature secretory granules, in a manner reminiscent of the secretory process in normal pancreatic acinar cells. The presence of approximately 40% of the label in association with secretory granules at 4 h postpulse indicates that GEF neoplastic cells retain (acquire) the essential regulatory controls of the secretory process. In GDF neoplastic acinar cells the drainage of label from RER is slower, but the peak label of approximately 20% in the Golgi apparatus is reached relatively rapidly (10 min postpulse). The movement of label from the Golgi to the post-Golgi vesicles is evident; further delineation of the secretory process in GDF neoplastic cells, however, was not possible due to lack of secretory granule differentiation. The movement of label from RER leads to the Golgi apparatus leads to the post-Golgi vesicles suggests that GDF neoplastic cells also synthesize secretory proteins, but to a lesser extent than the GEF cells. The reason(s) for the inability of GDF cells to concentrate and store exportable proteins remain to be elucidated.

Animals↗
PubMed1983

Ultrastructural localization of Ag-NOR proteins and nucleic acids in reticulated nucleoli.

Nucleoli with a thread-like fibrillar structure found in several human breast cancer cells were investigated for the localization of Ag-NOR proteins, RNA, DNA and associated nucleoproteins. Various cytochemical techniques were applied: the one-step silver method for Ag-NOR proteins, the Bernhard's staining preferential for RNP, the Feulgen-like osmium-ammine reaction for DNA and the RNase- and DNase-gold complexes for the demonstration of RNA and DNA respectively. Staining for Ag-NOR proteins was restricted to the edge of the threads or cords and to the nodes of the nucleolar network. The Bernhard's EDTA technique gave a positive staining of the entire nucleoli and on small peripheral granules which were linked to the nucleolar cords by fine RNP fibrils. Localization of RNA molecules by the RNase-gold complex showed a similar pattern, dense labeling by gold particles over the nucleoli and at the periphery of the cords. DNA molecules were revealed by the osmium-ammine technique in small nucleoplasmic clumps and at the edge of the nucleolar cords. Similar results were obtained by applying the DNase-gold complex which showed a labeling by gold particles over the dispersed chromatin and at the edge of the nucleolar cords. The consistency of the results obtained speaks in favor of their specificity and seems to demonstrate the simultaneous localization of certain Ag-NOR proteins, DNA and RNA molecules at the edge of the nucleolar cords. This edge of nucleolar cords becomes then the candidate for the site of nucleolar transcriptional activity.

Breast Neoplasms↗
PubMed1983

Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique.

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.

Amylases↗
PubMed1983

Immunocytochemical localization of fatty acid metabolizing heat-stable and heat-labile enoyl-coenzyme A (CoA) hydratases in liver and renal cortex.

Two enzymes, the heat-stable and the heat-labile enoyl-coenzyme A (CoA) hydratases, involved in the metabolism of fatty acids were localized in liver and renal cortex using specific antibodies, immunofluorescence, and the protein A-gold immunocytochemical technique. The qualitative and quantitative results have demonstrated that the heat-stable enoyl-CoA hydratase is a mitochondrial membrane-associated protein of hepatocytes and of epithelial cells in proximal and distal renal tubules. The hepatic sinusoidal cells, as well as the endothelial and epithelial cells of the glomeruli, fail to demonstrate any specific labeling. The heat-labile enoyl-CoA hydratase, on the other hand, was detected in the peroxisomal matrix of hepatocyte and proximal tubule epithelial cells. Its distribution was identical to that of catalase. The significance of the differential distribution of peroxisomal and mitochondrial enoyl-CoA hydratases is discussed in relation to their function.

Animals↗
PubMed1982

Contacts between endocrine and exocrine cells in the pancreas.

Close contacts between exocrine and endocrine cells were observed in human and rat pancreas. The presence of junctional specializations, including desmosomes, tight and gap junctions, as well as interdigitations between endocrine and exocrine cells, implies that these cells are structurally and functionally associated.

Cell Adhesion↗
PubMed1982

Alterations of exocrine pancreatic enzymes in virus-induced diabetic cattle as revealed by immunohistochemistry.

The pancreatic tissue of normal and virus-induced diabetic cattle was investigated by the indirect immunofluorescence technique. Seven secretory proteins (chymotrypsinogen A, trypsinogen, carboxypeptidase A, RNase, DNase, alpha-amylase and lipase) were localized in normal bovine pancreatic acinar cells but in diabetic animals amylase, lipase and carboxypeptidase were either not detectable or markedly diminished. Decrease in amylase content has been reported previously in other diabetic animals. The diminution of the three pancreatic enzymes may be related to the destruction of pancreatic endocrine tissue that occurs in these diabetic animals.

Amylases↗
PubMed1982

Ultrastructural localization of viral antigens using the protein A-gold technique.

HSV and DNV viral antigens have been localized by electron microscopy using the protein A-gold technique. The labelling of HSV antigens was detected over the (naked and enveloped) viral particles as well as on the cytoplasm and the nucleoplasm. In contrast, DNV antigens were revealed only over clusters of viral particles in the nucleus. The high sensitivity of the technique and the good ultrastructural preservation allowed a very fine identification of the labelled structures. Thus, the protein A-gold technique can be applied generally for the ultrastructural detection and identification of viral antigens and might be useful for diagnostic purposes.

Animals↗
PubMed1982

Double immunocytochemical labeling applying the protein A-gold technique.

In the present study we report the modifications and the different steps of the protein A-gold (pAg) technique that allow the simultaneous demonstration of two antigenic sites on the same tissue section. The labeling is carried out in the following manner: face A of the tissue section is incubated with an antiserum followed by a pAg complex prepared with large gold particles; face B of the same tissue section is then incubated with a second antiserum followed by a pAg complex prepared with small gold particles. Each of the pAg complexes reveals a different antigenic site on opposite faces of the tissue section. The transparency of the section in the electron beam allows the visualization of the gold particles present on both faces. The double labeling pAg technique was applied for the simultaneous demonstration of two secretory proteins in the same Golgi, condensing vacuoles, and zymogen granules of the rat pancreatic acinar cells.

Amylases↗
PubMed1982

Immunocytochemical localization of kallikrein in the rat exocrine pancreas.

The subcellular localization of kallikrein was studied in the rat pancreas using the immunocytochemical protein A-gold technique. Kallikrein was found at the level of the rough endoplasmic reticulum (RER), Golgi cisternae, condensing vacuoles, and zymogen granules of the pancreatic acinar cells as well as in the acinar lumen. The effect of various tissue processings on the immunocytochemical labeling of kallikrein was evaluated using pancreatic tissue fixed in glutaraldehyde and embedded in Epon, Lowicryl K4M, or glycol methacrylate (GMA). Compared to the results obtained with Epon, Lowicryl allowed improved resolution and specificity in the immunocytochemical labeling, while GMA retained greater amounts of kallikrein antigenicity leading to a higher intensity in the labeling; since it also gave a good ultrastructural preservation, GMA appeared to be the superior embedding medium for the localization of kallikrein. The quantitative evaluation of the labeling obtained under the three embedding conditions showed the presence of an increasing concentration gradient along the RER-Golgi-granule secretory pathway, suggesting that, like other pancreatic exocrine enzymes, kallikrein is synthesized in the RER, processed through the Golgi apparatus, and packed in the zymogen granules before being released into the acinar lumen.

Animals↗
PubMed1982

Immunocytochemical localization of actin in the pancreatic exocrine cell.

Immunoreactive actin molecules have been localized in pancreatic acinar cells by the protein A-gold technique. The labeling was found at the level of the filamentous cell web, and in close association with the Golgi cisternae, condensing vacuoles, and zymogen granules delimiting membranes as well as with the plasma membrane. A weak labeling was also present over the dense content of the zymogen granules. The association of actin with different membranes implicates that contractile proteins might constitute structural membrane proteins and, thus, might play an important role in protein secretion.

Actins↗
PubMed1982

Immunocytochemical localization of mitochondrial proteins in the rat hepatocyte.

Two mitochondrial proteins, carbamyl phosphate synthetase (CPS) and a structural membrane protein, OMM-35, were specifically localized in the hepatocyte mitochondrial matrix and inner and outer membranes, respectively, using the protein A-gold technique. Three embedding media, Epon, glycol methacrylate (GMA), and Lowicryl K4M, were tested for their ability to provide good ultrastructural preservation of mitochondrial membranes, while at the same time retaining protein antigenicity in embedded liver. Epon embedding proved to be relatively unsuitable, since mitochondrial membranes were poorly preserved. GMA and Lowicryl however gave excellent ultrastructural preservation and retained protein antigenicity sufficiently well to enable the localization of the structural membrane protein. Both qualitative and quantitative immunocytochemical demonstration of CPS have ascertained its localization to the rat hepatocyte mitochondrial matrix. The enzyme was undetectable, however, in the mitochondria of liver endothelial cells. OMM-35 was specifically located in the mitochondrial membranes and the quantitative evaluation confirms the biochemical data that OMM-35 is clearly enriched in the outer mitochondrial membrane. OMM-35 was detected in mitochondria of both hepatocytes and endothelial cells. The labeling of a relatively minor structural membrane protein such as OMM-35 gives and indication of the high sensitivity of the protein A-gold immunocytochemical technique.

Animals↗
PubMed1982

Immunocytochemical localization of catalase and heat-labile enoyl-CoA hydratase in the livers of normal and peroxisome proliferator-treated rats.

The intracellular localization of catalase and the heat-labile enoyl-CoA hydratase (second enzyme of the peroxisomal fatty acid beta-oxidation spiral) has been investigated using the protein A-gold immunocytochemical procedure in normal and peroxisome proliferator-treated rat livers. Peroxisome proliferation in rat liver was induced by the dietary administration of Wy-14,653 ([4-chloro-6-(2,3-xylidino)2-pyrimidinylthio]acetic acid). As expected, catalase was demonstrable exclusively in the matrix of all peroxisomes in hepatic parenchymal cells of normal and peroxisome proliferator-treated rats. The heat-labile enoyl-CoA hydratase, which was shown previously to be immunochemically identical with 80,000-molecular weight peroxisome proliferation-associated polypeptide, was also confined to the peroxisome matrix. The peroxisome nucleoids displayed no antigenic sites for any of these proteins. Both qualitative and quantitative evaluation of immunocytochemical labeling of catalase provide direct visual evidence for the decreased amount of this enzyme in proliferated peroxisomes when compared with normal peroxisomes. In contrast, the proliferated peroxisomes contained higher levels of heat-labile enoyl-CoA hydratase.

Animals↗
PubMed1981

Biogenesis of a 35-kilodalton protein associated with outer mitochondrial membrane in rat liver.

Biochemical analyses following subcellular fractionation of rat liver indicated that the outer mitochondrial membrane contains a number of membrane-specific proteins of which a 35-kilodalton species (OMM-35) is a prominent component. These results were confirmed and extended by electron microscope immunocytochemical tests based on the protein A-gold technique. OMM-35 is tightly bound to the outer mitochondrial membrane, e.g. it was not released by sonication in the presence of 1.5 M KCl and 0.1% sodium deoxycholate. However, it did not react with the photoaffinity probe azidopyrene, which indicates that OM-35 is located peripherally on the membrane rather than buried deep in the lipid bilayer as an intrinsic protein. Since low levels of detergent were required for OMM-35 in intact mitochondria to react with exogenous antibodies, OMM-35 is probably located on the side of the outer membrane which faces the interior of the organelle. When rat liver mRNA was translated in a messenger-dependent cell-free system derived from rabbit reticulocytes, antiserum against OMM-35 precipitated a single polypeptide product which migrated on sodium dodecyl sulfate-polyacrylamide gels with molecular weight characteristics of a protein slightly larger (by Mr = 500) than OMM-35 obtained from isolated outer mitochondrial membrane. The mRNA coding for OMM-35 was recovered exclusively from membrane-free polysomes. Thus, the route followed for synthesis and subsequent insertion of OMM-35 into the outer membrane of mitochondria is the post-translational pathway which has been previously described for proteins destined for the interior compartments of this organelle.

Animals↗
PubMed1981

Electron microscopical localization of nucleic acids by means of nuclease--gold complexes.

Nucleic acids can be specifically localized at the electron microscope level by means of enzyme--gold complexes. Two enzymes RNAase A and DNAase I were labelled with colloidal gold, and the enzyme--gold complexes obtained applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Using RNAase--gold, the rough endoplasmic reticulum and the nucleolus of different cells appeared densely labelled. With the DNAase--gold the labelling was present over the euchromatin and the mitochondria. The quantitative evaluation, performed on different cellular compartments of the pancreatic acinar cells, confirmed the qualitative observations and ascertained the specificity of the labelling. The application of this technique, for the demonstration of nucleic acids in different tissues, is illustrated.

Animals↗
PubMed1981

Ultrastructural localization of nuclei acids by the use of enzyme-gold complexes.

A cytochemical technique for the ultrastructural localization of substrates using enzyme-gold complexes is reported. RNase A and DNase I have been labeled with gold particles. The RNase-gold and dNase-gold complexes obtained were applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Different cellular compartments were labeled by these enzyme-gold complexes. Using the RNase-gold complex the rough endoplasmic reticulum appeared decorated with gold particles. The gold marker was also present over the nucleus, especially over the nucleolus; mitochondria were weakly labeled. Using the DNase-gold complex, gold particles were concentrated over the euchromatin of the nucleus and the mitochondria. The heterochromatin and the nucleolus showed a less intense labeling. For both enzyme-gold complexes, the Golgi area, the secretory granules and the extracellular space appeared free of label. In those control conditions where the substrates were added to the enzyme-gold complexes a major reduction in the labeling was observed. A quantitative evaluation of the labeling was performed. This evaluation confirmed the qualitative observations and the marked reduction of labeling occurring under the control conditions. The combination of the specificity of the enzyme-substrate interactions with the size and electron density of the gold particles and the good ultrastructural preservation of the tissues resulted in a very specific labeling with high resolution. These results demonstrate the possibility of detecting substrates by means of enzyme-gold complexes at the electron microscope level.

Animals↗
PubMed1981

Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue.

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.

Animals↗