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Biomedical subjects

M Bendayan

Publications and source records attributed to M Bendayan.

At least 37 records · Page 2Linked to original sources
PubMed1996

Impact of essential fatty acid deficiency on hepatic sterol metabolism in rats.

The major aim of the current investigation was to define whether essential fatty acid (EFA) deficiency modifies the intrahepatic metabolism and biliary output of sterols in rats. EFA-deficient diet caused an impoverishment in linoleic, arachidonic, and docosahexaenoic acids, and a marked enrichment in the eicosatrienoic acid of the plasma, liver, and hepatic microsomes. During a short term of biliary drainage, a significant decline of the pool size of biliary sterols was noted in EFA-deficient rats compared with control rats. To assess the biosynthesis of biliary components, the common bile duct was cannulated and the pool size depleted (24 hours). Subsequently, a 6-hour bile collection disclosed a significant decrease (nmoles/min/g liver) in bile acids (4.8 +/- 0.3 vs. 8.4 +/- 0.7, P < .005), cholesterol (0.26 +/- 0.01 vs. 0.34 +/- 0.02, P < .05), and phospholipids (1.49 +/- 0.11 vs. 2.82 +/- 0.32, P < .005) in EFA-deficient rats compared with controls (n = 6/group). When cholesterogenesis was measured by the incorporation of [14C]acetate and 3H20 into cholesterol, using liver slices, it was also found to be significantly (P < .001) reduced in EFA-deficient rats. The activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, was consistently lower (80 percent, P < .001) in EFA-deficient rats. In parallel experiments, the direct measurement of microsomal acyl-CoA:cholesterol acyl-transferase (ACAT) showed a decrease averaging 52 percent (P < .001). This is in striking contrast to the elevated activity (157 percent, P < .005) of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis. Current experiments also suggest that the enzyme regulation involving phosphorylation and dephosphorylation is modified by EFA deficiency. Among the structural alterations observed in the morphology of hepatocytes in EFA-deficient rats, the lumen of bile canaliculi was reduced in size. These results underline the importance of EFA in the major mechanisms involved in the maintenance of hepatocyte sterol balance.

Acetates↗
PubMed1996

The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments.

The proprotein convertase PC5 is encoded by multiple mRNAs, two of which give rise to the COOH-terminal variant isoforms PC5-A (915 amino acids [aa]) and PC5-B (1877 aa). To investigate the differences in biosynthesis and sorting between these two proteins, we generated stably transfected AtT-20 cell lines expressing each enzyme individually and examined their respective processing pattern and subcellular localization. Biosynthetic analyses coupled to immunofluorescence studies demonstrated that the shorter and soluble PC5-A is sorted to regulated secretory granules. In contrast, the COOH-terminally extended and membrane-bound PC5-B is located in the Golgi. The presence of a sorting signal in the COOH-terminal 38 amino acids unique to PC5-A was demonstrated by the inefficient entry into the regulated secretory pathway of a mutant lacking this segment. EM of pancreatic cells established the presence of immunoreactive PC5 in glucagon-containing granules, demonstrating the sorting of this protein to dense core secretory granules in endocrine cells. Thus, a single PC5 gene generates COOH-terminally modified isoforms with different sorting signals directing these proteins to distinct subcellular localization, thereby allowing them to process their appropriate substrates.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Presence of pancreatitis-associated protein in pancreatic acinar cells of rats treated with chlorophenylalanine methyl ester.

The biological significance and function of pancreatitis-associated protein (PAP), identified in 1984 as a new secretory protein appearing during pancreatitis, remain to be elucidated. The purpose of this study was to evaluate the presence of PAP in pancreatic tissue upon its exposure to chlorophenylalanine methyl ester (CPME), a drug known to disrupt the regulated secretory pathway of acinar cells, an experimental condition that differs from acute pancreatitis. Pancreatic tissues were processed either for immunocytochemistry or for Northern blot analysis at 17-72 h after a single intraperitoneal injection of CPME in rats. Pancreatic acinar cells displayed endoplasmic reticulum intracisternal crystals consisting of an abnormal aggregation of secretory proteins as well as a sub-population of small and aberrant secretory granules. PAP mRNA was strongly increased at 17-24 h after CPME treatment, and PAP immunoreactivity was detected along the regulated secretory pathway, particularly in the small and aberrant secretory granules at these same time points. Levels of PAP and PAP mRNA decreased gradually, to become undetectable after 72 h. Autoradiographic experiments demonstrated that these small aberrant granules stored preferentially newly synthesized proteins. Our results indicate that the induction of PAP is not exclusive to acute pancreatitis since it appears in pancreatic cells as a response to certain insults and is secreted preferentially through a particular population of small aberrant granules.

Acute-Phase Proteins↗
PubMed1996

Reabsorption of native and glycated albumin by renal proximal tubular epithelial cells.

The proximal tubule epithelial handling of native and glycated albumin was evaluated by quantitative immunocytochemistry. Native bovine serum albumin (BSA) and its glycated form (gBSA), tagged to different haptens, were simultaneously injected in anesthetized mice and maintained in circulation for 10 or 60 min. Both albumins were localized within the capillary lumen, in glomerular and peritubular basement membranes, the urinary space, and cellular compartments of the proximal tubular epithelial cells. In these cells, both forms of albumin were concomitantly found within the same endocytic-lysosomal system. Morphometric evaluations have indicated higher proportions of gBSA in the urinary space, reflecting probably a significant glomerular filtration of this form of albumin combined to a lesser reabsorptive clearance. Indeed, higher proportions of native BSA were found in the endocytic compartment of the tubular epithelial cells, suggesting its preferential reabsorption. The present study thus supports a preferential glomerular filtration of gBSA with a facilitated filtration of native BSA in the presence of the glycated one. It also demonstrates the tubular reabsorption of BSA and gBSA through a common endocytic pathway, in which the native BSA is preferentially reabsorbed with respect to its glycated form.

Absorption↗
PubMed1996

Possible association of chaperonin 60 with secretory proteins in pancreatic acinar cells.

Assembly and folding of newly synthesized polypeptides, acquisition of their biological active form, and their translocation in different cellular compartments are processes assisted by molecular chaperones. Because particular chaperones have been found to be present along the RER-Golgi-granule secretory pathway in pancreatic acinar cells, we presume that they should play important roles in secretion. In the present study, applying double immunogold labeling at the electron microscopic level on rat exocrine pancreas, we have revealed the existence of a topographical association between Hsp60 and particular pancreatic enzymes along the secretory pathway. The highest association was found for amylase, lipase, and chymotrypsinogen, whereas trypsinogen and carboxypeptidase B showed much lower association values. Immunoprecipitation of isolated zymogen granule content with an anti-Hsp60 antibody appears to confirm the morphological data, since amylase and lipase were found to co-precipitate with Hsp60. These findings support the hypothesis that Hsp60 is associated with certain pancreatic proteins along the secretory pathway. Hsp60 would assist the proper folding and assembly of pancreatic secretory proteins and could also prevent their autoactivation before secretion.

Amylases↗
PubMed1996

Transport of insulin and albumin by the microvascular endothelium of the rete mirabile.

Vascular permeability for albumin and insulin in the continuous capillary network of the rete mirabile of the eel swimbladder was evaluated by ultrastructural immunocytochemistry and countercurrent perfusion experiments. Upon perfusion of the rete capillaries with a buffer solution containing albumin and insulin, these serum proteins were revealed at the electron microscope level, by the Protein A-gold immunocytochemical technique on a post-embedding step. For the simultaneous detection of both proteins, the double labeling technique with different sized gold particles was used. Furthermore, labeling was performed with the mixture of anti-albumin and anti-insulin anti-bodies. The labelings obtained were morphometrically evaluated and demonstrate that: (1) serum proteins such as albumin and insulin are transported by the endothelial cells through their plasmalemmal vesicular system; (2) insulin is transported preferentially to albumin; and (3) this transport involves different populations of plasmalemmal vesicles. Measurements of diffusion permeability coefficients have confirmed the preferential transport of insulin, its coefficient being higher than that of albumin. Conversely, when compared to that of insulin or sucrose, which are assumed to be markers of the paracellular diffusion, it was found to be much lower, indicating that transcytosis through the vesicular system is less efficient than diffusion along the intercellular junctions. These results indicate that transcytosis of insulin and albumin occurs via different sets of plasmalemmal vesicles, probably through receptor-mediated mechanisms, and that the overall rate of transport across the rete capillaries, with respect to paracellular diffusion, is higher for insulin than for albumin.

Air Sacs↗
PubMed1995

Histones from diabetic rats contain increased levels of advanced glycation end products.

In a recent report we have demonstrated the in vitro formation of advanced glycation end products (AGEs) on histones in a time and sugar concentration dependent fashion. In the present work we examined histone advanced glycation in vivo. Diabetes was induced in rats by streptozotocin injection and the hyperglycemic state was maintained and surveyed for up to 24 weeks. Diabetic rats showed accumulation of early glycation products in plasma proteins and in hemoglobin. Histones from the liver of diabetic rats showed AGEs levels three-fold higher than those of their age-matched controls. Histone AGEs increased with the duration of diabetes and tended to increase with the age as well. Similar tendencies were apparent in skin collagen. Our data demonstrate that diabetes induces an increase in the accumulation of AGE products on histones. This reinforces the concept that advanced glycation occurs in intracellular proteins and suggests a possible role for intracellular glycation in the increased theratogeny associated with diabetes mellitus.

Aging↗
PubMed1995

Immunocytochemical investigation of the in vivo endocytosis by renal tubular epithelial cells.

The internalization and degradation of glomerular filtered serum proteins by the proximal tubular epithelium has been extensively studied by microperfusion methods. By using a cationic probe that easily traverses the glomerular wall into the urinary space, we have performed a morpho-cytochemical and quantitative study of the in vivo endocytotic activity of the proximal tubular epithelial cell. Bovine serum albumin (BSA) was tagged with dinitrophenol (DNP) and cationized to pI over 8. It was introduced into the circulation of normal mice for 5, 10, and 30 minutes and the distribution of the labeling was determined by protein A-gold immunocytochemistry, using specific antiDNP antibodies on tissue sections of routinely aldehyde-fixed, osmiumpostfixed, and Epon-embedded kidneys. Cationic BSA-DNP was detected at the endothelial and epithelial sides of the glomerular basement membrane, and over capillary and tubular basement membranes. In the proximal tubular epithelial cell, labeling was present over microvilli as well as over endosomal and lysosomal compartments, with labeling intensities varying from one compartment to the other. Morphometric evaluations of the labeling demonstrated a progressive incorporation of the probe from microvilli and endocytic compartments at 5 minutes to endocytic and lysosomal compartments at 10 and then 30 minutes. When considering labeling densities, no significant differences were found on microvilli and basolateral membranes between times of circulation; however, the labeling density over endosomal and lysosomal compartments was very intense at 10 minutes compared with 5 minutes, decreasing at 30 minutes. Results from this study validate the cationic albumin tagged with DNP as a tool in the study of the quantitative aspects of protein endocytosis at the ultrastructural level, in the kidney tubular epithelium.

Animals↗
PubMed1995

Presence of tumor necrosis factor alpha and interleukin-6 in renal mesangial cells of lupus nephritis patients.

Tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) antigenic sites were shown within the resident glomerular mesangial cells of lupus nephritis patients applying the colloidal gold immunocytochemical approach at the electron microscopic level. Using specific polyclonal antibodies against human recombinant (hr) TNF alpha and hrIL-6 in conjunction with the protein A-gold complex, TNF alpha and IL-6 were shown in the mesangial cells, being particularly associated with the membranes of the rough endoplasmic reticulum. In addition, IL-6 also was present in glomerular immune deposits and occasionally in glomerular epithelial cells. In normal renal tissue the TNF alpha and IL-6 immunoreactivities were undetectable. The specific presence of TNF alpha and IL-6 in pathological specimens was shown by several control experiments. Thus, our results offered morphological support that TNF alpha and IL-6 might play a role in human lupus nephritis. The data showed their synthesis by the mesangial cells and their possible participation in the progression to chronicity of the renal injury on secretion.

Glomerular Mesangium↗
PubMed1995

Presence of Chromatium vinosum chaperonins 10 and 60 in mitochondria and peroxisomes of rat hepatocytes.

In the present study we report the occurrence of chaperonins, cpn10 and cpn60, in Chromatium vinosum and rat hepatocytes, using specific polyclonal antibodies in conjunction with the protein A-gold immunocytochemical technique. As demonstrated by quantitative evaluations, the immunolabeling for cpn10 and cpn60 in C vinosum cells was associated primarily with the bacterial cell envelope. In rat liver homogenates, Western immunoblotting analysis has shown that antibodies to cpn10 from C vinosum recognize an unique 25-kDa protein that remains to be further characterized. On the other hand, the antibody to cpn60 from C vinosum revealed the presence of a 60-kDa protein in the rat liver homogenates. Immunofluorescence on rat liver tissue revealed an intracellular granular labeling for both chaperonins. On the other hand, using the post-embedding immunoelectron microscopy technique cpn10 and cpn60 were localized specifically in liver mitochondria and peroxisomes. Interestingly, further analysis of the labeling distribution confirmed the association of both proteins with the mitochondrial inner membrane whereas in the peroxisomes the chaperonins appeared to be located in the matrix, away from the limiting peroxisomal membrane. The colocalization of both chaperonins suggests that, as in other bacteria as well as eukaryotic cells, they may act in tandem for the proper folding of particular proteins.

Animals↗
PubMed1995

Glomerular handling of circulating glycated albumin in the normal mouse kidney.

In the present study, we have evaluated the glomerular handling of circulating glycated albumin in the normal mouse kidney by quantitative immunocytochemistry. Bovine serum albumin (BSA) was glycated in vitro and dinitrophenylated. Glycated and nonglycated probes were introduced into the circulation of anesthetized mice and traced by postembedding immunogold cytochemistry after 10 and 30 min of circulation. Endogenous albumin, as well as dinitrophenylated native BSA (DNP-BSA) and glycated albumins (DNP-gBSA), were localized within the capillary lumen, glomerular and peritubular basement membranes, and the mesangial matrix. Morphometric evaluation of the labeling over the glomerular basement membrane (GBM) revealed a peak of labeling in the endothelial side for either endogenous albumin or DNP-BSA. In contrast, the labeling distribution for DNP-gBSA showed a shift toward the epithelial side, suggesting a further penetration of the glycated probe into the GBM. When coinjected with gBSA, DNP-BSA was found to display a labeling distribution similar to that displayed by DNP-gBSA. These results indicate that the glycated tracer penetrates the normal glomerular wall deeper than the nonglycated one. Moreover, glycated albumin increases the infiltration of the nonglycated tracer through the normal glomerular wall. Circulating glycated serum proteins thus appear to play an important role in the onset of the glomerular dysfunction and proteinuria, which take place in long-term hyperglycemic states.

Animals↗
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↗