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Y Sambuy

Publications and source records attributed to Y Sambuy.

11 recordsLinked to original sources
PubMed1992

Epithelial cells in culture as a model for the intestinal transport of antimicrobial agents.

The bioavailabilities of orally administered drugs depend to a great extent on their capability of being transported across the intestinal mucosa. In an attempt to develop an in vitro model for studying the intestinal transport of drugs, we used an intestinal epithelial cell line (Caco 2) derived from a human colon adenocarcinoma. A renal epithelial cell line (MDCK) was also used to determine the tissue specificity of drug transport. These cell lines, which were grown on filters, form a monolayer of well-polarized cells coupled by tight junctions and can be used for transcellular transport experiments. We studied the transport of nine antimicrobial agents with different physicochemical and pharmacokinetic characteristics using these epithelial cell monolayers to determine whether this model could be predictive of oral bioavailability. The transepithelial passage was assayed from the apical (AP) to the basolateral (BL) side and in the opposite direction (BL to AP) in both cell lines. Radioactively labeled mannitol was used to monitor the intactness of the cell monolayer during drug passage. The results indicated that all antimicrobial agents tested tended to behave in vitro generally according to their known in vivo absorptive characteristics. In addition, the use of epithelia from different tissues enabled us to divide the drugs into four groups according to their behaviors and suggested the existence of different transport mechanisms. In particular, two antibiotics, gentamicin and teicoplanin, showed no passage in either direction or cell line, in accordance with their very poor in vivo absorbances after oral administration. In contrast, rifapentine, rifampin, and nalidixic acid passed very efficiently at similar rates in both directions and cell lines in a concentration-dependent, nonsaturable manner, which is suggestive of passive diffusion down a concentration gradient. Of the remaining drugs, isoniazid and novobiocin sodium showed some differences in passage between the two cell lines and, given their ionized state at the pH that was used, may use the paracellular route. Finally, trimethoprim and D-cycloserine exhibited differences in passage both with respect to polarity and cell line; in particular, trimethoprim had a faster rate of passage only in Caco 2 cells and in the BL to AP direction, while D-cycloserine was exclusively transported by Caco 2 cells in the AP to BL direction. In both cases it is possible that active transport mechanisms are involved.

Adenocarcinoma↗
PubMed1991

An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells.

A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site. Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour). Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis. Deletionof juxta-membrane attachment sites for a cluster of O-linked sugars did not alter apical localization. A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 amino acid deletion in the hNGFR tail that moved a cytoplasmic tyrosine (Tyr 308) closer to the membrane into a more charged environment resulted in a basolateral distribution of the mutant receptor and reversed vectorial (basolateral) targeting. The basolateral mutant receptor also internalized 125I-NGF rapidly (90% of surface bound NGF per hour), exhibited a larger intracellular fraction and displayed a considerably shortened half-life (approximately 3 h). We suggest that hNGFR with the internal cytoplasmic deletion expresses a basolateral targeting signal, related to endocytic signals, that is dominant over apical targeting information in the ecto/transmembrane domains. These results apparently contradict a current model that postulates that basolateral targeting is a default mechanism.

Amino Acid Sequence↗
PubMed1990

Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells.

We studied the cell-surface delivery pathways of newly synthesized membrane glycoproteins in MDCK cells and for this purpose we characterized an endogenous apical integral membrane glycoprotein. By combining a pulse-chase protocol with domain-selective cell-surface biotinylation, immune precipitation, and streptavidin-agarose precipitation (Le Bivic et al. 1989. Proc. Natl. Acad. Sci USA. 86:9313-9317), we followed the appearance at the cell surface of a major apical sialoglycoprotein, gp114, a basolateral protein, uvomorulin, and a transcytosing protein, the polyimmunoglobulin receptor (pIg-R). We determined that both gp114 and uvomorulin appeared to be delivered directly to their respective surface, with mistargeting levels of 8 and 2%, respectively. Using the same technique, the pIg-R was first detected on the basolateral domain and then on the apical domain, to be finally released into the apical medium, as described (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). To directly determine whether the gp114 pool present on the basolateral surface was a precursor of the apical gp114, we compared it with the equivalent pIg-R pool, by labeling with sulfo-NHS-SS-biotin, a cleavable, tight junction-impermeable probe, and by following the fraction of this probe that became resistant to basal glutathione and accessible to apical glutathione during incubation at 37 degrees C. We found that, contrary to pIg-R, basolateral gp114 was poorly endocytosed and was not transcytosed to the apical side. These results demonstrate that an endogenous apical integral membrane glycoprotein of Madin-Darby canine kidney cells is sorted intracellularly and is vectorially targeted to the apical surface.

Animals↗
PubMed1988

Isolation and characterization of the apical surface of polarized Madin-Darby canine kidney epithelial cells.

We have developed a fast and efficient method to isolate the apical surface of Madin-Darby canine kidney epithelial cells. After confluent cell monolayers were coated with alternate layers of cationized colloidal silica and a polyanion, 60% of the apical surface was recovered as large membrane sheets through the application of a polylysine-coated glass surface. Scanning electron microscopy of the cytoplasmic aspect of the apical surface revealed a honeycomb pattern given by the cell borders fractured at or above the level of the tight junctions. By transmission electron microscopy, the apical preparation appeared to be composed of plasma membrane and a thin layer of cytoplasm. Enzyme assays and immunoblots demonstrated a 6- to 7-fold enrichment of an apical marker and a low level of contamination by cytoplasmic and basolateral markers. After removal of cytosolic contaminants and peripheral membrane proteins by alkaline extraction, apical integral membrane proteins were characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). Lectin blotting and [3H]glucosamine labeling identified two major sialoglycoproteins of apparent Mr 200,000 and 100,000. The apical membrane sheets here described provide a useful model for systematic characterization of the molecular components of the membrane, for reconstitution of lipid and protein transport in cell-free systems, and for study of the interactions of submembranous cytoskeletal proteins with the apical plasma membrane domain.

Animals↗
PubMed1986

Formation of organoid structures and extracellular matrix production in an intestinal epithelial cell line during long-term in vitro culture.

During the long-term in vitro maintenance of an epithelial cell line established from rat duodenum (IEC-17) we have observed progressive morphological changes which, after approximately 4-5 months in culture, led to a loss of substrate adherence and to the formation of organoid structures characterized by organized layers of cells separated by continuous extracellular-like material and delimiting close lumina. The cells exhibited a defined polarity with deposition of extracellular matrix components on one side and development of microvilli on the opposite surface. The morphological changes observed did not appear to be the expression of spontaneous transformation since the cells retained a normal diploid rat karyotype and did not grow in soft agar. In this report we present the optical and electron microscopical characterization of the progressive organotypic differentiation of the cell line. Further studies are currently in progress to characterize the extracellular matrix during the process of differentiation.

Animals↗
PubMed1985

Age-related modulation of vascular smooth muscle cells proliferation following arterial wall damage.

The proliferative response of vascular smooth muscle cells (SMC) to an arterial wall injury has been investigated in young (3-6 months) and old (30-52 months) rabbits, employing two different experimental models. In the first model the lesion was produced by passing an inflated balloon catheter along the abdominal and descending thoracic aorta, while in the second, the endothelium was removed from a segment of the right carotid artery by air-drying. Both experimental models produced very heterogeneous lesions in which the de-endothelialization of the arteries was often accompanied by foci of cellular necrosis of variable extension throughout the vascular wall. The autoradiographic evaluation of the proliferative response of arterial SMCs following in vivo administration of 3H-thymidine revealed different patterns of labeling according to the type of lesion produced. A significant negative correlation (p less than 0.001) was found between the proliferative response and the cellular density of the vascular district. An age-related decrease in the proliferative response of SMCs was observed only in the thoracic aorta where the damage to the tunica media was less extensive as compared to the other vascular districts.

Aging↗
PubMed1985

Ultrastructural studies of spontaneous in vitro transformation of cultured marrow monocyte-macrophage cells from a patient with congenital hypoplastic anemia.

The CM-S cell line was established from the bone marrow of a patient suffering from congenital hypoplastic anemia (syndrome of Diamond-Blackfan). The cells grew in suspension in liquid culture and were dependent for their continuous replication in vitro on growth factors produced by the same cells seeded at high density. Initially, undifferentiated blasts, immature myeloid, megakaryocytic and, rarely, erythroid cells were observed. Eventually, a population of cells with characteristics of monocyte-macrophage precursors predominated. These cells could be induced to terminal macrophage differentiation by incubation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. During this period (over 150 continuous passages), the cells failed to form colonies in agar and to give rise to tumors when inoculated into athymic mice. On prolonged passages, however, the cells gradually increased their growth capacity in liquid culture and became capable of forming colonies in agar and tumors in animals. Ultrastructural studies revealed that the expression of differentiated traits markedly changed as a function of time: after 277 passages, the transformed cells, although displaying characteristics of monocyte precursors, appeared blocked at this stage and no longer responded to 12-O-tetradecanoylphorbol-13-acetate.

Anemia, Aplastic↗
PubMed1984

The effects of in vitro ageing on the exopeptidases of human diploid fibroblasts.

Six exopeptidases present in human diploid fibroblasts were identified by separation on polyacrylamide gel electrophoresis and their activity profiles against 17 dipeptides, two tripeptides and L-leucine-p-nitroanilide determined. No differences in relative activity or in the electrophoretic patterns of any of the six exopeptidases were detected with ageing. Aminoacylarylamidase activity assayed spectrophotometrically showed significantly increased activity in the middle age-group cells as opposed to the enzyme isolated from young and old cells. Heat-inactivation studies using the same substrate suggested the possibility of an increased proportion of heat-labile enzyme in the old cells but interpretation of the data was difficult because of the complex nature of the inactivation curves obtained. Overall, the results tended to refute the hypothesis that age-related changes in the free amino acid pool of human diploid fibroblasts were associated with significant alterations in the activities of cellular exopeptidases.

Cell Survival↗