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C Enrich

Publications and source records attributed to C Enrich.

At least 37 records · Page 2Linked to original sources
PubMed1997

Membrane transport in rat liver endocytic pathways: preparation, biochemical properties and functional roles of hepatic endosomes.

The endocytic compartment has emerged as a major regulator of the uptake and processing of circulating ligands, and has been extensively studied during the last decade. In this work, the polypeptides of the three endosomal fractions: compartment of uncoupling receptors and ligands (CURL), multivesicular bodies (MVB) and receptor recycling compartment (RRC), isolated from livers of estradiol-treated rats, were analyzed by two-dimensional gel electrophoresis. Silver-stained gels revealed that although the three endosomal fractions shared a generally similar pattern of approximately 120 components, qualitative and quantitative differences between the three endocytic fractions could be demonstrated. The polypeptide composition of the bile was also studied and compared with ligands and proteins identified in the different endosomal fractions. One- and two-dimensional gel electrophoresis and Western blotting were used to investigate the protein composition of the three isolated endocytic fractions and 39 proteins were identified. The distribution of identified receptors, ligands and structural proteins among the three endosomal fractions was in agreement with their expected functionalities and with the different endocytic pathways in the hepatocyte.

Animals↗
PubMed1997

[Immunohistochemical localization of annexin VI in the endocytic compartment of rat liver hepatocytes].

Annexin VI has been isolated from rat liver endosomes and affinity purified antibodies have been produced. By Western blotting, in rat liver subcellular fractions, anti-annexin VI was demonstrated to recognise a 68 kDa band in the three endosomal fractions. In the present study, immunogold labeling of ultrathin Lowicryl sections of rat liver has been used to get insights into the ultrastructural hepatocyte localization. Although at the immunofluorescence level the staining seemed located at the apical, canalicular plasma membrane, domain of the hepatocytes, the electron microscopy revealed that 80% of the labeling, with the anti-annexin VI antibody was specifically localized not at the plasma membrane but in the close subapical endocytic compartment surrounding the bile canalicular plasma membrane of the hepatocyte. Double immunogold labeling with an anti peptide antibody to Rab5 and anti-annexin VI showed that 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization of annexin VI and structures labeled with antibodies to the polymeric immunoglobulin receptor. The results suggest that annexin VI could be involved in regulating the functioning of this apical compartment in the hepatocyte.

Animals↗
PubMed1996

Calmodulin binds to the basolateral targeting signal of the polymeric immunoglobulin receptor.

We have identified a major calmodulin (CaM)-binding protein in rat liver endosomes using 125I-CaM overlays from two-dimensional protein blots. Immunostaining of blots demonstrates that this protein is the polymeric immunoglobulin receptor (pIgR). We further investigated the interaction between pIgR and CaM using Madin-Darby canine kidney cells stably expressing cloned wild-type and mutant pIgR. We found that detergent-solubilized pIgR binds to CaM-agarose in a Ca(2+)-dependent fashion, and binding is inhibited by the addition of excess free CaM or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide), suggesting that pIgR binding to CaM is specific. Furthermore, pIgR is the most prominent 35S-labeled CaM-binding protein in the detergent phase of Triton X-114-solubilized, metabolically labeled pIgR-expressing Madin-Darby canine kidney cells. CaM can be chemically cross-linked to both solubilized and membrane-associated pIgR, suggesting that binding can occur while the pIgR is in intact membranes. The CaM binding site is located in the membrane-proximal 17-amino acid segment of the pIgR cytoplasmic tail. This region of pIgR constitutes an autonomous basolateral targeting signal. However, binding of CaM to various pIgR mutants suggests that CaM binding is not necessary for basolateral targeting. We suggest that CaM may be involved in regulation of pIgR transcytosis and/or signaling by pIgR.

Amino Acid Sequence↗
PubMed1996

The polymeric immunoglobulin receptor is the major calmodulin-binding protein in an endosome fraction from rat liver enriched in recycling receptors.

Rat liver endosomes contain one major high-affinity calmodulin-binding protein (CaMBP) that now has been identified as the polymeric immunoglobulin receptor (pIgR). In isolated endosomes pIgR was enriched in the receptor-recycling compartment (RRC); lesser enrichment was found in 'early' endosome (CURL) and much less in 'late' endosome fractions (multivesicular bodies, MVB). The distribution of the major CaMBP, shown by Western blotting or by overlay with I125-calmodulin in the isolated fractions, was consistent with rapid accumulation of I125-immunoglobulin A (IgA) in RRC and CURL after intravenous injection into rats. The receptor was also found in sinusoidal plasma membranes but not in cell fractions containing apical (bile canalicular) or lateral plasma membrane domains of the hepatocyte. The interaction of pIgR with calmodulin was shown by direct binding assays and by affinity chromatography. Thus, calmodulin is the first cytoplasmic protein shown to interact with the pIgR. We postulate that calmodulin regulates pIgA trafficking in rat liver. In addition, the receptor recycling fraction emerges as an endosomal subcompartment involved in pIgA transport via pIgR.

Adult↗
PubMed1996

Calmodulin regulates the intracellular trafficking in epithelial cells.

In this study we have demonstrated the role of calmodulin in membrane trafficking. By means of specific calmodulin-antagonist (W13, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide) the transcytosis, recycling and receptor-mediated endocytosis of IgA were investigated. In W13 treated MDCK cells, exHpressing the polymeric immunoglobulin receptor (pIgR) and plated on polycarbonate filters (transwells), it has been shown that transcytosis of IgA was inhibited, the recycling was concomitantly increased and there were no modification on its internalization. Altogether, point to a role of calmodulin in regulating the function of endocytic compartment in epithelial cells.

Animals↗
PubMed1995

Functional identification of three major phosphoproteins in endocytic fractions from rat liver. A comparative in vivo and in vitro study.

Liver plasma membranes originating from the sinusoidal, lateral and canalicular domains and 'early' and 'late' endosomes were prepared from rats injected with [32P]orthophosphate. The phosphorylated polypeptides in these subcellular fractions, resolved by gel electrophoresis, were analysed and compared with those obtained by in vitro phosphorylation of the fractions by endogenous protein kinases. The polypeptides phosphorylated in vitro were different in plasma membranes, endosomes and lysosomes. Three of the major phosphoproteins in the endocytic membranes were shown to be the polymeric immunoglobulin receptor, the beta subunit of the insulin receptor and the 550-kDa low-density-lipoprotein-receptor-related protein (LRP). An additional 35-kDa polypeptide of unknown function was a major phosphorylated component and thus emerges as a candidate marker protein of hepatic endosomes. Phosphoserine was shown to be the major amino acid phosphorylated in vitro in the phosphoproteins of endocytic membranes. The subcellular distribution in liver tissue of protein kinase activity was also investigated and activity shown to be recovered mainly in blood-sinusoidal and lateral plasma membranes; bile canalicular plasma membranes and endosomes contained low protein kinase activities. The results show that receptor phosphorylation is an 'early' event in endocytosis and the trafficking of ligands that is sustained especially in early endosomes in liver, and emphasizes the biochemical and thus functional distinctiveness of the plasma membrane and the endosomal and lysosomal compartments with regard to their population of phosphorylated proteins.

Animals↗
PubMed1995

Differential expression of A and B laminin chains during rat liver regeneration.

We have studied the distribution and expression of laminin during rat liver regeneration by immunofluorescence and immunoblotting using affinity-purified laminin antibodies. Laminin was localized on sinusoidal surfaces in normal and regenerating hepatic parenchyma, but enhanced expression was detected during regeneration from 6 hours to 7 days after a partial hepatectomy. A and B laminin chains were specifically detected by Western blotting in highly purified plasma membrane fractions derived from the sinusoidal domain of hepatocytes. Analysis of laminin expression in whole homogenates from hepatectomized rats showed a differential expression of A and B laminin chains during regeneration: The A chain showed a progressive increase, whereas the B chain exhibited an early, prereplicative increase and then gradually declined to control levels at the end of restorative growth. The results suggest differential temporal requirements for A and B laminin chains during hepatic growth and tissue formation.

Animals↗
PubMed1994

The calmodulin antagonist, W-13, alters transcytosis, recycling, and the morphology of the endocytic pathway in Madin-Darby canine kidney cells.

The effect of the calmodulin antagonist N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13) on the endosomal system of Madin-Darby canine kidney cells was assessed. W-13 inhibited receptor-mediated IgA transcytosis, recycling of transferrin, and caused all material endocytosed from both surfaces of the cell to be delivered to exceptionally large, novel endosomal structures, which appear to be derived from early endosomes. Treatment with other calmodulin antagonists similarly inhibited transcytosis and caused large endosomes to form. These observations raise the possibility that calmodulin may be involved in regulating membrane trafficking through the endosomes of polarized epithelial cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Early induction of Na(+)-dependent uridine uptake in the regenerating rat liver.

Na(+)-dependent uridine transport into liver plasma membrane vesicles from partially hepatectomized and sham-operated rats was studied. Preparations purified 6 h after 70% hepatectomy exhibited an increased Vmax of uridine uptake (3.7 vs. 1.4 pmol/mg prot/3 s) without any change in Km (6 microM). Incubation of the vesicles in the presence of monensin decreased uridine uptake although the differences between both experimental groups remained identical. It is concluded that uridine transport is induced early after partial hepatectomy by a mechanism which does not involve changes in the transmembrane Na+ gradient. This is the first evidence in favor of modulation of nucleoside transport into liver cells.

Animals↗
PubMed1992

Differential expression of asialoglycoprotein receptor subunits in the endocytic compartment during liver regeneration.

Asialoglycoprotein receptors, responsible for the removal of circulating asialoglycoproteins by the liver, are located in at least two different membrane locations in hepatocytes. Receptors on the cell surface account only for a minor proportion (20-36%), for the majority of receptors in the liver are located intracellularly, mainly in the endocytic membrane networks. An understanding of the basis of receptor distribution and the underlying trafficking of receptors between the hepatocyte's polarised cell surface and the endocytic compartment would be aided if biochemical differences between the receptors in these pools were established. We now show, using three antibodies that recognise the receptor subunits in rat liver (RHL-1, RHL-2 and RHL-3), that the asialoglycoprotein receptors located in the plasma membrane domains and the endocytic compartment differ in oligomeric composition, sialic acid content, and solubility in Triton X-114 using two-phase systems. It is well established that the expression of the asialoglycoprotein receptor is down-regulated in livers regenerating after a partial hepatectomy. We demonstrate that the levels of the receptor subtype that is located mainly in the endocytic compartment (RHL-1, 42 kDa) was elevated in regenerating liver by agents that regulate cAMP production, whereas the levels of the other receptor subtypes remained unchanged. The asialoglycoprotein receptor subtypes that are present in different subcellular locations are thus regulated independently.

Animals↗
PubMed1992

Reorganization of the endocytic compartment in regenerating liver.

Antibodies raised to two membrane proteins present in rat liver endosomal fractions were used to study changes occurring in the endocytic compartment of hepatocytes during liver regeneration. Antibodies to the 42-kDa subunit (RHL-1) of the asialoglycoprotein receptor showed, by Western blotting of liver microsomes and endosomes, that there was a reduced expression of the receptor in liver 24 h following a partial hepatectomy. Immunocytochemical staining of thin sections of regenerating livers using these antibodies indicated that there was an intracellular relocation of endocytic structures in hepatocytes. The two main endocytic regions immunocytochemically stained in normal liver--one located beneath the sinusoidal plasma membrane and the other abutting the bile canaliculus--were replaced, in regenerating liver, by staining more closely associated with a region underlying the baso-lateral plasma membrane. A 140-kDa pI 4.3 calmodulin-binding protein located in endocytic and plasma membranes was also demonstrated, using a radio-iodinated calmodulin-binding assay, to be present at reduced levels in endosomes isolated from regenerating livers. Antibodies to this calmodulin-binding protein stained the hepatocyte's cytoplasm in a punctate manner. However, in regenerating liver, the staining was located in regions underlying the baso-lateral and apical plasma membrane of hepatocytes. Together, the results demonstrate that a reorganization of the endocytic compartment has occurred in hepatocytes 24 h following hepatectomy, with two endosomal proteins becoming relocated to a region below the baso-lateral-apical surface regions of hepatocytes.

Animals↗
PubMed1992

Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration.

We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell-matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E-cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.

Animals↗
PubMed1992

Changes in cell surface expression of fibronectin and fibronectin receptor during liver regeneration.

The surface expression of fibronectin and its major integrin receptor in liver, integrin alpha 5 beta 1, was studied during liver regeneration after partial hepatectomy. Using immunoblotting, plasma membranes isolated from livers at different regeneration stages were found to contain 6- to 8-fold elevated levels of fibronectin, alpha 5 and beta 1 at 12-24 h after the operation. Normal levels were gradually restored during the following 8-9 days. The membrane-associated fibronectin lacked the ED-A domain, suggesting that it consisted of plasma fibronectin. A prominent fibronectin fragment (180 kDa) was present at 12-24 h after surgery, possibly reflecting turnover of the pericellular matrix during cell division. Indirect immunohistochemical staining of liver sections revealed beta 1 and fibronectin mainly in the sinusoidal region of the hepatocyte plasma membrane. The distribution was not markedly altered during liver regeneration. The results suggest that the fibronectin-mediated contacts between the cells and the extracellular matrix increase during the pre-replicative and proliferative phases of liver regeneration. The significance of these results for the growth and for the structure of the liver during regeneration and development is discussed.

Animals↗
PubMed1991

Echinococcus granulosus: antigen characterization by chemical treatment and enzymatic deglycosylation.

Parasite antigenic fractions obtained by biochemical purification of sheep hydatid fluid were subjected to enzymatic digestion. The relative mobilities of the 5 and B antigens, before and after treatment, were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Antigenic fractions transferred to nitrocellulose were also treated with sodium metaperiodate and concanavalin A. The results indicate that antigen 5 contains a substantial amount of carbohydrates covalently linked to a polypeptide backbone, which strongly bind to concanavalin A and is removed by N-glycosidase F (PNGase F). Antigen 5 possesses complex N-linked oligosaccharides (PNGase F sensitive), without terminal N-acetyl-D-glucosamine residues (N-acetyl-D-glucosaminidase nonsensitive) and has no high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H nonsensitive). In contrast, the antigen B of low molecular weight is not susceptible to either enzymatic digestions (PNGase F, Endo H, and N-acetyl-D-glucosaminidase) or sodium metaperiodate oxidation and it does not bind to concanavalin A. Polyclonal antibodies prepared against the two antigens reacted with the deglycosylated antigen 5 in Western blot. The dominant epitopes are, therefore, polypeptides, although the presence of carbohydrate epitopes in the native glycoproteins cannot be excluded.

Acetylglucosaminidase↗
PubMed1991

Occurrence of hereditary leaky red cell syndrome and partial coagulation factor VII deficiency in a Spanish family.

A Spanish family was found to have the coexistence of a hereditary haemolytic syndrome associated with excessively leaky RBC membrane to sodium (Na+) and potassium (K+) cations and a partial coagulation factor VII deficiency. Haemolysis was mild in the propositus and the RBC membrane leak included a marked increase in passive permeability to Na+ and K+. This was associated with an increase in active Na+,K(+)-pump activity and in the ouabain-resistant fluxes: Na+, K(+)-cotransport and Na+, Li(+)-countertransport. Factor VII deficiency was of 50% and no clinical expression of the coagulation deficiency was observed. The family study revealed slightly abnormal RBC membrane cationic fluxes only in the father and decreased coagulation factor VII activity of 67% in the mother. Both parents were clinically and haematologically normal. It is suggested that the propositus has inherited the abnormal gene for leaky RBC syndrome from the father and the partial coagulation factor VII deficiency from the mother.

Anemia, Hemolytic, Congenital↗