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Apical Domain (apical + domain)
Selected AbstractsUpregulation of Brain Expression of P-Glycoprotein in MRP2-deficient TR - Rats Resembles Seizure-induced Up-regulation of This Drug Efflux Transporter in Normal RatsEPILEPSIA, Issue 4 2007Katrin Hoffmann Summary:,Purpose: The multidrug resistance protein 2 (MRP2) is a drug efflux transporter that is expressed predominantly at the apical domain of hepatocytes but seems also to be expressed at the apical membrane of brain capillary endothelial cells that form the blood,brain barrier (BBB). MRP2 is absent in the transport-deficient (TR,) Wistar rat mutant, so that this rat strain was very helpful in defining substrates of MRP2 by comparing tissue concentrations or functional activities of compounds in MRP2-deficient rats with those in transport-competent Wistar rats. By using this strategy to study the involvement of MRP2 in brain access of antiepileptic drugs (AEDs), we recently reported that phenytoin is a substrate for MRP2 in the BBB. However, one drawback of such studies in genetically deficient rats is the fact that compensatory changes with upregulation of other transporters can occur. This prompted us to study the brain expression of P-glycoprotein (Pgp), a major drug efflux transporter in many tissues, including the BBB, in TR, rats compared with nonmutant (wild-type) Wistar rats. Methods: The expression of MRP2 and Pgp in brain and liver sections of TR, rats and normal Wistar rats was determined with immunohistochemistry, by using a novel, highly selective monoclonal MRP2 antibody and the monoclonal Pgp antibody C219, respectively. Results: Immunofluorescence staining with the MRP2 antibody was found to label a high number of microvessels throughout the brain in normal Wistar rats, whereas such labeling was absent in TR, rats. TR, rats exhibited a significant up-regulation of Pgp in brain capillary endothelial cells compared with wild-type controls. No such obvious upregulation of Pgp was observed in liver sections. A comparable overexpression of Pgp in the BBB was obtained after pilocarpine-induced seizures in wild-type Wistar rats. Experiments with systemic administration of the Pgp substrate phenobarbital and the selective Pgp inhibitor tariquidar in TR, rats substantiated that Pgp is functional and compensates for the lack of MRP2 in the BBB. Conclusions: The data on TR, rats indicate that Pgp plays an important role in the compensation of MRP2 deficiency in the BBB. Because such a compensatory mechanism most likely occurs to reduce injury to the brain from cytotoxic compounds, the present data substantiate the concept that MRP2 performs a protective role in the BBB. Furthermore, our data suggest that TR, rats are an interesting tool to study consequences of overexpression of Pgp in the BBB on access of drugs in the brain, without the need of inducing seizures or other Pgp-enhancing events for this purpose. [source] Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscanaJOURNAL OF MORPHOLOGY, Issue 2 2005Godelieve R.J. Criel Abstract Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces. © 2004 Wiley-Liss, Inc. [source] Identifying natural substrates for chaperonins using a sequence-based approachPROTEIN SCIENCE, Issue 1 2005George Stan Abstract The Escherichia coli chaperonin machinery, GroEL, assists the folding of a number of proteins. We describe a sequence-based approach to identify the natural substrate proteins (SPs) for GroEL. Our method is based on the hypothesis that natural SPs are those that contain patterns of residues similar to those found in either GroES mobile loop and/or strongly binding peptide in complex with GroEL. The method is validated by comparing the predicted results with experimentally determined natural SPs for GroEL. We have searched for such patterns in five genomes. In the E. coli genome, we identify 1422 (about one-third) sequences that are putative natural SPs. In Saccharomyces cerevisiae, 2885 (32%) of sequences can be natural substrates for Hsp60, which is the analog of GroEL. The precise number of natural SPs is shown to be a function of the number of contacts an SP makes with the apical domain (NC) and the number of binding sites (NB) in the oligomer with which it interacts. For known SPs for GroEL, we find ,4 < NC < 5 and 2 , NB , 4. A limited analysis of the predicted binding sequences shows that they do not adopt any preferred secondary structure. Our method also predicts the putative binding regions in the identified SPs. The results of our study show that a variety of SPs, associated with diverse functions, can interact with GroEL. [source] Rotavirus impairs the biosynthesis of brush-border-associated dipeptidyl peptidase IV in human enterocyte-like Caco-2/TC7 cellsCELLULAR MICROBIOLOGY, Issue 3 2007Isabelle Beau Summary Rotavirus is the leading cause of severe dehydrating diarrhoea in infants and young children worldwide. This virus infects mature enterocytes in the small intestine, and induces structural and functional damage. In the present study, we have identified a new mechanism by which rotavirus impairs a brush border-associated intestinal protein. We show that infection of enterocyte-like Caco-2/TC7 cells by rhesus monkey rotavirus (RRV) impairs the biosynthesis of dipeptidyl peptidase IV (DPP IV), an important hydrolase in the digestion of dietary proline-rich proteins. We show that the enzyme activity of DPP IV was reduced, and that rearrangements of the protein occurred at the apical domain of the RRV-infected cells. Using pulse-chase experiments and cell surface immunoprecipitation, we have demonstrated that RRV infection did not affect the stability or apical targeting of DPP IV, but did induce a dramatic decrease in its biosynthesis. Using quantitative RT-PCR, we showed that RRV had no effect on the level of expression of DPP IV mRNA, suggesting that the observed decrease in the biosynthesis of the protein is related to an effect of the virus at the translational level. [source] Disruption of tight junction structure in salivary glands from Sjögren's syndrome patients is linked to proinflammatory cytokine exposureARTHRITIS & RHEUMATISM, Issue 5 2010Patricia Ewert Objective Disorganization of acinar cell apical microvilli and the presence of stromal collagen in the acinar lumen suggest that the labial salivary gland (LSG) barrier function is impaired in patients with Sjögren's syndrome. Tight junctions define cell polarity and regulate the paracellular flow of ions and water, crucial functions of acinar cells. This study was undertaken to evaluate the expression and localization of tight junction proteins in LSGs from patients with SS and to determine in vitro the effects of tumor necrosis factor , (TNF,) and interferon-, (IFN,) on tight junction integrity of isolated acini from control subjects. Methods Twenty-two patients and 15 controls were studied. The messenger RNA and protein levels of tight junction components (claudin-1, claudin-3, claudin-4, occludin, and ZO-1) were determined by semiquantitative reverse transcriptase,polymerase chain reaction and Western blotting. Tight junction protein localization was determined by immunohistochemistry. Tight junction ultrastructure was examined by transmission electron microscopy. Isolated acini from control subjects were treated with TNF, and IFN,. Results Significant differences in tight junction protein levels were detected in patients with SS. ZO-1 and occludin were strongly down-regulated, while claudin-1 and claudin-4 were overexpressed. Tight junction proteins localized exclusively to apical domains in acini and ducts of LSGs from controls. In SS patients, the ZO-1 and occludin the apical domain presence of decreased, while claudin-3 and claudin-4 was redistributed to the basolateral plasma membrane. Exposure of isolated control acini to TNF, and IFN, reproduced these alterations in vitro. Ultrastructural analysis associated tight junction disorganization with the presence of endocytic vesicles containing electron-dense material that may represent tight junction components. Conclusion Our findings indicate that local cytokine production in LSGs from SS patients may contribute to the secretory gland dysfunction observed in SS patients by altering tight junction integrity of epithelial cells, thereby decreasing the quality and quantity of saliva. [source] | |||||||||||||