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Biomedical subjects

M Bendayan

Publications and source records attributed to M Bendayan.

At least 73 records · Page 4Linked to original sources
PubMed1995

Effects of second messengers on the permeability and morphology of eel rete capillaries.

The effects of second-messenger concentration changes on capillary diffusion capacity (permeability-surface area product [PS]) to cellular and paracellular tracers and on capillary ultrastructure were studied during countercurrent perfusion of the rete of the eel swim bladder. Cyclic nucleotide effects were investigated with isoproterenol, forskolin, and dibutyryl cAMP. Isoproterenol (5 x 10(-6) mol/L) did not modify water and solute permeability or capillary structure. Forskolin (10(-4) mol/L) immediately raised the concentrations of cAMP in the rete and produced interstitial edema but did not change permeability. The addition of dibutyryl cAMP (10(-6) mol/L) to the perfusate had rapid effects: it reduced the PS of [3H]water and oxygen and increased the PS of [125I]albumin, [14C]sucrose, and 22Na. No structural changes were observed. Phosphoinositide effects were studied with 1,2-dioctanoyl-sn-glycerol (DG) and phorbol 12-myristate 13-acetate (PMA). DG (10(-5) mol/L) had no effect on the permeability of the rete to water and solutes, while inducing cell membrane vacuolization. PMA (10(-5) mol/L) progressively reduced the PS of [3H]water. In contrast, PS values of [125I]albumin, [14C]sucrose, and 22Na rose gradually. Membrane vacuoles bulging into the lumen and in the cytoplasm were a common feature. The Ca2+ effect was investigated with the Ca2+ ionophore A23187. At 5 x 10(-6) mol/L, unsteady permeability changes and extensive cytolysis were observed. At 5 x 10(-7) mol/L, the PS of [125I]albumin, [14C]sucrose, and 22Na rapidly increased. The PS values for water were not modified. No structural changes were identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumins↗
PubMed1995

Electron microscopic immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic beta-cells.

Endoproteolytic cleavage of pairs of basic amino acids is the key mechanism in the specific processing of precursor hormone molecules. Two endoproteases, PC1 (or PC3) and PC2, have recently been implicated in the conversion of proinsulin. Using antibodies against these proteases and proinsulin, followed by protein A-gold complex, we performed an immunocytochemical study for precise identification of the subcellular compartments involved in the processing of insulin. Both PC1 and PC2 immunoreactivities followed a pattern of gradually increasing density along the secretory pathway, being higher in the immature granules. Proinsulin labeling was detected in the Golgi apparatus and in the coated immature secretory granules located mainly in the Golgi area. Using double labeling, we demonstrated the presence of PC1 and/or PC2 in the majority of proinsulin-rich granules. In addition, we provided evidence that PC1 and PC2 are co-localized within the same granules. Co-expression of PC1 and PC2 with proinsulin in islet beta-cells indicates that these proteases are actively involved, probably in a sequential manner, in the conversion of proinsulin into insulin.

Animals↗
PubMed1995

Reaction of advanced glycation endproducts with renal tissue from normal and streptozotocin-induced diabetic rats: an ultrastructural study using colloidal gold cytochemistry.

Advanced glycation endproducts (AGEs) are believed to play important roles in the molecular basis of long-term diabetes complications. In the present study we have revealed direct reactivity of AGEs towards kidney tissue structures. Bovine serum albumin, modified by advanced glycation and tagged with colloidal gold (AGE-BSA-gold) was applied to renal tissue sections from normal and long-term diabetic rats by a post-embedding procedure at the electron microscopic level. The AGE-BSA probes were characterized by fluorometry, spectrophotometry, free amines analysis, measurement of early glycation products, electrophoresis, and isoelectrofocusing. When applied to renal tissue sections, AGE-BSA-gold exhibited strong labeling on nuclei. At the extracellular level, glomerular basement membranes and mesangial matrix were labeled. Probes having a higher content of AGEs yielded more intense labeling. Control experiments demonstrated the specificity of the labeling obtained. Quantitative evaluation indicated little variation in reactivity among tissues from rats of different ages. However, a significant increase in reactivity was established for tissues from streptozotocin-induced long-term diabetic rats. This study thus provides direct evidence that soluble AGEs bind to cell nuclei and to structures implicated in the development of diabetic glomerular nephropathy, demonstrating higher AGE reactivity in tissues from diabetic animals.

Animals↗
PubMed1995

Immunocytochemical investigation of insulin secretion by pancreatic beta-cells in control and diabetic Psammomys obesus.

Hyperproinsulinemia is a characteristic feature of non-insulin-dependent diabetes mellitus (NIDDM) caused by pancreatic beta-cell dysfunction through a secretion-related alteration or impaired proinsulin processing. We have investigated the insulin processing and secretion in Psammomys obesus fed with low- and high-energy diets, which represent a model for diet-induced NIDDM. With a high-energy diet the animals develop hyperglycemia and hyperinsulinemia, whereas those maintained on a low-energy diet remain normoglycemic. Although a large amount of insulin immunoreactivity was detected in beta-cells of the normoglycemic compared to hyperglycemic animals, in situ hybridization for insulin mRNA demonstrated a particularly high signal in the beta-cells of the hyperglycemic animals. By electron microscopy, the beta-cells of normoglycemic animals displayed large accumulations of secretory granules, whereas those of the hyperglycemic animals contained very few granules and large deposits of glycogen. These results reflect a secretory resting condition for the cells of the normoglycemic animals in contrast to stimulated synthetic and secretory activities in the cells of the hyperglycemic ones. Using colloidal gold immunocytochemistry at the electron microscopic level, we have examined subcellular proinsulin processing in relation to the convertases PC1 and PC2. Immunolabeling of proinsulin, insulin, C-peptide, PC1, and PC2 in different cell compartments involved in beta-cell secretion were evaluated. Both PC1 and PC2 antigenic sites were detected in beta-cells of hyperglycemic Psammomys, but their labeling intensity was weak compared to the cells of normoglycemic animals. In both groups of animals, higher levels of PC2 were found in the Golgi apparatus than in the immature granules. Major decreases in proinsulin, insulin, PC1, and PC2 immunoreactivity were recorded in beta-cells of the hyperglycemic Psammomys. In addition, all these antigenic sites were detected in lysosome-like structures, revealing a major degradation process. These results suggest that the insulin-secreting cells in hyperglycemic Psammomys obesus are in a chronic secretory state during which impaired processing of proinsulin appears to take place.

Animals↗
PubMed1995

Possibilities of false immunocytochemical results generated by the use of monoclonal antibodies: the example of the anti-proinsulin antibody.

The immunocytochemical application of a monoclonal antibody (MAb) against the Arg-Arg region at the junction of the C-peptide and the insulin beta-chain of the human proinsulin molecule on rat pancreatic tissue resulted in positive immunogold labeling over the insulin- as well as the glucagon-secreting cells. In the insulin cells, the Golgi cisternae and the immature clathrin-coated granules were intensely labeled, and in the glucagon cells all the granules were labeled. Control experiments using the anti-proinsulin MAb adsorbed with proinsulin, insulin, and glucagon have confirmed the crossreactivity of this proinsulin antibody with glucagon. Furthermore, the anti-proinsulin MAb appears to crossreact not only with rat but also with human glucagon and with bovine and porcine insulin and glucagon. Examination of the amino acid sequences of proinsulin and proglucagon has shown that both molecules display this Arg-Arg dipeptide sequence, which could explain the labeling obtained on both cell types. In addition, other propeptides also display such a sequence and, indeed, positive cells were detected in rat pituitary. These results demonstrate that by immunocytochemistry this MAb, although providing very specific results, reveals non-related molecules. This is due to the fact that these molecules exhibit similar short determinants. MAbs raised against very short amino acid sequences can reveal this determinant in various molecules, generating false-positive results.

Animals↗
PubMed1994

Involvement of molecular chaperones in the aberrant aggregation of secretory proteins in pancreatic acinar cells.

Molecular chaperones have recently been shown to be accurately located along distinct cellular compartments of the secretory pathway of pancreatic acinar cells. Since the aberrant aggregation of secretory proteins leading to the formation of RER intracisternal crystals induced by DL-p-chlorophenylalanine methyl ester (CPME) comprises major changes in the sorting, selective transport, and/or posttranslational modifications of secretory proteins, we decided to investigate the possible involvement of chaperones in this phenomenon by applying the protein A-gold immunocytochemical approach. In addition to their presence in the cellular compartments of the secretory pathway, the chaperonins cpn10 and cpn60 were found to also be concentrated in the RER intracisternal crystals. In contrast, the hsp70 protein remained confined to the trans-Golgi network and was absent from the crystals. In both control and experimental conditions the three chaperones were present in mitochondria. Quantitative evaluations confirmed these observations and revealed an overall decrease in the labeling, particularly for hsp70 after CPME treatment. These labeling patterns suggest a participation of the chaperonins cpn10 and cpn60 but not of the hsp70 in the aberrant aggregation of secretory proteins leading to RER crystal formation. The role played by chaperones in this process, however, remains to be elucidated.

Amylases↗
PubMed1994

Biochemical and morpho-cytochemical evidence for the intestinal absorption of insulin in control and diabetic rats. Comparison between the effectiveness of duodenal and colon mucosa.

A combined biochemical and morpho-cytochemical investigation was carried out in order to assess insulin absorption by the duodenal and colon epithelium. Insulin was introduced in the lumen of the rat duodenum or colon in combination with sodium cholate and aprotinin. Blood analysis made at several time points has demonstrated a rapid increase in circulating levels of insulin followed by significant and consistent decreases in blood glucose. This indicates that biologically active insulin is absorbed by the intestinal mucosa and transferred to the circulation. Because of the initial high blood glucose levels, the lowering of the glycaemic values was more significant in diabetic animals. Also, levels of circulating insulin remained higher for longer time when the administration was performed in the colon. The integrity of the intestinal wall after insulin administration, evaluated morphologically, was retained. Application of protein A-gold immunocytochemistry has established the pathway for insulin absorption. In both duodenal and colon epithelial cells the labelling for insulin was detected in the endosomal compartment, in the Golgi apparatus and in association with the baso-lateral plasma membrane interdigitations. Some labelling was also present in the interstitial space and in capillary endothelial plasmalemmal vesicles. Insulin introduced in the lumen of the rat duodenum and colon appears thus to be rapidly internalized by the epithelial cells and transferred through a transcytotic pathway to the interstitial space from which it reaches the blood circulation. This exogenous insulin then induces significant decreases in plasma glucose levels which lasts for several hours. The results obtained support the possibility for the clinical development of an oral preparation of insulin.

Animals↗
PubMed1994

Compartmentalization of secretory proteins in pancreatic zymogen granules as revealed by immunolabeling on cryo-fixed and molecular distillation processed tissue.

Immunogold labeling of amylase obtained over zymogen granules of rat pancreatic acinar cells processed through cryofixation, molecular distillation drying and embedding in resins was found to be of high intensity and displayed a particular pattern. Indeed, it was concentrated in certain areas of the granules leaving others devoid of gold particles. This pattern of labeling reflects a strong compartmentalization of the secretory proteins within each granule. In order to assess this phenomenon, we have compared the intensities and the pattern of distribution of the labelings in tissues processed through: chemical fixation with embedding in various resins, cryo-ultramicrotomy and cryo-fixation followed by molecular distillation drying. Serials sections and double labeling experiments were performed for further evaluation of the results and for assessing artefactual displacement of proteins during tissue preparation. The results obtained indicate that the secretory proteins are indeed segregated within the granule which appears thus as a well organized structure. Cryo-fixation combined with molecular distillation appears thus to be superior in terms of preservation of protein antigenicity and retention of cellular components close to their living state.

Amylases↗
PubMed1994

Endocrine cells in the rat pancreatic and bile duct system: alteration in diabetes.

Endocrine cells of the pancreatic and bile duct system of the diabetic rat were characterized with reference to their influence on duct function. In streptozotocin-induced diabetic rats, the endocrine cells showed various changes in number and topographic distribution along the epithelial lining of the duct system. With the exception of insulin cells, which demonstrated a marked decrease, the number of duct endocrine cells generally increased in the duct system of the diabetic animal, particularly in the terminal portion of both the common hepatic and the accessory pancreatic ducts encompassed by the muscle sphincters. Among them, the cells secreting somatostatin, a potential peptide inducing contraction of the muscle sphincter, showed a remarkable increase in the opening portion of the common hepatic and the accessory pancreatic ducts of the diabetic animal. The duct cells producing glucagon and pancreatic polypeptide, the hormones exerting an inhibitory effect on exocrine secretion of duct and acinar cells, also increased significantly in the duct system of the diabetic animal. These results suggest that the duct endocrine cells are closely related, not only to functional properties of the duct system, but also to disorders of the pancreas and biliary tract in diabetes.

Animals↗
PubMed1994

Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells.

Three chaperones, the chaperonins cpn10 and cpn60, and the hsp70 protein, were revealed by immunochemistry and cytochemistry in pancreatic rat acinar cells. Western immunoblotting analysis of rat pancreas homogenates has shown that antibodies against cpn10, cpn60 and hsp70 protein recognize single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpn10 and cpn60 were also detected in pancreatic juice. Immunofluorescence studies on rat pancreatic tissue revealed a strong positive signal in the apical region of the acinar cells for cpn10 and cpn60, while an immunoreaction was detected at the juxtanuclear Golgi region with the anti-hsp70 antibody. Immunocytochemical gold labeling confirmed the presence of these three chaperones in distinct cell compartments of pancreatic acinar cells. Chaperonin 10 and cpn60 were located in the endoplasmic reticulum, Golgi apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 followed the increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway. On the contrary, the labeling for hsp70 was mainly concentrated in the endoplasmic reticulum and the Golgi apparatus. In the latter, the hsp70 was found to be primary located in the trans-most cisternae and to colocalize with acid phosphatase in the trans-Golgi network. The three chaperones were also present in mitochondria. In view of the role played by the chaperones in the proper folding, sorting and aggregation of proteins, we postulate that hsp70 assists the adequate sorting and packaging of proteins from the ER to the trans-Golgi network while cpn10 and cpn60 play key roles in the proper packaging and aggregation of secretory proteins as well as, most probably, in the prevention of early enzyme activation in secretory granules.

Animals↗
PubMed1993

Absence of trypsinogen autoactivation and immunolocalization of pancreatic secretory trypsin inhibitor in acinar cells in vitro.

To establish the significance of the addition of trypsin inhibitors to pancreatic acinar cells maintained in vitro, cells were cultured in the presence or absence of soybean trypsin inhibitor. Both cultures exhibited similar growth pattern, ultrastructural appearance, as well as secretory properties. Moreover, there was no evidence of trypsinogen activation in the culture medium. Using the immunocytochemical approach, pancreatic secretory trypsin inhibitor antigenic sites were revealed with specific polyclonal and monoclonal antibodies. The results obtained demonstrated that this trypsin inhibitor is in fact a typical pancreatic secretory protein being processed through the endoplasmic reticulum-Golgi-granule secretory pathway of the acinar cells in rat and human tissues. While the polyclonal antibody yield labelings of increasing intensities along the secretory pathway, the monoclonal one probably due to the molecular nature of its specific antigenic determinant, gave higher labelings in the endoplasmic reticulum. In conclusion the present study has shown that pancreatic acinar cells secrete a specific pancreatic trypsin inhibitor which most probably is involved in the mechanism to prevent trypsinogen activation.

Animals↗
PubMed1993

Pathway of insulin in pancreatic tissue on its release by the B-cell.

Insulin was revealed in the extracellular space and blood capillaries of the rat pancreas by applying protein A-gold immunocytochemistry. On the discharge by the B-cell, insulin diffuses in the extracellular space and interacts with the plasma membrane of all pancreatic cells. For the B-cell, the interstitial microvillar plasma membrane domain was preferentially labeled compared with the flat domain. In contrast, the digitations and flat domains of the basal plasma membrane of the acinar cells were equally labeled. In the endothelium, the labeling was superior in fenestrated areas than in the high cytoplasmic ones, the fenestrae, the luminal and abluminal plasma membranes being labeled. In the cytoplasmic areas the plasmalemmal vesicles were significantly labeled; the intercellular junctions were not. These results indicate that upon binding to the membrane, the transfer of insulin across the capillaries occurs through both the fenestrations and the vesicular system. The labeling of insulin in the subendothelial space and blood capillaries decreased significantly from the insular to the peri-insular and further to the teleinsular regions, demonstrating that insulin levels, high in insular tissue, decrease very rapidly as insulin circulates through and out of the pancreas. The pancreatic cells are thus exposed to very high levels of insulin that vary according to the regions, probably contributing to the topographical partition of the acinar parenchyma into peri-insular and teleinsular tissues.

Animals↗
PubMed1993

Intracisternal crystals in pancreatic acinar cells: failure in the distinct aggregation of secretory proteins.

Mechanisms leading to the formation of crystalline inclusions in the cisternal space of the rough endoplasmic reticulum are poorly understood. This phenomenon was investigated in pancreatic acinar cells using two different experimental models: 1) Intraperitoneal injection of DL-p-chlorophenylalanine methyl ester, and 2) culture of isolated acinar cells within the Matrigel basement membrane in the presence of 2% dimethyl sulfoxide. Features and composition of induced crystals were analyzed by protein A-gold and lectin-gold cytochemistry, electron microscope autoradiography, electron energy loss spectroscopic imaging and energy dispersive X-ray analysis. Crystal formation occurred in ribosome partially free rough endoplasmic reticulum (RER) regions and was similar in both experimental protocols. The protein A-gold revealed the presence of nine major pancreatic enzymes in the crystals. However, the labeling intensities varied among enzymes with higher concentrations of amylase than chymotrypsinogen when compared to the secretory granules. Concanavalin A and Helix pomatia labelings were weak over the crystals and did not correspond to those of RER or secretory granules. Sulfur contents in crystals were lower than phosphorus and their ratio was opposite to the one found in secretory granules. Electron microscope autoradiography demonstrated incorporation of radiolabeled leucine and presence of newly synthesized proteins in the crystals. Furthermore, cells containing both crystals and secretory granules displayed silver grains in most of the cellular compartments involved in secretion. Thus, failure in the normal concentration and sorting process of secretory proteins leading to crystal formation includes changes in protein glycosylation and decrease of disulfide bond formation while retaining secretory capabilities.

Amylases↗
PubMed1993

Development of the endocrine cells in the rat pancreatic and bile duct system.

Morphological features of the endocrine cells in the duct system of the pancreas and the biliary tract have been recently characterized in the adult animal with respect to their physiological roles. In the present study, we have investigated their chronological appearance as well as their developmental progress at various stages of the rat fetal and postnatal life. On day 12 of gestation, glucagon and insulin, as well as CCK cells, were identified in the pancreatic primordium. On day 14, glucagon and CCK cells were first detected in the epithelial lining of the common hepatic and the hepatic ducts. These cells remained the dominant endocrine type in the duct system during the fetal period. Insulin and pancreatic polypeptide cells were first observed in the common hepatic duct only on days 16 and 18 of gestation respectively. In spite of their presence in the islets, somatostatin cells were not detected in the duct system during fetal life. They started to appear in the accessory pancreatic duct of the neonate, and subsequently in the common hepatic duct as well as in the small pancreatic ones on day 7 after birth. During postnatal development, the endocrine cells showed progressive or retrogressive changes in different portions of the duct system according to the cell type. In general, somatostatin, CCK and pancreatic polypeptide cells showed an increase, while glucagon and insulin cells gradually dwindled in number up to the adult stage. Somatostatin cells exhibited a significant increase in number, becoming the highest population among the duct endocrine cells in the adult. Throughout the developmental progress, the endocrine cells appear to be allocated in regions relevant to their possible influence modulating the exocrine secretion as well as the drainage of the pancreatic and bile fluid.

Aging↗
PubMed1993

Differentiation of pancreatic acinar cells into duct-like cells in vitro.

BACKGROUND: Previous studies have shown that a combination of both the extracellular matrix and secretagogues plays critical roles in the maintenance of well-differentiated pancreatic acinar cells in culture. In the present study, we have shown that, upon proper stimulation, acinar cells change their growth pattern and morphologic appearance to a duct like phenotype. EXPERIMENTAL DESIGN: Both rat and guinea pig acinar cells were cultured on or embedded into the Matrigel basement membrane, in the presence of differentiating agents such as dimethylsulfoxide (DMSO), hexamethyl-bis-acetamide, dimethylformamide, triiodothyronine, and butyric acid. The growth patterns, cell proliferation, ultrastructural appearance, intracellular contents, secretion and immunolocalization of amylase, as well as the expression of the ductal marker carbonic anhydrase II and lectin-binding specific sites were analyzed. Moreover, the effects of metabolic inhibitors such as cycloheximide and actinomycin D on the DMSO induced action were also examined. RESULTS: Isolated acinar cells from both rat and guinea pig pancreas showed an important modification of their growth pattern and morphologic appearance when culture embedded into Matrigel in the presence of 2% DMSO. They reaggregate and form isolated branched tubular structures lined by a single cell layer. These tubules can be maintained in culture for over a period of 21 days. The cells lining the tubules were originally acinar cells that became elongated and progressively lost their secretory granules. They displayed a lower number of apical microvilli and established long junctional complexes with elaborated interdigitations. The immunocytochemical localization and biochemical determination of intracellular and secreted amylase revealed a progressive decrease reaching minimal values by the 12th day of culture. The cells further expressed the duct cell marker carbonic anhydrase II and lost the Helix pomatia lectin-binding affinity characteristic of acinar cells. Cell proliferation by modified cells as measured by thymidine incorporation and the autoradiographic labeling index, was significantly lower than in control cultured acinar cells. The DMSO differentiating action was mimicked, but to a lesser extent, by the other agents except butyric acid. Since cycloheximide and actinomycin D inhibited the DMSO-induced changes, protein synthesis and DNA transcription seem to be required. CONCLUSIONS: Our results demonstrate that normal pancreatic acinar cells retain a morphogenetic plasticity and, upon particular stimulation, can change their differentiation commitment pattern toward that of the duct cell phenotype.

3,4-Methylenedioxyamphetamine↗
PubMed1993

Ultrastructural localization of DNA in immune deposits of human lupus nephritis.

DNA molecules were revealed in the glomerular wall of lupus nephritis patients by applying two specific colloidal gold cytochemical approaches at the electron microscope level: immunocytochemistry using a monoclonal anti-DNA antibody in conjunction with protein A-gold and enzyme-gold cytochemistry using DNAse-gold complexes. Application of both techniques has demonstrated that DNA molecules are preferentially located over the electron-dense deposits found in the glomerular basement membrane and mesangial matrix of SLE patients, as well as over the nuclei. Their distribution within the glomerular wall was correlated with electron-dense immune deposits revealed by anti-light chain antibodies. In normal control kidney, DNA labeling was restricted to the cell nuclei. Several control experiments have demonstrated the high specificity of the results. These data thus suggest a possible role for DNA as an antigenic component in the formation of immune complexes.

Adolescent↗
PubMed1993

Poly-L-lysine-gold probe for the detection of anionic sites in normal glomeruli and in idiopathic and experimentally-induced nephrosis. A comparative ultrastructural study.

Anionic sites play a key role in the charge selectivity of glomerular filtration as well as in the maintenance of the structural integrity of the visceral epithelium and podocytes. Alterations in these sites are believed to be a major factor underlying human idiopathic nephrosis and puromycin-nephrosis in the rat. The poly-L-lysine-gold complex was used for the ultrastructural detection of anionic sites in renal glomeruli of patients with idiopathic nephrosis as well as of rats with puromycin-induced nephrosis, allowing for study of the changes occurring in the anionic sites during nephrosis and for the comparison between human disease and this experimental model. In both normal human and rat controls, the probe was detected on epithelial and endothelial cell surfaces and on the glomerular basement membrane, mainly in both laminae rarae. In proteinuric rats, a decrease in labeling intensity was noted on podocyte membranes and in the lamina rara externa, with a corresponding increase in the more central areas of the glomerular basement membrane. These changes were not as evident in proteinuric humans. Furthermore, a reduction in labeling density was noted in the glomerular basement membrane of proteinuric animals, although this could not be substantiated in human tissues. Poly-L-lysine-gold is a useful probe for anionic sites in fixed tissues, and allows for comparison between human disease and its experimental counterpart.

Adolescent↗