Biomedical subjects

M Bendayan

Publications and source records attributed to M Bendayan.

At least 163 records · Page 9Linked to original sources

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↗

PubMed1985

Alteration in the distribution of type IV collagen in glomerular basal laminae in diabetic rats as revealed by immunocytochemistry and morphometrical approach.

The glomerular basal laminae of normoglycaemic and long-term streptozotocin-induced hyperglycaemic rats was studied by morphometrical and immunocytochemical approaches. Using the orthogonal intercept method, we have confirmed that in long-term diabetes, the thickness of the glomerular basal laminae increases significantly. Applying the high resolution protein A-gold immunocytochemical technique, type IV collagen was localized in the glomerular basal laminae. In tissues from normoglycaemic animals the labelling was present over the central lamina densa. However, the labelling obtained over the thickened glomerular basal laminae of the hyperglycaemic animals was restricted to the subendothelial site of the lamina. Thus major alteration in the distribution of type IV collagen occurs during the development of diabetic microangiopathy in hyperglycaemic animals.

Animals↗

PubMed1985

Simultaneous labelling of basal lamina components and acetylcholinesterase at the neuromuscular junction.

A double labelling technique has been developed which permits the concomitant localization of basal lamina constituents together with acetylcholinesterase in mouse skeletal muscles. First, using the protein A-gold technique, type IV collagen and laminin were revealed on basal laminae ensheathing skeletal muscle fibres. The immunolabelling for both proteins was higher in synaptic than extrasynaptic regions. At synaptic sites the anti-type IV collagen immunolabelling exhibited an asymmetry; it was more intense on the portion of basal lamina closest to the postsynaptic membrane, whereas the anti-laminin immunolabelling was more uniformly distributed. It was also observed that the laminin immunoreactivity associated with Schwann and perineural cells was higher than that of skeletal muscle fibres. Secondly, the two basal lamina antigens were revealed simultaneously with another synaptic protein, acetylcholinesterase, using a refined cytochemical technique prior to the immunolabelling. The cytochemical reaction, which facilitates the location of endplates, did not alter the immunolabelling pattern. This double labelling procedure permits ready comparison of the distributions of type IV collagen and laminin with that of acetylcholinesterase, and may prove to be a useful approach in studies on synaptic components in developing and diseased muscle.

Acetylcholinesterase↗

PubMed1985

PSL, an S phase-related p55 nuclear antigen, associates transiently with chromatin.

An S phase-related nuclear 55K antigen, also designated PSL, has been characterized in various mammalian cells, using a human serum from a patient with autoimmune disorders (Barque et al., EMBO j 2 (1983) 743). In this report, we show by immunoelectron microscopy that the p55 protein associates in situ with the chromatin of rat hepatocytes. This association is a transient one, as indirect immunofluorescence studies show that PSL does not bind to individualized metaphase chromosomes. Furthermore, immunoprecipitation tests indicate that the majority of PSL is in the non-chromosomal cell fraction. These results suggest that this nuclear antigen is directly involved in the DNA replication process.

Animals↗

PubMed1985

Morphometrical and immunocytochemical studies on rat pancreatic acinar cells under control and experimental conditions.

Process of amylase and chymotrypsinogen secretion by acinar cells has been studied applying morphological and biochemical approaches. Three conditions were investigated; resting (fed control), cholinergic stimulation and fasting. Morphometrical evaluations have shown that under stimulation, the volume density of zymogen granules decreases drastically while that of the Golgi apparatus increases. This may result from the enhancement in protein processing and the rapid discharge. Quantitation of amylase and chymotrypsinogen immunolabelings present over the cellular compartments has shown that there is no difference in the intensities between tissues from control and stimulated animals. These results imply that total amounts of protein processed by the Golgi apparatus are markedly enhanced primarily because of the increase in size of the organelle, the amounts of protein processed per unit surface remaining unchanged. Under starvation where reduction of secretion occurs, there is a significant decrease in the volume density of the Golgi apparatus but no variation in that of the zymogen granules. However, the morphological aspect of these was markedly altered since many of them present an electron luscent periphery which was devoid of immunolabeling for amylase and chymotrypsinogen. Quantitation of amylase and chymotrypsinogen immunolabelings has shown significant diminution for both enzymes. In both experimental conditions, the volume density of lysosomes was enhanced, however in none of these conditions evidence of crinophagy was observed. The morphometrical and immunocytochemical results were consistent with those obtained from biochemical determination of amylase and chymotrypsinogen contents in tissues. Correlations between results obtained from morphometric and immunocytochemical studies were made leading to a better understanding of the cellular secretory activity during experimental conditions.

Amylases↗

PubMed1985

Ultrastructural localization of cytoskeletal proteins in pancreatic secretory cells.

Actin, myosin, and keratin immunoreactive sites have been localized with high resolution in pancreatic exocrine cells, by applying the protein A-gold technique on tissues processed at low temperature conditions. The labeling by gold particles was found at the level of the cell web and closely associated with the limiting membranes of the immature and mature secretory granules, as well as those of the "trans" cisternae of the Golgi apparatus. These results, together with those obtained in the study on the localization of secretory proteins in exocrine pancreatic cells, demonstrate that cytoskeletal proteins are present at sites where maturation and (or) concentration of the secretory proteins occur. Thus, besides the role that cytoskeletal proteins must play in the transport of the secretory granules from the Golgi to the plasma membrane, they may also be involved in the process of protein maturation and (or) concentration.

Actins↗

PubMed1985

Enamel protein biosynthesis and secretion in mouse incisor secretory ameloblasts as revealed by high-resolution immunocytochemistry.

Mouse secretory ameloblasts express a number of enamel proteins, which have been divided into amelogenin and enamelin subfamilies. We have used polyclonal antibodies to murine amelogenins to reveal enamel proteins in mouse ameloblasts using the protein A-gold immunocytochemical technique. Specific immunolabeling was detected over the extracellular enamel matrix and over the rough endoplasmic reticulum, the saccules of the Golgi apparatus, and the secretory granules of the ameloblasts. In addition, some lysosome-like granules were also labeled. Only background labeling was obtained over mitochondria, nuclei, cytosol, adjacent odontoblasts, and dentin. Quantitation of the intensity of labeling showed the presence of an increasing gradient along the secretory pathway, which may correspond to the concentration or the maturation of these proteins as they are processed by the cell. These findings indicate that the ameloblast displays an intracellular distribution of its secretory products similar to that of other merocrine secreting cells. The presence of enamel proteins in lysosomes suggests that crinophagy and/or resorption occurs in these cells.

Ameloblasts↗

PubMed1985

Morphometrical and immunocytochemical characterization of peri-insular and tele-insular acinar cells in the rat pancreas.

Morphometrical and immunocytochemical techniques have been applied in order to characterize the pancreatic acinar cells located in peri-insular and tele-insular regions of the pancreas. The results obtained, have shown that the acinar cells of the peri-insular regions are twice as large as those of the tele-insular. On the other hand, the volume density of all organelles, except that of the zymogen granules, remains constant implying that the larger the cell, the larger are its organelles. For the zymogen granules however, their volume density was found to be higher in peri-insular acinar cells. The immunofluorescence technique applied for the demonstration of amylase and chymotrypsinogen has confirmed the presence of an inhomogeneity in the staining. Acinar cells in peri-insular regions show a brighter fluorescent staining. At the electron microscope level, both amylase and chymotrypsinogen were demonstrated in all organelles of acinar cells involved in protein secretion. Quantitative evaluations demonstrate no major differences in the intensity of labeling per micron2 between organelles of peri-insular and tele-insular cells. These results put together demonstrate that peri-insular acinar cells contain higher amounts of secretory proteins because their organelles are larger and their zymogen granules are more numerous. The partition of the exocrine pancreas into peri- and tele-insular regions, confirmed herein through morphometrical and cytochemical techniques, is discussed in relation to the possible influence of the endocrine secretion arising from the islets of Langerhans on the surrounding acinar cells.

Amylases↗

PubMed1985

Immunocytochemical localization of a non-vitellogenin, estrogen-induced plasma protein in the hepatocyte of the American bullfrog, Rana catesbeiana.

Recently, a non-vitellogenin, estrogen-induced frog plasma protein of unknown function and site of synthesis, which has been given the temporary name, protein-RcX, was isolated and partially characterized by Mitchell et al. In the present study, the protein A-gold immunocytochemical technique was applied to investigate its site of secretion; this was found to be the hepatocyte of the estradiol-17 beta-treated adult, male American bullfrog, Rana catesbeiana. Specific immunolabeling for protein-RcX was present over those intracellular compartments involved in protein secretion, i.e., the rough endoplasmic reticulum, Golgi apparatus and secretory granules, in addition, lysosomes were also labeled. No specific labeling for this protein was observed on hepatocytes of normal, non-estrogen treated, adult, male bullfrogs. Further, the labeling was abolished when plasma containing protein-RcX was added to the antibody prior to incubation but remained when purified vitellogenin was added. These observations support the hypothesis that protein-RcX is a non-vitellogenin, estrogen-induced plasma protein which is synthesized and secreted in parallel with vitellogenin by the hepatocyte of the estrogen-treated frog.

Animals↗

PubMed1984

Concentration of amylase along its secretory pathway in the pancreatic acinar cell as revealed by high resolution immunocytochemistry.

The modified protein A-gold immunocytochemical technique was applied to the localization of amylase in rat pancreatic acinar cells. Due to the good ultrastructural preservation of the cellular organelles obtained on glutaraldehyde-fixed, osmium tetroxide-postfixed tissue, the labelling was detected with high resolution over the cisternae of the rough endoplasmic reticulum (RER), the Golgi apparatus, the condensing vacuoles, the immature 'pre-zymogen' granules, and the mature zymogen granules. Over the Golgi area, the labelling was present over the transitional elements of the endoplasmic reticulum, some of the smooth vesicular structures at the cis- and trans-faces and all the different Golgi cisternae. The acid phosphatase-positive rigid trans-cisternae as well as the coated vesicles were either negative or weakly labelled. Quantitative evaluations of the degree of labelling demonstrated an increasing intensity which progresses from the RER, through the Golgi, to the zymogen granules and have identified the sites where protein concentration occurs. The results obtained have thus demonstrated that amylase is processed through the conventional RER-Golgi-granule secretory pathway in the pancreatic acinar cells. In addition a concomitance has been found between some sites where protein concentration occurs: the trans-most Golgi cisternae, the condensing vacuoles, the pre- and the mature zymogen granules, and the presence of actin at the level of the limiting membranes of these same organelles as reported previously (Bendayan, 1983). This suggests that beside their possible role in transport and release of secretory products, contractile proteins may also be involved in the process of protein concentration.

Acid Phosphatase↗

PubMed1984

Pituitary adenomas: patterns of hPRL and hGH secretion as revealed by high resolution immunocytochemistry.

Seven human pituitary adenomas obtained by transphenoidal surgery were investigated for the intracellular localization of PRL and GH, using the protein A-gold immunocytochemical technique. Among the seven cases two were prolactinomas, two were GH-secreting adenomas and three were mixed PRL and GH-secreting adenomas. When PRL or GH were revealed, immunoreactivity was found in the cellular compartments involved in protein secretion, RER, Golgi apparatus and secretory granules of corresponding secreting cells. An increasing gradient in the intensity of labeling was observed from the RER to the Golgi and to the granules which may correspond to the increasing concentration of the proteins occurring along their secretory pathway. In addition, crinophagy or destruction of secretory granules by the lysosomal system was observed for both secretory cells. Cells displaying simultaneously PRL and GH reactivity were never found, neither in pure nor in mixed adenomas demonstrating that in the different adenomas studied, secreting cells have retained their specificity and differentiation for the secretion of a single hormone.

Adenoma↗

PubMed1984

Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues: comparative evaluation of the different labelings.

Several cytochemical techniques, such as sodium tungstate, acid hydrolysis phosphotungstic acid (HAPTA), ethylenediaminetetraacetic acid (EDTA), RNase-gold, and osmium-ammine, have been applied for the ultrastructural demonstration of nucleic acids on sections of tissues fixed in glutaraldehyde postfixed with osmium tetroxide and embedded in Epon. In order to obtain specific results, the sections had to be treated with sodium metaperiodate prior to performing the labeling protocol. The results for each method were identical to those obtained on nonosmicated tissues; the main difference being the enhancement in the ultrastructural preservation, which allowed for higher resolution. In addition to these techniques, and for comparative evaluations, DNA was also revealed by the DNase-gold approach on nonosmicated tissue sections. The consistency in the results, obtained over the nucleus with either EDTA or the RNase-gold complex for revealing RNA and those obtained with either osmium-ammine or DNase-gold for revealing DNA, supports the high specificity of the RNase-gold, DNase-gold, and osmium-ammine techniques. Furthermore, these results demonstrate the possibility of performing various cytochemical techniques on tissues processed for routine electron microscopy.

Animals↗

PubMed1984

The intracellular pathway of vitellogenin secretion in the frog hepatocyte as revealed by protein A-gold immunocytochemistry.

The protein A-gold immunocytochemical technique was applied to the localization of vitellogenin in the hepatocyte of the bullfrog, Rana catesbeiana, eight days after treatment with estradiol-17 beta. Specific labeling was present in cellular compartments involved in protein secretion and was shown to progress in sequence through RER, Golgi apparatus, immature secretory granules, and mature secretory granules. Labeling intensities were quantitated and the values ranged from 34.6 to 172 gold particles/micron 2. In contrast, low background labeling was observed over mitochondria, nuclei, lipid droplets, and bile canaliculi. These observations support the hypothesis that vitellogenin synthesis and secretion in the frog hepatocyte lies exclusively along the RER-Golgi-granule secretory pathway. In addition to the cellular compartments involved in protein secretion, labeling was found over the majority of the lysosomes. The intensity of lysosomal labeling was intermediate between that of RER and Golgi apparatus. This labeling of lysosomes may be a consequence of the high blood plasma concentrations of vitellogenin that occur in the frog model, or to the well-known crinophagy phenomenon present in secretory cells.

Animals↗

PubMed1984

Immunocytochemical localization of exocrine enzymes in rat pancreatic acinar carcinoma.

Ten pancreatic secretory proteins have been demonstrated in differentiated pancreatic acinar carcinoma cells by the protein A-gold immunocytochemical approach. The high resolution of the technique has allowed for the localization of the different proteins in the cellular compartments involved in protein secretion: RER, Golgi and secretory granules. The quantitative evaluation of the labeling for amylase has demonstrated the presence of an increasing gradient in the intensity from the RER to the Golgi and to the secretory granules which may reflect the process of protein concentration along the secretory pathway. These results, together with those obtained using the pulse-labeling autoradiographic approach, demonstrate that differentiated acinar carcinoma cells are capable of processing secretory proteins. When intensities of labeling obtained for different proteins on acinar carcinoma cells were compared to those obtained on normal pancreatic acinar cells, major differences were observed for some proteins. In addition, studies performed on the pancreatic tissue of the tumor-bearing animals have shown the presence of morphological alterations in the acinar cells.

Animals↗

PubMed1983

Ultrastructural localization of actin in muscle, epithelial and secretory cells by applying the protein A-gold immunocytochemical technique.

Actin-immunoreactive sites have been localized at the electron microscope level by the protein A-gold technique in striated and smooth muscle cells as well as in epithelial and secretory cells. The combination of the highly sensitive protein A-gold technique with the good ultrastructural preservation and retention of antigenicity obtained using low-temperature embedding conditions has allowed a very precise identification of the labelled structures with high resolution. In striated muscle cells the labelling was obtained over the myofilaments and the Z-band, mainly at its periphery. Labelling was also observed at the edge of the intercalated discs of the cardiac muscle cells. In smooth muscle cells the labelling was present over the myofilaments; the dense plaques associated with the plasma membrane were labelled at their periphery where actin filaments have been reported to anchor. In epithelial cells of the duodenum and the renal convoluted proximal tubule, the labelling occurred over the filamentous core of the microvilli and over the cell web. Gold particles were often present over, or closely associated with, the cell membrane at the tip of the microvilli or of invaginations and vesicular structures. At the level of the junctional complexes the gold particles were aligned at the edge of the dense zones. In pancreatic endocrine and exocrine secretory cells, actin-immunoreactive sites were revealed over the Golgi apparatus, mainly at the level of the inner cisternae in the maturing face over or closely associated with the membranes of the condensing vacuoles and secretory granules, and also over the plasma membrane. Microvilli and cell web were also labelled. Finally, in fibroblasts, gold particles were associated with the membrane of vesicular structures. The consistent finding of actin-immunoreactive sites closely associated with membranes of secretory granules and vesicular structures brings support to the proposal that contractile proteins might play an important role in transcellular transport and protein secretion.

Actins↗

PubMed1983

Ultrastructural detection of RNA: complementarity of high-resolution autoradiography and of RNAase-gold method.

The recently developed RNAase-gold cytochemical method and the more classical high-resolution autoradiography following incorporation of tritiated uridine, were applied for the localization of RNA molecules in thin sections of isolated liver cells cultured under control conditions or submitted to drugs known to alter the distribution of nuclear RNA. The similar pattern of labeling obtained with both techniques under the three experimental conditions studied (control, treatments with CdCl2, or actinomycin D), together with the results obtained after RNAase digestion, are a good indication of the high specificity and sensitivity of the RNAase-gold method and provide a demonstration of the complementarity of these two methods for the study of the ultrastructural distribution of nuclear RNA.

Animals↗