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Sample records for divergens apical membrane

  1. Babesia divergens apical membrane antigen-1 (BdAMA-1): A poorly polymorphic protein that induces a weak and late immune response.

    PubMed

    Moreau, E; Bonsergent, C; Al Dybiat, I; Gonzalez, L M; Lobo, C A; Montero, E; Malandrin, L

    2015-08-01

    Babesiosis is an important veterinary and zoonotic tick borne disease caused by the hemoprotozoan Babesia spp. which infects red blood cell of its vertebrate host. In order to control the infection, vaccination that targets molecules involved in the invasion process of red blood cells could provide a good alternative to chemotherapy. Among these molecules, Apical Membrane Antigen-1 (AMA-1) has been described as an excellent vaccine candidate in Plasmodium spp. In this paper, we have investigated AMA-1 of Babesia divergens (BdAMA-1) as vaccine candidate by evaluating its polymorphism and by studying the humoral response against BdAMA-1 of sheep experimentally infected with B. divergens. Polymorphism of BdAMA-1 was investigated by sequencing the corresponding gene of 9 B. divergens isolates from different geographical areas in France. Two Bdama-1 haplotypes (A and B) could be defined based on 2 non-synonymous point mutations. In silico prediction of linear epitopes revealed that the antigenicity of the 2 haplotypes is very similar. Antibody production against the extracellular domain of BdAMA-1 is weak and late, between 1 and 5 months after the inoculation of parasites. Both IgG1 and IgG2 are components of the anti-BdAMA-1 response. These results indicate that while BdAMA-1 may not be an immuno-dominant antigen, it could induce a mixed type 1 and type 2 immune response. In light of these results, the potential of BdAMA-1 as vaccine candidate is discussed. PMID:25956948

  2. Hypercompliant Apical Membranes of Bladder Umbrella Cells

    PubMed Central

    Mathai, John C.; Zhou, Enhua H.; Yu, Weiqun; Kim, Jae Hun; Zhou, Ge; Liao, Yi; Sun, Tung-Tien; Fredberg, Jeffrey J.; Zeidel, Mark L.

    2014-01-01

    Urinary bladder undergoes dramatic volume changes during filling and voiding cycles. In the bladder the luminal surface of terminally differentiated urothelial umbrella cells is almost completely covered by plaques. These plaques (500 to 1000 nm) are made of a family of proteins called uroplakins that are known to form a tight barrier to prevent leakage of water and solutes. Electron micrographs from previous studies show these plaques to be interconnected by hinge regions to form structures that appear rigid, but these same structures must accommodate large changes in cell shape during voiding and filling cycles. To resolve this paradox, we measured the stiffness of the intact, living urothelial apical membrane and found it to be highly deformable, even more so than the red blood cell membrane. The intermediate cells underlying the umbrella cells do not have uroplakins but their membranes are an order of magnitude stiffer. Using uroplakin knockout mouse models we show that cell compliance is conferred by uroplakins. This hypercompliance may be essential for the maintenance of barrier function under dramatic cell deformation during filling and voiding of the bladder. PMID:25229135

  3. GLUT2 Accumulation in Enterocyte Apical and Intracellular Membranes

    PubMed Central

    Ait-Omar, Amal; Monteiro-Sepulveda, Milena; Poitou, Christine; Le Gall, Maude; Cotillard, Aurélie; Gilet, Jules; Garbin, Kevin; Houllier, Anne; Château, Danièle; Lacombe, Amélie; Veyrie, Nicolas; Hugol, Danielle; Tordjman, Joan; Magnan, Christophe; Serradas, Patricia; Clément, Karine; Leturque, Armelle; Brot-Laroche, Edith

    2011-01-01

    OBJECTIVE In healthy rodents, intestinal sugar absorption in response to sugar-rich meals and insulin is regulated by GLUT2 in enterocyte plasma membranes. Loss of insulin action maintains apical GLUT2 location. In human enterocytes, apical GLUT2 location has not been reported but may be revealed under conditions of insulin resistance. RESEARCH DESIGN AND METHODS Subcellular location of GLUT2 in jejunal enterocytes was analyzed by confocal and electron microscopy imaging and Western blot in 62 well-phenotyped morbidly obese subjects and 7 lean human subjects. GLUT2 locations were assayed in ob/ob and ob/+ mice receiving oral metformin or in high-fat low-carbohydrate diet–fed C57Bl/6 mice. Glucose absorption and secretion were respectively estimated by oral glucose tolerance test and secretion of [U-14C]-3-O-methyl glucose into lumen. RESULTS In human enterocytes, GLUT2 was consistently located in basolateral membranes. Apical GLUT2 location was absent in lean subjects but was observed in 76% of obese subjects and correlated with insulin resistance and glycemia. In addition, intracellular accumulation of GLUT2 with early endosome antigen 1 (EEA1) was associated with reduced MGAT4a activity (glycosylation) in 39% of obese subjects on a low-carbohydrate/high-fat diet. Mice on a low-carbohydrate/high-fat diet for 12 months also exhibited endosomal GLUT2 accumulation and reduced glucose absorption. In ob/ob mice, metformin promoted apical GLUT2 and improved glucose homeostasis. Apical GLUT2 in fasting hyperglycemic ob/ob mice tripled glucose release into intestinal lumen. CONCLUSIONS In morbidly obese insulin-resistant subjects, GLUT2 was accumulated in apical and/or endosomal membranes of enterocytes. Functionally, apical GLUT2 favored and endosomal GLUT2 reduced glucose transepithelial exchanges. Thus, altered GLUT2 locations in enterocytes are a sign of intestinal adaptations to human metabolic pathology. PMID:21852673

  4. Fatty Acids Inhibit Apical Membrane Chloride Channels in Airway Epithelia

    NASA Astrophysics Data System (ADS)

    Anderson, Matthew P.; Welsh, Michael J.

    1990-09-01

    Apical membrane Cl^- channels control the rate of transepithelial Cl^- secretion in airway epithelia. cAMP-dependent protein kinase and protein kinase C regulate Cl^- channels by phosphorylation; in cystic fibrosis cells, phosphorylation-dependent activation of Cl^- channels is defective. Another important signaling system involves arachidonic acid, which is released from cell membranes during receptor-mediated stimulation. Here we report that arachidonic acid reversibly inhibited apical membrane Cl^- channels in cell-free patches of membrane. Arachidonic acid itself inhibited the channel and not a cyclooxygenase or lipoxygenase metabolite because (i) inhibitors of these enzymes did not block the response, (ii) fatty acids that are not substrates for the enzymes had the same effect as arachidonic acid, and (iii) metabolites of arachidonic acid did not inhibit the channel. Inhibition occurred only when fatty acids were added to the cytosolic surface of the membrane patch. Unsaturated fatty acids were more potent than saturated fatty acids. Arachidonic acid inhibited Cl^- channels from both normal and cystic fibrosis cells. These results suggest that fatty acids directly inhibit apical membrane Cl^- channels in airway epithelial cells.

  5. An Apical-Membrane Chloride Channel in Human Tracheal Epithelium

    NASA Astrophysics Data System (ADS)

    Welsh, Michael J.

    1986-06-01

    The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.

  6. Dlg5 maintains apical polarity by promoting membrane localization of Crumbs during Drosophila oogenesis

    PubMed Central

    Luo, Jun; Wang, Heng; Kang, Di; Guo, Xuan; Wan, Ping; Wang, Dou; Chen, Jiong

    2016-01-01

    Apical-basal polarity plays critical roles in the functions of epithelial tissues. However, the mechanisms of epithelial polarity establishment and maintenance remain to be fully elucidated. Here we show that the membrane-associated guanylate kinase (MAGUK) family protein Dlg5 is required for the maintenance of apical polarity of follicle epithelium during Drosophila oogenesis. Dlg5 localizes at the apical membrane and adherens junction (AJ) of follicle epithelium in early stage egg chambers. Specifically, we demonstrate that the major function of Dlg5 is to promote apical membrane localization of Crumbs, since overexpression of Crumbs but not other major apical or AJ components could rescue epithelial polarity defects resulted from loss of Dlg5. Furthermore, we performed a structure-function analysis of Dlg5 and found that the C-terminal PDZ3 and PDZ4 domains are required for all Dlg5’s functions as well as its ability to localize to apical membrane. The N-terminal coiled-coil motif could be individually targeted to the apical membrane, while the central linker region could be targeted to AJ. Lastly, the MAGUK core domains of PDZ4-SH3-GUK could be individually targeted to apical, AJ and basolateral membranes. PMID:27211898

  7. Dlg5 maintains apical polarity by promoting membrane localization of Crumbs during Drosophila oogenesis.

    PubMed

    Luo, Jun; Wang, Heng; Kang, Di; Guo, Xuan; Wan, Ping; Wang, Dou; Chen, Jiong

    2016-01-01

    Apical-basal polarity plays critical roles in the functions of epithelial tissues. However, the mechanisms of epithelial polarity establishment and maintenance remain to be fully elucidated. Here we show that the membrane-associated guanylate kinase (MAGUK) family protein Dlg5 is required for the maintenance of apical polarity of follicle epithelium during Drosophila oogenesis. Dlg5 localizes at the apical membrane and adherens junction (AJ) of follicle epithelium in early stage egg chambers. Specifically, we demonstrate that the major function of Dlg5 is to promote apical membrane localization of Crumbs, since overexpression of Crumbs but not other major apical or AJ components could rescue epithelial polarity defects resulted from loss of Dlg5. Furthermore, we performed a structure-function analysis of Dlg5 and found that the C-terminal PDZ3 and PDZ4 domains are required for all Dlg5's functions as well as its ability to localize to apical membrane. The N-terminal coiled-coil motif could be individually targeted to the apical membrane, while the central linker region could be targeted to AJ. Lastly, the MAGUK core domains of PDZ4-SH3-GUK could be individually targeted to apical, AJ and basolateral membranes. PMID:27211898

  8. Sulfate transport in apical membrane vesicles isolated from tracheal epithelium

    SciTech Connect

    Elgavish, A.; DiBona, D.R.; Norton, P.; Meezan, E.

    1987-09-01

    Sulfate uptake in apical membrane vesicles isolated from bovine tracheal epithelium is shown to occur into an osmotically sensitive intravesicular space, via a carrier-mediated system. This conclusion is based on three lines of evidence: 1) saturation kinetics: 2) substrate specificity; and 3) inhibition by the anion transport inhibitors SITS and DIDS. The affinity of the transport system is highest in low ionic strength media and decreases in the presence of gluconate. Chloride appears to cis-inhibit sulfate uptake and to trans-stimulate sulfate efflux. Cis-inhibition and trans-stimulation studies with a variety of anions indicate that this exchange system may be shared by HCO/sub 3//sup -/, S/sub 2/O/sub 3//sup 2 -/, SeO/sub 4//sup 2 -/, and MoO/sub 4//sup 2 -/ but not by H/sub 2/PO/sub 4//sup -/ or HAsO/sub 4//sup 2/. Studies indicate that protons may play two distinct roles in sulfate transport in this system. These studies show that the carrier-mediated system can function in the absence of chloride. The overshoot observed in the presence of a proton gradient indicates that under those conditions the mechanism of transport may be a SO/sub 4//sup 2 -/-OH/sup -/ exchange.

  9. Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane

    PubMed Central

    Gerl, Mathias J.; Sampaio, Julio L.; Urban, Severino; Kalvodova, Lucie; Verbavatz, Jean-Marc; Binnington, Beth; Lindemann, Dirk; Lingwood, Clifford A.; Shevchenko, Andrej; Schroeder, Cornelia

    2012-01-01

    The influenza virus (IFV) acquires its envelope by budding from host cell plasma membranes. Using quantitative shotgun mass spectrometry, we determined the lipidomes of the host Madin–Darby canine kidney cell, its apical membrane, and the IFV budding from it. We found the apical membrane to be enriched in sphingolipids (SPs) and cholesterol, whereas glycerophospholipids were reduced, and storage lipids were depleted compared with the whole-cell membranes. The virus membrane exhibited a further enrichment of SPs and cholesterol compared with the donor membrane at the expense of phosphatidylcholines. Our data are consistent with and extend existing models of membrane raft-based biogenesis of the apical membrane and IFV envelope. PMID:22249292

  10. Transepithelial water flow regulates apical membrane retrieval in antidiuretic hormone-stimulated toad urinary bladder.

    PubMed

    Harris, H W; Wade, J B; Handler, J S

    1986-09-01

    Antidiuretic hormone (ADH) increases the osmotic water permeability (Posm) of toad urinary bladder. This increase is believed to be produced by fusion of intracellular vesicles called aggrephores with the granular cell apical plasma membrane. Aggrephores contain intramembrane particle aggregates postulated to be water channels. ADH-stimulated Posm is decreased by osmotic gradient exposure, which is termed flux inhibition. We studied flux inhibition by exposing ADH-stimulated bladders to various osmotic gradients. Osmotic water flow was initially proportional to the applied osmotic gradient, but Posm decreased with time. Ultrastructural and quantitative studies of endocytosis demonstrate that apical membrane retrieval was a direct function of the transepithelial osmotic gradient. Posm remained unchanged when apical membrane retrieval was blocked by incubation of bladders at 2 degrees C, or under low water-flow conditions. These effects were reversed by increases in temperature or the applied osmotic gradient. We conclude that apical membrane retrieval causes the phenomenon of flux inhibition. PMID:2427542

  11. Characteristics of Kcnn4 channels in the apical membranes of an intestinal epithelial cell line

    PubMed Central

    Basalingappa, Kanthesh M.; Wonderlin, William F.

    2011-01-01

    Intermediate-conductance K+ (Kcnn4) channels in the apical and basolateral membranes of epithelial cells play important roles in agonist-induced fluid secretion in intestine and colon. Basolateral Kcnn4 channels have been well characterized in situ using patch-clamp methods, but the investigation of Kcnn4 channels in apical membranes in situ has been hampered by a layer of mucus that prevents seal formation. In the present study, we used patch-clamp methods to characterize Kcnn4 channels in the apical membrane of IEC-18 cells, a cell line derived from rat small intestine. A monolayer of IEC-18 cells grown on a permeable support is devoid of mucus, and tight junctions enable selective access to the apical membrane. In inside-out patches, Ca2+-dependent K+ channels observed with iberiotoxin (a Kcnma1/large-conductance, Ca2+-activated K+ channel blocker) and apamin (a Kcnn1–3/small-conductance, Ca2+-activated K+ channel blocker) present in the pipette solution exhibited a single-channel conductance of 31 pS with inward rectification. The currents were reversibly blocked by TRAM-34 (a Kcnn4 blocker) with an IC50 of 8.7 ± 2.0 μM. The channels were not observed when charybdotoxin, a peptide inhibitor of Kcnn4 channels, was added to the pipette solution. TRAM-34 was less potent in inhibiting Kcnn4 channels in patches from apical membranes than in patches from basolateral membranes, which was consistent with a preferential expression of Kcnn4c and Kcnn4b isoforms in apical and basolateral membranes, respectively. The expression of both isoforms in IEC-18 cells was confirmed by RT-PCR and Western blot analyses. This is the first characterization of Kcnn4 channels in the apical membrane of intestinal epithelial cells. PMID:21868633

  12. Diluting segment in kidney of dogfish shark. II. Electrophysiology of apical membranes and cellular resistances.

    PubMed

    Hebert, S C; Friedman, P A

    1990-02-01

    Diluting segments from the bundle zone of the dogfish shark kidney were perfused in vitro and the electrophysiological characteristics of this segment investigated using conventional microelectrodes and cable analysis. In 21 tubules perfused with symmetrical Ringer solutions the average transepithelial voltage (Vte), transepithelial conductance (Gte), and equivalent short circuit current (Isc) were 8.7 +/- 0.6 mV, 91.3 +/- 10.2 mS/cm2, and 641 +/- 48 microA/cm2, respectively. Microelectrode impalements in 52 cells yielded values for the basolateral membrane voltage (Vb) and an estimated apical membrane fractional resistance (fRa) of -57.5 +/- 1.3 mV and 0.896 +/- 0.008, respectively. All of these parameters were distributed in a Gaussian manner. Liminal furosemide (10(-4) M) abolished Isc, hyperpolarized apical membrane voltage (Va) and Vb, increased Gte, and reduced fRa. The apical membrane was predominantly conductive to K+: increasing luminal K+ from 5 to 49.7 mM resulted in an apical depolarization of 41.2 mV and a fall in fRa and luminal Ba2+ (1 mM) depolarized Va by 14.3 mV and increased fRa. The apical transference number for K+ was 0.74 +/- 0.07. The cellular and paracellular resistances were estimated from the effects of luminal Ba2+ on fRa and Gte. The cell conductance represented approximately 45% of Gte, with the primary resistance barrier located at the apical membrane: apical membrane resistance was 59.7 +/- 16.0 and basolateral membrane resistance was 5.9 +/- 2.3 omega.cm2. From these resistance values together with the passive permeability (PNa/PCl) of 2.5 determined previously, the ratio of net Cl- absorption to net transcellular Na+ absorption was determined to be 2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2309934

  13. Effects of ADH on the apical and basolateral membranes of toad urinary bladder epithelial cells.

    PubMed

    Donaldson, P J; Leader, J P

    1993-11-01

    Short-circuited urinary bladders from Bufo marinus were supported on their apical surface by an agar mounting method and impaled with microelectrodes via their basolateral membrane. This arrangement provided stable and long-lasting impalements of epithelial cells and yielded reliable membrane potentials and voltage divider ratios (Ra/Rb), where Ra and Rb are apical and basolateral membrane resistances respectively. The membrane potential under short-circuit conditions (Vsc) was -51.4 +/- 2.2 mV (n = 59), while under open-circuit conditions apical membrane potential (Va) and basolateral membrane potential (Vb) were -31.0 +/- 2.4 and 59.5 +/- 2.4 mV, respectively. This yields a "well-shaped" potential profile across the toad urinary bladder, where Va is inversely related to the rate of transport, Isc. Antidiuretic hormone (ADH) produced a hyperpolarisation of Vsc and Vb but had no significant effect on Va. In addition, Ra/Rb was significantly increased by ADH (4.6 +/- 0.5 to 10.2 +/- 3.6). Calculation of individual membrane resistances following the addition of amiloride showed that ADH produced a parallel decrease in Ra and Rb membrane resistance, with the observed increase in Ra/Rb being due to a greater percentage decrease in Rb than in Ra. The ability of ADH to effect parallel changes in apical and basolateral membrane conductance helps to maintain a constant cellular volume despite an increase in transepithelial transport. PMID:8309781

  14. Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion

    PubMed Central

    Bargieri, Daniel Y.; Andenmatten, Nicole; Lagal, Vanessa; Thiberge, Sabine; Whitelaw, Jamie A.; Tardieux, Isabelle; Meissner, Markus; Ménard, Robert

    2013-01-01

    Apicomplexan parasites invade host cells by forming a ring-like junction with the cell surface and actively sliding through the junction inside an intracellular vacuole. Apical membrane antigen 1 is conserved in apicomplexans and a long-standing malaria vaccine candidate. It is considered to have multiple important roles during host cell penetration, primarily in structuring the junction by interacting with the rhoptry neck 2 protein and transducing the force generated by the parasite motor during internalization. Here, we generate Plasmodium sporozoites and merozoites and Toxoplasma tachyzoites lacking apical membrane antigen 1, and find that the latter two are impaired in host cell attachment but the three display normal host cell penetration through the junction. Therefore, apical membrane antigen 1, rather than an essential invasin, is a dispensable adhesin of apicomplexan zoites. These genetic data have implications on the use of apical membrane antigen 1 or the apical membrane antigen 1–rhoptry neck 2 interaction as targets of intervention strategies against malaria or other diseases caused by apicomplexans. PMID:24108241

  15. Apical and basal membrane ion transport mechanisms in bovine retinal pigment epithelium.

    PubMed

    Joseph, D P; Miller, S S

    1991-04-01

    1. Intracellular voltage recordings using conventional and double-barrelled chloride-selective microelectrodes have been used to identify several transport mechanisms at the apical and basolateral membranes of the isolated bovine retinal pigment epithelium (RPE)-choroid preparation. Intracellular recordings were obtained from two cell populations, melanotic (pigmented) and amelanotic (non-pigmented). The electrical properties of these two populations are practically identical. For melanotic cells the average apical resting membrane potential (VA) is -61 +/- 2 mV (mean +/- S.E.M., n = 49 cells, thirty-three eyes). For these cells the ratio of apical to basolateral membrane resistance (a) was 0.22 +/- 0.02. The mean transepithelial voltage and resistance were 6 +/- 1 mV and 138 +/- 7 omega cm2, respectively. 2. The apical membrane, which faces the distal retina, contains a Ba(2+)-inhibitable K+ conductance and a ouabain-inhibitable, electrogenic Na(+)-K+ pump. In addition it contains a bumetanide-sensitive mechanism, the putative Na(+)-K(+)-Cl- cotransporter. The basolateral membrane contains a DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid)-inhibitable chloride channel. The relative conductances of the apical and basolateral membranes to K+ and Cl- are TK approximately 0.9 and TCl approximately 0.7, respectively. 3. The ouabain-induced fast phase of apical membrane depolarization (0-30 s) was used to calculate the equivalent resistances of the apical (RA) and basolateral (RB) cell membranes, as well as the paracellular or shunt resistance (RS). They are: 3190 +/- 400, 17920 +/- 2730 and 2550 +/- 200 omega (mean +/- S.E.M., n = 9 tissues), respectively. From these data the equivalent electromotive forces (EMF) at the apical (EA) and basolateral (EB) membranes were also calculated. They are: -69 +/- 5.0 and -24 +/- 5.0 mV, respectively. 4. Intracellular Cl- activity (aiCl) was measured using double-barreled ion-selective microelectrodes. In the steady state

  16. Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells

    PubMed Central

    Gálvez-Santisteban, Manuel; Rodriguez-Fraticelli, Alejo E.; Bryant, David M.; Vergarajauregui, Silvia; Yasuda, Takao; Bañón-Rodríguez, Inmaculada; Bernascone, Ilenia; Datta, Anirban; Spivak, Natalie; Young, Kitty; Slim, Christiaan L.; Brakeman, Paul R.; Fukuda, Mitsunori; Mostov, Keith E.; Martín-Belmonte, Fernando

    2012-01-01

    SUMMARY The formation of epithelial tissues requires both the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to formation of a singular apical membrane, resulting in contribution of each cell to only a single lumen. Here, from a functional screen for genes required for 3D epithelial architecture we identify key roles for Synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PI(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE Syntaxin-3. Together, Slp2-a/4-a co-ordinate the spatiotemporal organization of vectorial apical transport to ensure only a single apical surface, and thus formation of a single lumen, occurs per cell. PMID:22820376

  17. Homeostasis of the apical plasma membrane during regulated exocytosis in the salivary glands of live rodents

    PubMed Central

    Masedunskas, Andrius; Sramkova, Monika; Weigert, Roberto

    2011-01-01

    In exocrine organs such as the salivary glands, fluids and proteins are secreted into ductal structures by distinct mechanisms that are tightly coupled. In the acinar cells, the major secretory units of the salivary glands, fluids are secreted into the acinar canaliculi through paracellular and intracellular transport, whereas proteins are stored in large granules that undergo exocytosis and fuse with the apical plasma membranes releasing their content into the canaliculi. Both secretory processes elicit a remodeling of the apical plasma membrane that has not been fully addressed in in vitro or ex vivo models. Recently, we have studied regulated exocytosis in the salivary glands of live rodents, focusing on the role that actin and myosin plays in this process. We observed that during exocytosis both secretory granules and canaliculi are subjected to the hydrostatic pressure generated by fluid secretion. Furthermore, the absorption of the membranes of the secretory granules contributes to the expansion and deformation of the canaliculi. Here we suggest that the homeostasis of the apical plasma membranes during exocytosis is maintained by various strategies that include: (1) membrane retrieval via compensatory endocytosis, (2) increase of the surface area via membrane folds and (3) recruitment of a functional actomyosin complex. Our observations underscore the important relationship between tissue architecture and cellular response, and highlight the potential of investigating biological processes in vivo by using intravital microscopy. PMID:22754613

  18. Apical membrane limits urea permeation across the rat inner medullary collecting duct.

    PubMed Central

    Star, R A

    1990-01-01

    Urea diffuses across the terminal inner medullary collecting duct (IMCD) via a facilitated transport pathway. To examine the mechanism of transcellular urea transport, membrane-apparent urea (Purea) and osmotic water (Pf) permeabilities of IMCD cells were measured by quantitative light microscopy in isolated IMCD-2 tubules perfused in the absence of vasopressin. Basolateral membrane Pf, determined by addition of raffinose to the bath, was 69 microns/s. Basolateral membrane Purea, determined by substituting urea for raffinose without change in osmolality, was 14 X 10(-5) cm/s. Bath phloretin inhibited basolateral Purea by 85% without a significant effect on Pf. The basolateral reflection coefficient for urea, determined by addition of urea in the presence of phloretin, was 1.0. These results indicate that urea crosses the basolateral membrane by diffusion, and not by solvent drag. In perfused tubules, the rate of cell swelling following substitution of urea for mannitol was significantly greater with bath than lumen changes. After correcting for membrane surface area, the basolateral membrane was twofold more permeable than the apical membrane. Conclusions: (a) in the absence of vasopressin, urea permeation across the IMCD cell is limited by the apical membrane; (b) the basolateral membrane contains a phloretin-sensitive urea transporter; (c) transepithelial urea transport occurs by movement of urea through the IMCD cell. PMID:2212006

  19. Does apical membrane GLUT2 have a role in intestinal glucose uptake?

    PubMed Central

    Naftalin, Richard J

    2014-01-01

    It has been proposed that the non-saturable component of intestinal glucose absorption, apparent following prolonged exposure to high intraluminal glucose concentrations, is mediated via the low affinity glucose and fructose transporter, GLUT2, upregulated within the small intestinal apical border. The evidence that the non-saturable transport component is mediated via an apical membrane sugar transporter is that it is inhibited by phloretin, after exposure to phloridzin. Since the other apical membrane sugar transporter, GLUT5, is insensitive to inhibition by either cytochalasin B, or phloretin, GLUT2 was deduced to be the low affinity sugar transport route. As in its uninhibited state, polarized intestinal glucose absorption depends both on coupled entry of glucose and sodium across the brush border membrane and on the enterocyte cytosolic glucose concentration exceeding that in both luminal and submucosal interstitial fluids, upregulation of GLUT2 within the intestinal brush border will usually stimulate downhill glucose reflux to the intestinal lumen from the enterocytes; thereby reducing, rather than enhancing net glucose absorption across the luminal surface. These states are simulated with a computer model generating solutions to the differential equations for glucose, Na and water flows between luminal, cell, interstitial and capillary compartments. The model demonstrates that uphill glucose transport via SGLT1 into enterocytes, when short-circuited by any passive glucose carrier in the apical membrane, such as GLUT2, will reduce transcellular glucose absorption and thereby lead to increased paracellular flow. The model also illustrates that apical GLUT2 may usefully act as an osmoregulator to prevent excessive enterocyte volume change with altered luminal glucose concentrations. PMID:25671087

  20. LLC-PK sub 1 cells express Na sup + -lactate cotransport in apical membranes after confluency

    SciTech Connect

    Poustis-Delpont, C.; Mengual, R.; Sudaka, P. )

    1988-12-01

    L-({sup 3}H)lactate uptake was characterized in LLC-PK{sub 1} cell apical membrane vesicles obtained by intensive culture on microcarrier beads. The apical membrane preparation technique involved MgCl{sub 2} precipitation. Na{sup +}-dependent L-({sup 3}H)lactate uptake was present only after confluency; its appearance paralleled the subcellular localization of aminopeptidase in apical membranes. L-({sup 3}H)lactate uptake was Na{sup +}-dependent and electrogenic. Only the Na{sup +}-dependent component of L({sup 3}H)lactate uptake was saturable with one family of independent carriers. The apparent affinity constant was 1.1 {plus minus} 0.25 mM and the apparent maximal velocity was 29 {plus minus} 3 nmol{center dot}mg{sup {minus}1}{center dot}min{sup {minus}1}. The Na{sup +}-lactate cotransport stoichiometry was 2 Na{sup +} for 1 lactate. The specificity of the L-lactate transport system was compatible with that of the monocarboxylic acid pathway described previously brush-border membranes of kidney cortex and discrete from the tricarboxylic acid carrier, the D-glucose transporter, and the general pathway for anions. The LLC-PK{sub 1} cell line appears to be a useful tool for study of the regulation of L-lactate uptake and biosynthesis of the renal monocarboxylic acid transporter.

  1. TRP Channels Localize to Subdomains of the Apical Plasma Membrane in Human Fetal Retinal Pigment Epithelium

    PubMed Central

    Zhao, Peter Y.; Gan, Geliang; Peng, Shaomin; Wang, Shao-Bin; Chen, Bo; Adelman, Ron A.; Rizzolo, Lawrence J.

    2015-01-01

    Purpose. Calcium regulates many functions of the RPE. Its concentration in the subretinal space and RPE cytoplasm is closely regulated. Transient receptor potential (TRP) channels are a superfamily of ion channels that are moderately calcium-selective. This study investigates the subcellular localization and potential functions of TRP channels in a first-passage culture model of human fetal RPE (hfRPE). Methods. The RPE isolated from 15- to 16-week gestation fetuses were maintained in serum-free media. Cultures were treated with barium chloride (BaCl2) in the absence and presence of TRP channel inhibitors and monitored by the transepithelial electrical resistance (TER). The expression of TRP channels was determined using quantitative RT-PCR, immunoblotting, and immunofluorescence confocal microscopy. Results. Barium chloride substantially decreased TER and disrupted cell–cell contacts when added to the apical surface of RPE, but not when added to the basolateral surface. The effect could be partially blocked by the general TRP inhibitor, lanthanum chloride (LaCl3, ~75%), or an inhibitor of calpain (~25%). Family member-specific inhibitors, ML204 (TRPC4) and HC-067047 (TRPV4), had no effect on basal channel activity. Expression of TRPC4, TRPM1, TRPM3, TRPM7, and TRPV4 was detected by RT-PCR and immunoblotting. The TRPM3 localized to the base of the primary cilium, and TRPC4 and TRPM3 localized to apical tight junctions. The TRPV4 localized to apical microvilli in a small subset of cells. Conclusions. The TRP channels localized to subdomains of the apical membrane, and BaCl2 was only able to dissociate tight junctions when presented to the apical membrane. The data suggest a potential role for TRP channels as sensors of [Ca2+] in the subretinal space. PMID:25736794

  2. Golgi sorting regulates organization and activity of GPI-proteins at apical membranes

    PubMed Central

    Tivodar, Simona; Formiggini, Fabio; Ossato, Giulia; Gratton, Enrico; Tramier, Marc; Coppey-Moisan, Maïté; Zurzolo, Chiara

    2014-01-01

    Here, we combined classical biochemistry with novel biophysical approaches to study with high spatial and temporal resolution the organization of GPI-anchored proteins (GPI-APs) at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, following sorting in the Golgi, each GPI-AP reaches the apical surface in homo-clusters. Golgi-derived homo-clusters are required for their subsequent plasma membrane organization into cholesterol-dependent hetero-clusters. By contrast, in non-polarized MDCK cells GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form hetero-clusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, different from fibroblasts, in polarized epithelial cells a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and the function of GPI-APs at the apical surface. PMID:24681536

  3. Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane

    PubMed Central

    1995-01-01

    The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells. PMID:7896870

  4. Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion.

    PubMed

    Saslowsky, David E; Thiagarajah, Jay R; McCormick, Beth A; Lee, Jean C; Lencer, Wayne I

    2016-04-01

    The apical brush border membrane (BBM) of intestinal epithelial cells forms a highly structured and dynamic environmental interface that serves to regulate cellular physiology and block invasion by intestinal microbes and their products. How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function. We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin. Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network. Accumulation of apical membrane ceramide is necessary and sufficient to induce the actin phenotype, and this coincides with altered membrane structure and augmented innate immune function as evidenced by resistance to invasion by Salmonella. PMID:26864627

  5. Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion

    PubMed Central

    Saslowsky, David E.; Thiagarajah, Jay R.; McCormick, Beth A.; Lee, Jean C.; Lencer, Wayne I.

    2016-01-01

    The apical brush border membrane (BBM) of intestinal epithelial cells forms a highly structured and dynamic environmental interface that serves to regulate cellular physiology and block invasion by intestinal microbes and their products. How the BBM dynamically responds to pathogenic and commensal bacterial signals can define intestinal homeostasis and immune function. We previously found that in model intestinal epithelium, the conversion of apical membrane sphingomyelin to ceramide by exogenous bacterial sphingomyelinase (SMase) protected against the endocytosis and toxicity of cholera toxin. Here we elucidate a mechanism of action by showing that SMase induces a dramatic, reversible, RhoA-dependent alteration of the apical cortical F-actin network. Accumulation of apical membrane ceramide is necessary and sufficient to induce the actin phenotype, and this coincides with altered membrane structure and augmented innate immune function as evidenced by resistance to invasion by Salmonella. PMID:26864627

  6. Purine and pyrimidine nucleotide receptors in the apical membranes of equine cultured epithelia

    PubMed Central

    Ko, W H; Wilson, S M; Wong, P Y D

    1997-01-01

    The short circuit current (ISC) technique was used to quantify electrolyte transport by equine cultured sweat gland epithelia. Adenosine 5′-triphosphate (ATP) and certain related compounds, caused transient increases in ISC when added to the apical solution. The order of potency was uridine triphosphate (UTP)>ATP>ADP>>AMP=adenosine.The responses to apical nucleotides were due to chloride and bicarbonate secretion and were reduced in pertussis toxin-treated cells. P2-receptors sensitive to uridine 5′-triphosphate (UTP), that interact with inhibitory G proteins, therefore appear to be present in the apical membrane.Responses to ATP and UTP were reduced in cells loaded with BAPTA, a calcium chelator. BAPTA attenuated the response to ATP more than the response to UTP suggesting that these nucleotides may not act via a common pathway.Cross-desensitization experiments indicated that two populations of UTP-sensitive receptor were present. One was sensitive to UTP and ATP, whereas the second was sensitive only to UTP. Uridine diphosphate appeared to activate the ATP-insensitive receptor population selectively.These data suggest that apical pyrimidinoceptors may be expressed by these cells. The physiological role of these receptors is unknown but they may allow the autocrine regulation of epithelial function. PMID:9146899

  7. Human cytomegalovirus glycoprotein B contains autonomous determinants for vectorial targeting to apical membranes of polarized epithelial cells.

    PubMed

    Tugizov, S; Maidji, E; Xiao, J; Zheng, Z; Pereira, L

    1998-09-01

    We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Delta717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the N- and O-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBDelta646-906) or the TM anchor alone (gBDelta751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBDelta772-906) or the last 73 amino acids (gBDelta834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural

  8. Human Cytomegalovirus Glycoprotein B Contains Autonomous Determinants for Vectorial Targeting to Apical Membranes of Polarized Epithelial Cells

    PubMed Central

    Tugizov, Sharof; Maidji, Ekaterina; Xiao, Jianqiao; Zheng, Zhenwei; Pereira, Lenore

    1998-01-01

    We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Δ717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the N- and O-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBΔ646-906) or the TM anchor alone (gBΔ751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBΔ772-906) or the last 73 amino acids (gBΔ834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural elements that

  9. An inwardly rectifying potassium channel in apical membrane of Calu-3 cells.

    PubMed

    Wu, Jin V; Krouse, Mauri E; Rustagi, Arjun; Joo, Nam Soo; Wine, Jeffrey J

    2004-11-01

    Patch clamp methods and reverse transcription-polymerase chain reaction (RT-PCR) were used to characterize an apical K+ channel in Calu-3 cells, a widely used model of human airway gland serous cells. In cell-attached and excised apical membrane patches, we found an inwardly rectifying K+ channel (Kir). The permeability ratio was PNa/PK = 0.058. In 30 patches with both cystic fibrosis transmembrane conductance regulator and Kir present, we observed 79 cystic fibrosis transmembrane conductance regulator and 58 Kir channels. The average chord conductance was 24.4 +/- 0.5 pS (n = 11), between 0 and -200 mV, and was 9.6 +/- 0.7 pS (n = 8), between 0 and 50 mV; these magnitudes and their ratio of approximately 2.5 are most similar to values for rectifying K+ channels of the Kir4.x subfamilies. We attempted to amplify transcripts for Kir4.1, Kir4.2, and Kir5.1; of these only Kir4.2 was present in Calu-3 lysates. The channel was only weakly activated by ATP and was relatively insensitive to internal pH. External Cs+ and Ba2+ blocked the channel with Kd values in the millimolar range. Quantitative modeling of Cl- secreting epithelia suggests that secretion rates will be highest and luminal K+ will rise to 16-28 mm if 11-25% of the total cellular K+ conductance is placed in the apical membrane (Cook, D. I., and Young, J. A. (1989) J. Membr. Biol. 110, 139-146). Thus, we hypothesize that the K+ channel described here optimizes the rate of secretion and is involved in K+ recycling for the recently proposed apical H+ -K+ -ATPase in Calu-3 cells. PMID:15328350

  10. Patch-clamp evidence for calcium channels in apical membranes of rabbit kidney connecting tubules.

    PubMed Central

    Tan, S; Lau, K

    1993-01-01

    To test the hypothesis that Ca channel plays a role in renal epithelial Ca transport, we exposed and patched apical membranes of freshly microdissected rabbit connecting tubules (CNTs). Single channel Ca currents were recorded with Ba as the charge carrier. In the cell-attached mode, 8-Br-cAMP increased the open-state probability (Po) to 0.6%. In excised, inside-out patches, Po was low spontaneously and remained low during either bath protein kinase A catalytic subunit (PKAcs) or Bay K 8644. Exposure to both agonists, however, unmasked Ca channels previously latent with only one, raising Po by 1.05% at membrane potential of -70 mV. Mean Po for 14 seals (2.57%) peaked at -70 mV, declining with either hyperpolarization or depolarization. The slope conductance was 25 pS. The extrapolated reversal potential (138 mV) agrees with the calculated equilibrium potential for Ca (158 mV). The Ca to Na permeability ratio exceeded 2,800. In four patches stimulated by Bay K 8644 and PKAcs, bath nifedipine reduced Po from 1.03 to 0.15% at -63 mV. These patch-clamp data demonstrate a selective, 25-pS, cAMP/PKAcs-sensitive Ca channel in apical membranes of CNT. Po is stimulated by PKAcs and dihydropyridine (DHP) agonist, but inhibited by DHP antagonist and by depolarization. The data are consistent with the potential role of apical membrane Ca channel in epithelial Ca transport. PMID:7504693

  11. Comparative infectivity of Babesia divergens and a zoonotic Babesia divergens-like parasite in cattle.

    PubMed

    Holman, Patricia J; Spencer, Angela M; Telford, Sam R; Goethert, Heidi K; Allen, Andrew J; Knowles, Donald P; Goff, Will L

    2005-11-01

    Babesia divergens-like parasites identified in human babesiosis cases in Missouri and Kentucky and in eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts, share identical small subunit ribosomal RNA gene sequences. This sequence is 99.8% identical to that of Babesia divergens, suggesting that the U.S. parasite may be B. divergens, a causative agent of human and bovine babesiosis in Europe. Holstein-Friesian calves were inoculated with cultured Nantucket Island Babesia sp. (NR831) and B. divergens parasites and monitored by clinical signs, Giemsa-stained blood films, PCR, and culture. The NR831 recipients did not exhibit clinical signs of infection and remained negative for all assays. The B. divergens recipients developed clinical infections and became positive by all assays. NR831 recipients were fully susceptible upon challenge inoculation with B. divergens. This study confirms that the Nantucket Island Babesia sp. is not conspecific with B. divergens based on host specificity for cattle. PMID:16282295

  12. Transport characteristics of L-citrulline in renal apical membrane of proximal tubular cells.

    PubMed

    Mitsuoka, Keisuke; Shirasaka, Yoshiyuki; Fukushi, Akimasa; Sato, Masanobu; Nakamura, Toshimichi; Nakanishi, Takeo; Tamai, Ikumi

    2009-04-01

    L-Citrulline has diagnostic potential for renal function, because its plasma concentration increases with the progression of renal failure. Although L-citrulline extracted by glomerular filtration in kidney is mostly reabsorbed, the mechanism involved is not clearly understood. The present study was designed to characterize L-citrulline transport across the apical membranes of renal epithelial tubular cells, using primary-cultured rat renal proximal tubular cells, as well as the human kidney proximal tubular cell line HK-2. L-Citrulline was transported in a Na(+)-dependent manner from the apical side of both cell types cultured on permeable supports with a microporous membrane. Kinetic analysis indicated that the transport involves two distinct Na(+)-dependent saturable systems and one Na(+)-independent saturable system in HK-2 cells. The uptake was competitively inhibited by neutral and cationic, but not anionic amino acids. Relatively large cationic and anionic compounds inhibited the uptake, but smaller ones did not. In HK-2 cells, mRNA expression of SLC6A19 and SLC7A9, which encode B(0)AT1 and b(0,+)AT, respectively, was detected by RT-PCR. In addition, L-citrulline transport was significantly decreased in HK-2 cells in which either SLC6A19 or SLC7A9 was silenced. Hence, these results suggest that amino acid transporters B(0)AT1 and b(0,+)AT are involved in the reabsorption of L-citrulline in the kidney, at least in part, by mediating the apical membrane transport of L-citrulline in renal tubule cells. PMID:19322909

  13. Mapping symplasmic fields at the shoot apical meristem using iontophoresis and membrane potential measurements.

    PubMed

    van der Schoot, Christiaan; Rinne, Päivi L H

    2015-01-01

    Microinjections of fluorescent dyes have revealed that the shoot apical meristem (SAM) is dynamically partitioned into symplasmic fields (SFs), implying that plasmodesmata (Pd) are held shut at specific locations in the proliferating cellular matrix. The SFs are integrated into a coherent morphogenetic unit by exchange of morphogens and transcription factors via gating Pd between adjacent SFs, and by ligand-receptor interactions that operate across the extracellular space. We describe a method for the real-time mapping of SF in the SAM by iontophoresis and membrane potential measurements. PMID:25287203

  14. Sorting of membrane and fluid at the apical pole of polarized Madin-Darby canine kidney cells.

    PubMed

    Leung, S M; Ruiz, W G; Apodaca, G

    2000-06-01

    When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (apical early endosome but is excluded from the subapical elements of the Rab11-positive recycling compartment. We propose that the term ARE be used to describe the subapical Rab11-positive compartment and that the ARE is distinct from both the transferrin-rich common recycling endosome and the fluid-rich apical early endosome. PMID:10848634

  15. Sorting of Membrane and Fluid at the Apical Pole of Polarized Madin-Darby Canine Kidney Cells

    PubMed Central

    Leung, Som-Ming; Ruiz, Wily G.; Apodaca, Gerard

    2000-01-01

    When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (≤2.5 min at 37°C) and are then rapidly segregated from one another. The dextran remains in the large supranuclear EEA1-positive early endosomes while recycling polymeric immunoglobulin receptor–bound immunoglobulin A is delivered to a Rab11-positive subapical recycling compartment. This latter step requires an intact microtubule cytoskeleton. Receptor-bound transferrin, a marker of the basolateral recycling pathway, has limited access to the fluid-rich apical early endosome but is excluded from the subapical elements of the Rab11-positive recycling compartment. We propose that the term ARE be used to describe the subapical Rab11-positive compartment and that the ARE is distinct from both the transferrin-rich common recycling endosome and the fluid-rich apical early endosome. PMID:10848634

  16. Apical membrane sodium and chloride entry during osmotic swelling of renal (A6) epithelial cells.

    PubMed

    Crowe, W E; Ehrenfeld, J; Brochiero, E; Wills, N K

    1995-03-01

    To assess the role of chloride in cell volume and sodium transport regulation, we measured cell height changes (CH), transepithelial chloride and sodium fluxes, and intracellular chloride content during challenge with hyposmotic solutions under open circuit (OC) conditions. CH maximally increased following hyposmotic challenge within approximately 5 minutes. The change in CH was smaller under short circuit (SC) conditions or following replacement of chloride in the mucosal solution by gluconate or cyclamate (Cl(-)-freem). When corrected for the osmotically inactive cell volume (30 +/- 2%), delta CH for controls (OC) were greater than predicted for an ideal osmometer. In contrast, delta CH for Cl(-)-freem or SC conditions were similar to that predicted for an ideal osmometer. Na+ and Cl- mucosa-to-serosa fluxes increased following hyposmotic challenge. Chloride fluxes increased maximally within 5 min, then decreased. In contrast, the Na+ flux increased slowly and reached a steady state after approximately 25 min. Under isosmotic conditions, exposure to Cl(-)-freem solutions led to decreases in the transepithelial conductance, Na+ flux, and CH. Chloride permeabilities in the apical and basolateral membranes were detected using the fluorescent intracellular chloride indicator MQAE. The results indicate that during osmotic swelling, the entry of both sodium and chloride is increased. The time courses of these increases differ, suggesting distinct mechanisms for the osmotic regulation of these apical membrane transport processes. PMID:7541082

  17. Evidence for basolateral but not apical membrane localization of outwardly rectifying depolarization-induced Cl(-) channel in airway epithelia.

    PubMed

    Hwang, T H; Lee, H J; Lee, N K; Choi, Y C

    2000-08-01

    The rat primary cultured-airway monolayer had been an excellent model for deciphering the ion channel after nystatin permeabilization of its basolateral or apical membrane (Hwang et al., 1996). After apical membrane permeabilization of rat primary cultured-airway monolayer, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive outwardly rectifying depolarization-induced Cl(-) (BORDIC) currents were observed across the basolateral membrane in symmetrical NMG-Cl solution in this study. No significant Cl(-) current induced by the application of voltage clamping was observed across the apical membrane in symmetrical NMG-Cl solution after basolateral membrane permeabilization. The halide permeability sequence for BORDIC current was Br(-) = I(-) > Cl(-). BORDIC current was not affected by basolaterally applied bumetanide (0.5 mm). Basolateral DIDS (0.2 mm) but not apical DIDS inhibited CFTR mediated short-circuit current (I(sc)) in an intact monolayer of rat airway epithelia, a T84 human colonal epithelial cell line, and a Calu-3 human airway epithelial cell line. This is the first report showing that depolarization induced Cl(-) current is present on the basolateral membrane of airway epithelia. PMID:10931973

  18. Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area

    PubMed Central

    Harrison, Theresa A.; He, Zhiguo; Boggs, Kristin; Thuret, Gilles; Liu, Hong-Xiang

    2016-01-01

    Purpose The corneal endothelium is widely believed to consist of geometrically regular cells interconnected by junctional complexes. However, while en face visualization of the endothelial apical surface reveals characteristic polygonal borders, the overall form of the component cells has rarely been observed. Methods To visualize the shape of individual endothelial cells within the native monolayer, two independent Cre/LoxP-based cell labeling approaches were used. In the first, a P0-Cre mouse driver strain was bred to an R26-tdTomato reporter line to map neural crest–derived endothelial cells with cytosolic red fluorescent protein. In the second, HPRT-Cre induction of small numbers of green and red fluorescent protein–filled cells within a background of unlabeled cells was achieved using a dual-color reporter system, mosaic analysis with double markers (MADM). Selective imaging of the endothelial lateral membranes at different apicobasal levels was accomplished after staining with antibodies to ZO-1 and the neural cell adhesion molecule (NCAM). Results When viewed in their entirety in whole-mount preparations, fluorescent protein–filled cells appear star-shaped, extending multiple dendritic processes that radiate outward in the plane of the monolayer. Examination of rare cases where cells expressing different fluorescent proteins lie directly adjacent to one another reveals that these long processes undergo extensive interdigitation. The resulting overlap allows individual cells to extend over a greater area than if the cell boundaries were mutually exclusive. Anti-NCAM staining of these interlocking peripheral cell extensions reveals an elaborate system of lateral membrane folds that, when viewed in optical sections, increase in complexity from the apical to the basal pole. This not only produces a substantial increase in the basolateral, relative to the apical, membrane but also greatly extends the paracellular pathway as a highly convoluted space

  19. A Cre-inducible fluorescent reporter for observing apical membrane dynamics.

    PubMed

    Pan, Xinchao; Schnell, Ulrike; Karner, Courtney M; Small, Erin V; Carroll, Thomas J

    2015-01-01

    The ability to image living tissues with fluorescent proteins has revolutionized the fields of cell and developmental biology. Fusions between fluorescent proteins and various polypeptides are allowing scientists to image tissues with sub-cellular resolution. Here, we describe the generation and activity of a genetically engineered mouse line expressing a fusion between the green fluorescent protein (GFP) and the apically localized protein Crumbs3 (Crb3). This reporter drives Cre-inducible expression of Crb3-GFP under control of the EF1a regulatory domains. The fusion protein is broadly expressed in embryonic and adult tissues and shows apical restriction in the majority of epithelial cell types. It displays a variably penetrant gain of function activity in the neural tube. However, in several cell types, over-expression of Crb3 does not appear to have any effect on normal development or maintenance. Detailed analysis of kidneys expressing this reporter indicates normal morphology and function highlighting the utility for live imaging. Thus, the EF1a(Crb3-GFP) mouse line will be of broad use for studying membrane and/or tissue dynamics in living tissues. PMID:25809849

  20. Cystic fibrosis decreases the apical membrane chloride permeability of monolayers cultured from cells of tracheal epithelium.

    PubMed

    Widdicombe, J H; Welsh, M J; Finkbeiner, W E

    1985-09-01

    The tracheal mucosa from a 12-year-old girl was digested with collagenase 4 hr after her death from cystic fibrosis. Forty million viable cells were obtained. The cells, plated at 10(6) per cm2 onto four Nuclepore filters coated with human placental collagen, formed confluent monolayers after 1 day. Their ultrastructure was similar to that of normal human cells. They were studied in conventional Ussing chambers or with intracellular microelectrodes on days 5-7 after plating. The monolayers displayed resistance of 380 +/- 50 omega X cm2 and short-circuit current (Isc) of 1.8 +/- 0.4 microA X cm-2. This resistance is similar to that obtained for dog or normal human monolayers. The Isc is less than normal human (approximately 3 microA X cm-2) or dog (approximately 10 microA X cm-2) cells. The cystic fibrosis cells resembled normal monolayers in that serosal ouabain and mucosal amiloride inhibited Isc, while mucosal ouabain or serosal amiloride had no effect. They differed from normal human or dog cells in that Isc was not inhibited by bumetanide and the stimulation of Isc by isoproterenol or prostaglandin E2 was greatly reduced or abolished. Addition of isoproterenol depolarized apical membrane potential (psi a) and decreased fractional resistance (fR) in normal human and dog but had no effect on psi a or fR in cystic fibrosis cells. Reduction of mucosal chloride from 120 to 5 mM by replacement with gluconate increased fR of dog and normal human monolayers and depolarized psi a by 22 (dog) or 30 (human) mV. In cystic fibrosis monolayers, chloride replacement hyperpolarized psi a by 2 mV and had little effect on fR. These results suggest that the primary defect in cystic fibrosis is reduced apical membrane chloride conductance. PMID:3862125

  1. Identification of two partners from the bacterial Kef exchanger family for the apical plasma membrane V-ATPase of Metazoa.

    PubMed

    Day, Jonathan P; Wan, Susan; Allan, Adrian K; Kean, Laura; Davies, Shireen A; Gray, Joe V; Dow, Julian A T

    2008-08-01

    The vital task of vectorial solute transport is often energised by a plasma membrane, proton-motive V-ATPase. However, its proposed partner, an apical alkali-metal/proton exchanger, has remained elusive. Here, both FlyAtlas microarray data and in situ analyses demonstrate that the bacterial kefB and kefC (members of the CPA2 family) homologues in Drosophila, CG10806 and CG31052, respectively, are both co-expressed with V-ATPase genes in transporting epithelia. Immunocytochemistry localises endogenous CG10806 and CG31052 to the apical plasma membrane of the Malpighian (renal) tubule. YFP-tagged CG10806 and CG31052 both localise to the plasma membrane of Drosophila S2 cells, and when driven in principal cells of the Malpighian tubule, they localise specifically to the apical plasma membrane. V-ATPase-energised fluid secretion is affected by overexpression of CG10806, but not CG31052; in the former case, overexpression causes higher basal rates, but lower stimulated rates, of fluid secretion compared with parental controls. Overexpression also impacts levels of secreted Na+ and K+. Both genes rescue exchanger-deficient (nha1 nhx1) yeast, but act differently; CG10806 is driven predominantly to the plasma membrane and confers protection against excess K+, whereas CG31052 is expressed predominantly on the vacuolar membrane and protects against excess Na+. Thus, both CG10806 and CG31052 are functionally members of the CPA2 gene family, colocalise to the same apical membrane as the plasma membrane V-ATPase and show distinct ion specificities, as expected for the Wieczorek exchanger. PMID:18628302

  2. The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth

    PubMed Central

    Riquelme, Meritxell; Bredeweg, Erin L.; Callejas-Negrete, Olga; Roberson, Robert W.; Ludwig, Sarah; Beltrán-Aguilar, Alejandro; Seiler, Stephan; Novick, Peter; Freitag, Michael

    2014-01-01

    Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis. PMID:24523289

  3. Determination of apical membrane polarity in mammary epithelial cell cultures: The role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions

    SciTech Connect

    Parry, G.; Beck, J.C.; Moss, L.; Bartley, J. ); Ojakian, G.K. )

    1990-06-01

    The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. The authors have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembranous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. They suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS0O while colchicine and acrylamide did not. They hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.

  4. Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane.

    PubMed

    McShane, Adam J; Bajrami, Bekim; Ramos, Alex A; Diego-Limpin, Pamela A; Farrokhi, Vahid; Coutermarsh, Bonita A; Stanton, Bruce A; Jensen, Tim; Riordan, John R; Wetmore, Diana; Joseloff, Elizabeth; Yao, Xudong

    2014-11-01

    Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel's function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy. PMID:25227318

  5. Targeted Proteomic Quantitation of the Absolute Expression and Turnover of Cystic Fibrosis Transmembrane Conductance Regulator in the Apical Plasma Membrane

    PubMed Central

    2015-01-01

    Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel’s function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy. PMID:25227318

  6. Current-voltage relations of sodium-coupled sugar transport across the apical membrane of Necturus small intestine.

    PubMed

    Lapointe, J Y; Hudson, R L; Schultz, S G

    1986-01-01

    The current-voltage (I-V) relations of the rheogenic Na-sugar cotransport mechanism at the apical membrane of Necturus small intestine were determined from the relations between the electrical potential difference across the apical membrane, psi mc, and that across the entire epithelium, psi ms, when the latter was varied over the range +/- 200 mV, under steady conditions in the presence of galactose and after the current across the apical membrane carried by the cotransporter, ImSNa, is blocked by the addition of phloridzin to the mucosal solution. ImSNa was found to be strongly dependent upon psi mc over the range -50 mV less than psi mc less than EmSNa where EmSNa is the "zero current" or "reversal" potential. Over the range of values of psi mc encountered under physiological conditions the cotransporter may be modeled as a conductance in series with an electromotive force so that ImSNa = gmSNa (EmSNa - psi mc) where gmSNa is the contribution of this mechanism to the conductance of the apical membrane and is "near constant." In several instances ImSNa "saturated" at large hyperpolarizing or depolarizing values of psi mc. The values of EmSNa determined in the presence of 1, 5, and 15 mM galactose strongly suggest that if the Na-galactose cotransporters are kinetically homogeneous, the stoichiometry of this coupled process is unity. Finally, the shapes of the observed I-V relations are consistent with the predictions of a simple kinetic model which conforms with current notions regarding the mechanico-kinetic properties of this cotransport process. PMID:3820278

  7. [Determination, using a piezo-impulse method, of iso-osmotic permeability of the apical membrane of epithelium].

    PubMed

    Eyraud, C; Dubief, M C; Charmasson, R

    1985-01-01

    A frog skin, mechanically held on the mucosal side separates two Ringer solutions. It is submitted to an hydrostatic pressure difference delta P varying between 2 and 120 mb. Water permeability P (delta P) delta pi = 0 is determined with a piezo-impulse method. The rapid variation of permeability within the 2-25 mb range indicates a reversible closing of the junctions. The limiting value Pisol for high delta P is the isoosmotic permeability of the apical membrane. PMID:3922570

  8. Multiple apical plasma membrane constituents are associated with susceptibility to meconium ileus in individuals with cystic fibrosis.

    PubMed

    Sun, Lei; Rommens, Johanna M; Corvol, Harriet; Li, Weili; Li, Xin; Chiang, Theodore A; Lin, Fan; Dorfman, Ruslan; Busson, Pierre-François; Parekh, Rashmi V; Zelenika, Diana; Blackman, Scott M; Corey, Mary; Doshi, Vishal K; Henderson, Lindsay; Naughton, Kathleen M; O'Neal, Wanda K; Pace, Rhonda G; Stonebraker, Jaclyn R; Wood, Sally D; Wright, Fred A; Zielenski, Julian; Clement, Annick; Drumm, Mitchell L; Boëlle, Pierre-Yves; Cutting, Garry R; Knowles, Michael R; Durie, Peter R; Strug, Lisa J

    2012-05-01

    Variants associated with meconium ileus in cystic fibrosis were identified in 3,763 affected individuals by genome-wide association study (GWAS). Five SNPs at two loci near SLC6A14 at Xq23-24 (minimum P = 1.28 × 10(-12) at rs3788766) and SLC26A9 at 1q32.1 (minimum P = 9.88 × 10(-9) at rs4077468) accounted for ~5% of phenotypic variability and were replicated in an independent sample of affected individuals (n = 2,372; P = 0.001 and 0.0001, respectively). By incorporating the knowledge that disease-causing mutations in CFTR alter electrolyte and fluid flux across surface epithelium into a hypothesis-driven GWAS (GWAS-HD), we identified associations with the same SNPs in SLC6A14 and SLC26A9 and established evidence for the involvement of SNPs in a third solute carrier gene, SLC9A3. In addition, GWAS-HD provided evidence of association between meconium ileus and multiple genes encoding constituents of the apical plasma membrane where CFTR resides (P = 0.0002; testing of 155 apical membrane genes jointly and in replication, P = 0.022). These findings suggest that modulating activities of apical membrane constituents could complement current therapeutic paradigms for cystic fibrosis. PMID:22466613

  9. Interaction of apical and basal membrane ion channels underlies electroreception in ampullary epithelia of skates.

    PubMed Central

    Lu, J.; Fishman, H. M.

    1994-01-01

    The exquisite sensitivity of elasmobranch fishes to electric fields is thought to reside in electroreceptive organs called ampullae of Lorenzini. We measured the stimulus-response behavior of ampullary organs excised from skates. Under open-circuit conditions, the ampullary organ showed three distinct response states: spontaneous repetitive spikes, evoked spikes, and small, damped oscillatory responses. Under short-circuit conditions, the amplitude range for a linear current response to a sinusoidal (0.5 Hz) voltage clamp of an organ (assessed by spectral analysis of the harmonics generated) was 7-200 microV rms. Changes in the spike firing rate of the afferent nerve innervating the organ were evident for voltage clamps of the ampullary epithelium of 3 microV and the spike rate saturated for clamp steps exceeding 100 microV. Thus, the linear response range of the ampullary epithelium exceeded the range in spike firing rate of the afferent nerve. The steady-state transorgan electrical properties under voltage clamp conditions were obtained by analysis of complex admittance determinations in the frequency range 0.05-20 Hz for perturbations (< 100 microV rms) in the linear range. Admittance functions were distinctly related to the preparation states observed under open-circuit conditions. A negative real part in the organ admittance (i.e., a steady-state negative conductance generated by the preparation) was a common characteristic of the two (open-circuit) excitable states. The negative conductance was also confirmed by the direction of current flow through the ampullary epithelium in response to step voltage clamps. We conclude that the steady state-negative conductance is an essential property of the ampullary epithelium,and we suggest that the interplay of negative and positive conductances generated by ion channels in apical and basal membranes of receptor cells results in signal amplification that may contribute significantly to the electric field sensitivity of

  10. Characterisation of a Babesia orientalis apical membrane antigen, and comparison of its orthologues among selected apicomplexans.

    PubMed

    He, Lan; Fan, Lizhe; Hu, Jinfang; Miao, Xiaoyan; Huang, Yuan; Zhou, Yanqin; Hu, Min; Zhao, Junlong

    2015-04-01

    In the present study, we identified and characterised the complete coding sequence of Babesia orientalis apical membrane antigen 1 (designated Bo-ama1); it is 1803bp in length and encodes a polypeptide of 601 amino acids (aa). The Bo-ama-1 gene product (Bo-AMA1) is predicted to be 67kDa in size and contains a signal peptide. Mature Bo-AMA1 is predicted to have one transmembrane region and a short cytoplasmic tail (C-terminal domain). The extracellular part of Bo-AMA1 has three functional domains (DI, DII and DIII) with 14 conserved cysteine residues. A Bo-AMA1 fragment containing all three of these domains (designated Bo-AMA1-DI/II/III) was cloned into the plasmid vector pET-28a and expressed as a recombinant (His-fusion) protein of 53kDa. Antibodies in the serum from a B. orientalis-infected water buffalo specifically recognised this protein in immunoblotting analysis. Rabbit antibodies raised against the recombinant protein were able to detect native Bo-AMA1 (67kDa) from erythrocytes of B. orientalis-infected water buffalo. Bo-AMA1 is a new member of the AMA1 family and might be a good antigen for the specific detection of antibodies produced in B. orientalis infected cattle. This protein is likely to play critical roles during host cell adherence and invasion by B. orientalis, as the AMA1s reported in other organisms such as Plasmodium falciparum and Toxoplasma gondii. Further research is required to explore the biological functions of this protein and to determine whether its immunisation can induce protective effects in water buffalo against B. orientalis infection. PMID:25732411

  11. Small GTPases promote actin coat formation on microsporidian pathogens traversing the apical membrane of Caenorhabditis elegans intestinal cells.

    PubMed

    Szumowski, Suzannah C; Estes, Kathleen A; Popovich, John J; Botts, Michael R; Sek, Grace; Troemel, Emily R

    2016-01-01

    Many intracellular pathogens co-opt actin in host cells, but little is known about these interactions in vivo. We study the in vivo trafficking and exit of the microsporidian Nematocida parisii, which is an intracellular pathogen that infects intestinal cells of the nematode Caenorhabditis elegans. We recently demonstrated that N. parisii uses directional exocytosis to escape out of intestinal cells into the intestinal tract. Here, we show that an intestinal-specific isoform of C. elegans actin called ACT-5 forms coats around membrane compartments that contain single exocytosing spores, and that these coats appear to form after fusion with the apical membrane. We performed a genetic screen for host factors required for actin coat formation and identified small GTPases important for this process. Through analysis of animals defective in these factors, we found that actin coats are not required for pathogen exit although they may boost exocytic output. Later during infection, we find that ACT-5 also forms coats around membrane-bound vesicles that contain multiple spores. These vesicles are likely formed by clathrin-dependent compensatory endocytosis to retrieve membrane material that has been trafficked to the apical membrane as part of the exocytosis process. These findings provide insight into microsporidia interaction with host cells, and provide novel in vivo examples of the manner in which intracellular pathogens co-opt host actin during their life cycle. PMID:26147591

  12. NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles. Measurement by induced osmosis and solvent drag.

    PubMed

    Pearce, D; Verkman, A S

    1989-06-01

    Two independent methods, induced osmosis and solvent drag, were used to determine the reflection coefficients for NaCl (sigma NaCl) in brush border and basolateral membrane vesicles isolated from rabbit proximal tubule. In the induced osmosis method, vesicles loaded with sucrose were subjected to varying inward NaCl gradients in a stopped-flow apparatus. sigma NaCl was determined from the osmolality of the NaCl solution required to cause no initial osmotic water flux as measured by light scattering (null point). By this method sigma NaCl was greater than 0.92 for both apical and basolateral membranes with best estimates of 1.0. sigma NaCl was determined by the solvent drag method using the Cl-sensitive fluorescent indicator, 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ), to detect the drag of Cl into vesicles by inward osmotic water movement caused by an outward osmotic gradient. sigma NaCl was determined by comparing experimental data with theoretical curves generated using the coupled flux equations of Kedem and Katchalsky. By this method we found that sigma NaCl was greater than 0.96 for apical and greater than 0.98 for basolateral membrane vesicles, with best estimates of 1.0 for both membranes. These results demonstrate that sigma NaCl for proximal tubule apical and basolateral membranes are near unity. Taken together with previous results, these data suggest that proximal tubule water channels are long narrow pores that exclude NaCl. PMID:2765660

  13. Multiple apical plasma membrane constituents are associated with susceptibility to meconium ileus in individuals with cystic fibrosis

    PubMed Central

    Sun, Lei; Rommens, Johanna M; Corvol, Harriet; Li, Weili; Li, Xin; Chiang, Theodore; Lin, Fan; Dorfman, Ruslan; Busson, Pierre-François; Parekh, Rashmi V; Zelenika, Diana; Blackman, Scott; Corey, Mary; Doshi, Vishal; Henderson, Lindsay; Naughton, Kathleen; O'Neal, Wanda K; Pace, Rhonda G; Stonebraker, Jaclyn R; Wood, Sally D; Wright, Fred A; Zielenski, Julian; Clement, Annick; Drumm, Mitchell L; Boëlle, Pierre-Yves; Cutting, Garry R; Knowles, Michael R; Durie, Peter R; Strug, Lisa J

    2012-01-01

    Variants associated with meconium ileus in cystic fibrosis (CF) were identified in 3,763 patients by GWAS. Five SNPs at two loci near SLC6A14 (min P=1.28×10−12 at rs3788766), chr Xq23-24 and SLC26A9 (min P=9.88×10−9 at rs4077468), chr 1q32.1 accounted for ~5% of the phenotypic variability, and were replicated in an independent patient collection (n=2,372; P=0.001 and 0.0001 respectively). By incorporating that disease-causing mutations in CFTR alter electrolyte and fluid flux across epithelia into an hypothesis-driven genome-wide analysis (GWAS-HD), we identified the same SLC6A14 and SLC26A9 associated SNPs, while establishing evidence for the involvement of SNPs in a third solute carrier gene, SLC9A3. In addition, GWAS-HD provided evidence of association between meconium ileus and multiple constituents of the apical plasma membrane where CFTR resides (P=0.0002, testing 155 apical genes jointly and replicated, P=0.022). These findings suggest that modulating activities of apical membrane constituents could complement current therapeutic paradigms for cystic fibrosis. PMID:22466613

  14. Efficient Trafficking of MDR1/P-Glycoprotein to Apical Canalicular Plasma Membranes in HepG2 Cells Requires PKA-RIIα Anchoring and Glucosylceramide

    PubMed Central

    Wojtal, Kacper A.; de Vries, Erik; Hoekstra, Dick

    2006-01-01

    In hepatocytes, cAMP/PKA activity stimulates the exocytic insertion of apical proteins and lipids and the biogenesis of bile canalicular plasma membranes. Here, we show that the displacement of PKA-RIIα from the Golgi apparatus severely delays the trafficking of the bile canalicular protein MDR1 (P-glycoprotein), but not that of MRP2 (cMOAT), DPP IV and 5′NT, to newly formed apical surfaces. In addition, the direct trafficking of de novo synthesized glycosphingolipid analogues from the Golgi apparatus to the apical surface is inhibited. Instead, newly synthesized glucosylceramide analogues are rerouted to the basolateral surface via a vesicular pathway, from where they are subsequently endocytosed and delivered to the apical surface via transcytosis. Treatment of HepG2 cells with the glucosylceramide synthase inhibitor PDMP delays the appearance of MDR1, but not MRP2, DPP IV, and 5′NT at newly formed apical surfaces, implicating glucosylceramide synthesis as an important parameter for the efficient Golgi-to-apical surface transport of MDR1. Neither PKA-RIIα displacement nor PDMP inhibited (cAMP-stimulated) apical plasma membrane biogenesis per se, suggesting that other cAMP effectors may play a role in canalicular development. Taken together, our data implicate the involvement of PKA-RIIα anchoring in the efficient direct apical targeting of distinct proteins and glycosphingolipids to newly formed apical plasma membrane domains and suggest that rerouting of Golgi-derived glycosphingolipids may underlie the delayed Golgi-to-apical surface transport of MDR1. PMID:16723498

  15. Concentrative nucleoside transporter (rCNT1) is targeted to the apical membrane through the hepatic transcytotic pathway.

    PubMed

    Duflot, Sylvie; Calvo, Maria; Casado, F Javier; Enrich, Carlos; Pastor-Anglada, Marçal

    2002-11-15

    The Na+-dependent nucleoside transporter CNT1 has been identified in a caveolin-enriched plasma membrane fraction (CEF), in transcytotic endosomes, and in canalicular membranes isolated from quiescent rat liver in which the transporter appears to be biologically active. CNT1 was also detected, albeit in small amounts, in the early/sorting endosomes. Plasma membrane preparations enriched in basolateral markers showed Na+-dependent nucleoside transport activity that is mostly, if not exclusively, accounted for by CNT2, a transporter protein which was not detected in CEF nor in the endosomal fractions. These data are consistent with different localization and trafficking pathways of the two isoforms in hepatocytes. CNT1 is the first transporter which is reported to follow the transcytotic pathway to be inserted on the apical side of liver parenchymal cells. PMID:12441131

  16. Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells

    PubMed Central

    Komaba, Shigeru; Coluccio, Lynne M.

    2015-01-01

    Amino acid transporters (AATers) in the brush border of the apical plasma membrane (APM) of renal proximal tubule (PT) cells mediate amino acid transport (AAT). We found that the membrane-associated class I myosin myosin 1b (Myo1b) localized at the apical brush border membrane of PTs. In opossum kidney (OK) 3B/2 epithelial cells, which are derived from PTs, expressed rat Myo1b-GFP colocalized in patched microvilli with expressed mouse V5-tagged SIT1 (SIT1-V5), which mediates neutral amino acid transport in OK cells. Lentivirus-mediated delivery of opossum Myo1b-specific shRNA resulted in knockdown (kd) of Myo1b expression, less SIT1-V5 at the APM as determined by localization studies, and a decrease in neutral AAT as determined by radioactive uptake assays. Myo1b kd had no effect on Pi transport or noticeable change in microvilli structure as determined by rhodamine phalloidin staining. The studies are the first to define a physiological role for Myo1b, that of regulating renal AAT by modulating the association of AATers with the APM. PMID:26361046

  17. Surface heat shock protein 90 serves as a potential receptor for calcium oxalate crystal on apical membrane of renal tubular epithelial cells.

    PubMed

    Fong-Ngern, Kedsarin; Sueksakit, Kanyarat; Thongboonkerd, Visith

    2016-07-01

    Adhesion of calcium oxalate monohydrate (COM) crystals on renal tubular epithelial cells is a crucial step in kidney stone formation. Finding potential crystal receptors on the apical membrane of the cells may lead to a novel approach to prevent kidney stone disease. Our previous study identified a large number of crystal-binding proteins on the apical membrane of MDCK cells. However, their functional role as potential crystal receptors had not been validated. The present study aimed to address the potential role of heat shock protein 90 (HSP90) as a COM crystal receptor. The apical membrane was isolated from polarized MDCK cells by the peeling method and recovered proteins were incubated with COM crystals. Western blot analysis confirmed the presence of HSP90 in the apical membrane and the crystal-bound fraction. Immunofluorescence staining without permeabilization and laser-scanning confocal microscopy confirmed the surface HSP90 expression on the apical membrane of the intact cells. Crystal adhesion assay showed that blocking surface HSP90 by specific anti-HSP90 antibody and knockdown of HSP90 by small interfering RNA (siRNA) dramatically reduced crystal binding on the apical surface of MDCK cells (by approximately 1/2 and 2/3, respectively). Additionally, crystal internalization assay revealed the presence of HSP90 on the membrane of endocytic vesicle containing the internalized COM crystal. Moreover, pretreatment of MDCK cells with anti-HSP90 antibody significantly reduced crystal internalization (by approximately 1/3). Taken together, our data indicate that HSP90 serves as a potential receptor for COM crystals on the apical membrane of renal tubular epithelial cells and is involved in endocytosis/internalization of the crystals into the cells. PMID:27115409

  18. Ca-activated K channels in apical membrane of mammalian CCT, and their role in K secretion.

    PubMed

    Frindt, G; Palmer, L G

    1987-03-01

    High conductance, Ca-activated K channels were studied in the apical membrane of the rat cortical collecting tubule (CCT) using the patch-clamp technique. In cell-attached patches the channels were found mainly in the closed state at the spontaneous apical membrane potential. They spent progressively more time in the open state as the pipette potential was made negative relative to the bath. In excised patches these channels had a high selectivity for K over Na and were activated by micromolar concentrations of Ca2+ on the cytoplasmic side of the membrane in a voltage-dependent manner. They had a low conductance to Rb and were blocked by Ba (1-100 microM) from the cytoplasmic side and tetraethylammonium (TEA) (0.2-1 mM) from the luminal side. Block by external TEA and small conductance to Rb were used to investigate the role of these channels in K transport by the isolated perfused rabbit CCT. Ba (2.5 mM), a well-studied blocker of apical K conductance in this segment, hyperpolarized the transepithelial voltage (VT) by 3.7 +/- 0.9 mV when added to the luminal solution of the perfused tubule. Addition of TEA (5 mM) to the luminal solution has no effect on VT. When Na transport was abolished by luminal amiloride, perfusion with 30 mM K (replacing Na) resulted in a lumen-negative VT (18-34 mV). Under these conditions, VT was reduced by 6.0 +/- 1.5 mV by 2.5 mM Ba, whereas TEA had no effect. Perfusion with 30 mM Rb (replacing Na) also caused a lumen-negative VT that was approximately 50% of that observed with 30 mM K. The apical K conductance of the perfused CCT appears to be insensitive to luminal TEA and only modestly selective for K over Rb. This conductance, at least under the conditions of our studies, is probably not mediated by the high conductance Ca-activated K channel. PMID:2435175

  19. Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials.

    PubMed

    Krishnarjuna, Bankala; Lim, San Sui; Devine, Shane M; Debono, Cael O; Lam, Raymond; Chandrashekaran, Indu R; Jaipuria, Garima; Yagi, Hiromasa; Atreya, Hanudatta S; Scanlon, Martin J; MacRaild, Christopher A; Scammells, Peter J; Norton, Raymond S

    2016-06-01

    Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26804042

  20. Calcium is not involved in the cAMP-mediated stimulation of Cl- conductance in the apical membrane of Necturus gallbladder epithelium.

    PubMed

    Kottra, G

    1995-03-01

    The permeability properties of the forskolin-stimulated Cl- conductance in the apical membrane of Necturus gallbladder epithelium and the possible participation of intracellular Ca2+ in its stimulation have been investigated. The anion selectivity sequence as derived from biionic potential measurements (SCN- > I- approximately NO3- > Br- > Cl- > ISE-) differed from the sequence derived from measurements of apical membrane resistance (NO3- approximately Br- approximately Cl- > SCN- > I- approximately ISE-). Accordingly, the conductance was inhibited by SCN- and I- which, from the potential measurements, appeared to be more permeable than Cl-. This finding agrees with observations of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel reported recently. However, none of the commonly used Cl- channel blockers, such as 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and glibenclamide reduced this conductance in Necturus gallbladder. In contrast to the situation in most other epithelia, elevation of intracellular Ca2+ concentration ([Ca2+]i) by ionomycin stimulated only K+ conductance and not that of Cl- in the apical cell membrane. Chelation of intracellular Ca2+ did not prevent the stimulation of Cl- conductance by forskolin. This indicates that [Ca2+]i does not have even a permissive role in the cyclic adenosine monophosphate-(cAMP)-mediated stimulation process, as would have been expected if exocytosis was involved. Further evidence against the involvement of exocytosis in the stimulation process came from the observation that the stimulation was not associated with an increase in apical membrane capacitance and was not suppressed by disruption of the cytoskeleton by preincubation of the tissue with cytochalasin D. The data indicate that Necturus gallbladder epithelium contains homologues of the CFTR Cl- channel which reside permanently in the

  1. The apicomplexan parasite Babesia divergens internalizes band 3, glycophorin A and spectrin during invasion of human red blood cells.

    PubMed

    Repnik, Urska; Gangopadhyay, Preetish; Bietz, Sven; Przyborski, Jude M; Griffiths, Gareth; Lingelbach, Klaus

    2015-07-01

    Plasmodium falciparum invades human red blood cells (RBC), while Babesia divergens infects bovine and, occasionally, human RBC. The mammalian RBC is normally unable to endocytose or phagocytose and the events leading to invasion are incompletely understood. Initially, both parasites are surrounded by the RBC plasma membrane-derived parasitophorous vacuolar membrane (PVM) that is formed during invasion. In P. falciparum-infected RBC, the PVM persists at least until parasite replication is completed whereas it has been proposed that the B. divergens PVM is disintegrated soon upon invasion. Here, we have used a B. divergens strain adapted to human RBC to investigate the formation and fate of the PVM. Using ultrastructural analysis and whole-mount or on-section immunofluorescence and immunogold labelling, we demonstrate that the initial vacuolar membrane is formed from protein and lipid components of the RBC plasma membrane. Integral membrane proteins band 3 and glycophorin A and the cytoskeletal protein spectrin are associated with the PVM of the B. divergens, but are absent from the PVM of P. falciparum at the ring or the trophozoite stage. Our results provide evidence that the biophysical properties of the RBC cytoskeleton per se do not preclude the internalization of cytoskeletal proteins by invading parasites. PMID:25628009

  2. An Extended Surface Loop on Toxoplasma gondii Apical Membrane Antigen 1 (AMA1) Governs Ligand Binding Selectivity

    PubMed Central

    Parker, Michelle L.; Boulanger, Martin J.

    2015-01-01

    Apicomplexan parasites are the causative agents of globally prevalent diseases including malaria and toxoplasmosis. These obligate intracellular pathogens have evolved a sophisticated host cell invasion strategy that relies on a parasite-host cell junction anchored by interactions between apical membrane antigens (AMAs) on the parasite surface and rhoptry neck 2 (RON2) proteins discharged from the parasite and embedded in the host cell membrane. Key to formation of the AMA1-RON2 complex is displacement of an extended surface loop on AMA1 called the DII loop. While conformational flexibility of the DII loop is required to expose the mature RON2 binding groove, a definitive role of this substructure has not been elucidated. To establish a role of the DII loop in Toxoplasma gondii AMA1, we engineered a form of the protein where the mobile portion of the loop was replaced with a short Gly-Ser linker (TgAMA1ΔDIIloop). Isothermal titration calorimetry measurements with a panel of RON2 peptides revealed an influential role for the DII loop in governing selectivity. Most notably, an Eimeria tenella RON2 (EtRON2) peptide that showed only weak binding to TgAMA1 bound with high affinity to TgAMA1ΔDIIloop. To define the molecular basis for the differential binding, we determined the crystal structure of TgAMA1ΔDIIloop in complex with the EtRON2 peptide. When analyzed in the context of existing AMA1-RON2 structures, spatially distinct anchor points in the AMA1 groove were identified that, when engaged, appear to provide the necessary traction to outcompete the DII loop. Collectively, these data support a model where the AMA1 DII loop serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove. These data also highlight the importance of considering the functional implications of the DII loop in the ongoing development of therapeutic intervention strategies targeting the AMA1-RON2 invasion

  3. Immunization with Apical Membrane Antigen 1 Confers Sterile Infection-Blocking Immunity against Plasmodium Sporozoite Challenge in a Rodent Model

    PubMed Central

    Schussek, Sophie; Trieu, Angela; Apte, Simon H.; Sidney, John; Sette, Alessandro

    2013-01-01

    Apical membrane antigen 1 (AMA-1) is a leading blood-stage malaria vaccine candidate. Consistent with a key role in erythrocytic invasion, AMA-1-specific antibodies have been implicated in AMA-1-induced protective immunity. AMA-1 is also expressed in sporozoites and in mature liver schizonts where it may be a target of protective cell-mediated immunity. Here, we demonstrate for the first time that immunization with AMA-1 can induce sterile infection-blocking immunity against Plasmodium sporozoite challenge in 80% of immunized mice. Significantly higher levels of gamma interferon (IFN-γ)/interleukin-2 (IL-2)/tumor necrosis factor (TNF) multifunctional T cells were noted in immunized mice than in control mice. We also report the first identification of minimal CD8+ and CD4+ T cell epitopes on Plasmodium yoelii AMA-1. These data establish AMA-1 as a target of both preerythrocytic- and erythrocytic-stage protective immune responses and validate vaccine approaches designed to induce both cellular and humoral immunity. PMID:23836827

  4. Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites

    PubMed Central

    Yap, Alan; Azevedo, Mauro F; Gilson, Paul R; Weiss, Greta E; O’Neill, Matthew T; Wilson, Danny W; Crabb, Brendan S; Cowman, Alan F

    2014-01-01

    Summary Malaria is caused by obligate intracellular parasites, of which Plasmodium falciparum is the most lethal species. In humans, P. falciparum merozoites (invasive forms of the parasite) employ a host of parasite proteins to rapidly invade erythrocytes. One of these is the P. falciparum apical membrane antigen 1 (PfAMA1) which forms a complex with rhoptry neck proteins at the tight junction. Here, we have placed the Pfama1 gene under conditional control using dimerizable Cre recombinase (DiCre) in P. falciparum. DiCre-mediated excision of the loxP-flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle. This reduces growth by 40%, due to decreased invasion efficiency characterized by a post-invasion defect in sealing of the parasitophorous vacuole. These results show that PfAMA1 is an essential protein for merozoite invasion in P. falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event. PMID:24571085

  5. A promising new ELISA diagnostic test for cattle babesiosis based on Babesia bigemina Apical Membrane Antigen-1.

    PubMed

    Torina, Alessandra; Cordaro, Antonio; Blanda, Valeria; D'Agostino, Rosalia; Scimeca, Salvatore; Scariano, Maria E; Sireci, Guido; Lelli, Rossella

    2016-01-01

    Babesiosis due to Babesia bigemina is a relevant tick-borne disease, affecting cattle worldwide. Many surface proteins of the pathogen including the Apical Membrane Antigen 1 (AMA-1) - have been analysed for vaccine and diagnostic purposes. This study focused on B. bigemina AMA-1 and on its use for the assessment of diagnostic tests. After bioinformatic analyses, AMA-1 codifying region was amplified and cloned into an expression vector used to induce protein synthesis in Escherichia coli cells. AMA-1 was purified by affinity chromatography and used to set up the best condition for an ELISA protocol. Bovine field sera positive to B. bigemina were used to evaluate the presence of anti-AMA-1 antibodies. In order to verify the assay specificity, sera positive to Babesia bovis or to the piroplasm Theileria annulata were also included. Significant differences were obtained between sera negative to both B. bigemina and B. bovis and samples positive to B. bigemina, to B. bovis or to both pathogens. No significant reaction was observed with T. annulata positive sera. The results showed that AMA-1 protein is suitable to be used as antigen in diagnostic assays for babesiosis diagnosis in cattle, as it does not show any cross reaction with anti-T. annulata antibodies. PMID:27033532

  6. Molecular Insights into the Interaction between Plasmodium falciparum Apical Membrane Antigen 1 and an Invasion-Inhibitory Peptide

    PubMed Central

    Wang, Geqing; MacRaild, Christopher A.; Mohanty, Biswaranjan; Mobli, Mehdi; Cowieson, Nathan P.; Anders, Robin F.; Simpson, Jamie S.; McGowan, Sheena; Norton, Raymond S.; Scanlon, Martin J.

    2014-01-01

    Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1∶1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction. PMID:25343578

  7. Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project.

    PubMed

    Escalante, A A; Grebert, H M; Chaiyaroj, S C; Magris, M; Biswas, S; Nahlen, B L; Lal, A A

    2001-04-01

    We have investigated the genetic diversity of the gene encoding the apical membrane antigen-1 (AMA-1) in natural populations of Plasmodium falciparum from western Kenya and compared it with parasite populations from other geographic regions. A total of 28 complete sequences from Kenya, Thailand, India, and Venezuela field isolates were obtained. The genetic polymorphism is not evenly distributed across the gene, which is in agreement with the pattern reported in earlier studies. The alleles from Kenya exhibit 20 and 30% more polymorphism than that found in Southeast Asia and Venezuelan alleles, respectively. Based on the gene genealogies derived from sequencing data, no evidence for allele families was found. We have found evidence supporting limited gene flow between the parasite populations, specifically, between the Southeast Asian and Venezuelan isolates; however, no alleles could be linked to a specific geographic region. This study reveals that positive natural selection is an important factor in the maintenance of genetic diversity for AMA-1. We did not find conclusive evidence indicating intragenic recombination is important in the generation of the AMA-1 allelic diversity. The study provides information on the genetic diversity of the AMA-1 gene that would be useful in vaccine development and testing, as well as in assessing factors that are involved in the generation and maintenance of the genetic diversity in P. falciparum. PMID:11295182

  8. The secretion-stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli.

    PubMed Central

    Hanzel, D; Reggio, H; Bretscher, A; Forte, J G; Mangeat, P

    1991-01-01

    Stimulation of gastric acid secretion in parietal cells involves the translocation of the proton pump (H,K-ATPase) from cytoplasmic tubulovesicles to the apical membrane to form long, F-actin-containing, microvilli. Following secretion, the pump is endocytosed back into tubulovesicles. The parietal cell therefore offers a system for the study of regulated membrane recycling, with temporally separated endocytic and exocytic steps. During cAMP-mediated stimulation, an 80 kDa peripheral membrane protein becomes phosphorylated on serine residues. This protein is a major component, together with actin and the pump, of the isolated apical membrane from stimulated cells, but not the resting tubulovesicular membrane. Here we show that the gastric 80 kDa phosphoprotein is closely related or identical to ezrin, a protein whose phosphorylation on serine and tyrosine residues was recently implicated in the induction by growth factors of cell surface structures on cultured cells [Bretscher, A. (1989) J. Cell Biol., 108, 921-930]. Light and electron microscopy reveal that ezrin is associated with the actin filaments of the microvilli of stimulated cells, but not with the filaments in the terminal web. In addition, a significant amount of ezrin is present in the basolateral membrane infoldings of both resting and stimulated cells. Extraction studies show that ezrin is a cytoskeletal protein in unstimulated and stimulated cells, and its association with the cytoskeleton is more stable in stimulated cells. These studies indicate that ezrin is a membrane cytoskeletal linker that may play a key role in the control of the assembly of secretory apical microvilli in parietal cells and ultimately in the regulation of acid secretion. Taken together with the earlier studies, we suggest that ezrin might be a general substrate for kinases involved in the regulation of actin-containing cell surface structures. Images PMID:1831124

  9. Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

    PubMed Central

    Bueno, Lilian Lacerda; Lobo, Francisco Pereira; Morais, Cristiane Guimarães; Mourão, Luíza Carvalho; de Ávila, Ricardo Andrez Machado; Soares, Irene Silva; Fontes, Cor Jesus; Lacerda, Marcus Vinícius; Olórtegui, Carlos Chavez; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio; Braga, Érika Martins

    2011-01-01

    Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies. PMID:21713006

  10. Comparative sequence analysis of domain I of Plasmodium falciparum apical membrane antigen 1 from Saudi Arabia and worldwide isolates.

    PubMed

    Al-Qahtani, Ahmed A; Abdel-Muhsin, Abdel-Muhsin A; Bin Dajem, Saad M; AlSheikh, Adel Ali H; Bohol, Marie Fe F; Al-Ahdal, Mohammed N; Putaporntip, Chaturong; Jongwutiwes, Somchai

    2016-04-01

    The apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) plays a crucial role in erythrocyte invasion and is a target of protective antibodies. Although domain I of PfAMA1 has been considered a promising vaccine component, extensive sequence diversity in this domain could compromise an effective vaccine design. To explore the extent of sequence diversity in domain I of PfAMA1, P. falciparum-infected blood samples from Saudi Arabia collected between 2007 and 2009 were analyzed and compared with those from worldwide parasite populations. Forty-six haplotypes and a novel codon change (M190V) were found among Saudi Arabian isolates. The haplotype diversity (0.948±0.004) and nucleotide diversity (0.0191±0.0008) were comparable to those from African hyperendemic countries. Positive selection in domain I of PfAMA1 among Saudi Arabian parasite population was observed because nonsynonymous nucleotide substitutions per nonsynonymous site (dN) significantly exceeded synonymous nucleotide substitutions per synonymous site (dS) and Tajima's D and its related statistics significantly deviated from neutrality in the positive direction. Despite a relatively low prevalence of malaria in Saudi Arabia, a minimum of 17 recombination events occurred in domain I. Genetic differentiation was significant between P. falciparum in Saudi Arabia and parasites from other geographic origins. Several shared or closely related haplotypes were found among parasites from different geographic areas, suggesting that vaccine derived from multiple shared epitopes could be effective across endemic countries. PMID:26867816

  11. Crystal Structure of Plasmodium knowlesi Apical Membrane Antigen 1 and Its Complex with an Invasion-Inhibitory Monoclonal Antibody

    PubMed Central

    van der Eijk, Marjolein; Thomas, Alan W.; Singh, Balbir; Kocken, Clemens H. M.

    2015-01-01

    The malaria parasite Plasmodium knowlesi, previously associated only with infection of macaques, is now known to infect humans as well and has become a significant public health problem in Southeast Asia. This species should therefore be targeted in vaccine and therapeutic strategies against human malaria. Apical Membrane Antigen 1 (AMA1), which plays a role in Plasmodium merozoite invasion of the erythrocyte, is currently being pursued in human vaccine trials against P. falciparum. Recent vaccine trials in macaques using the P. knowlesi orthologue PkAMA1 have shown that it protects against infection by this parasite species and thus should be developed for human vaccination as well. Here, we present the crystal structure of Domains 1 and 2 of the PkAMA1 ectodomain, and of its complex with the invasion-inhibitory monoclonal antibody R31C2. The Domain 2 (D2) loop, which is displaced upon binding the Rhoptry Neck Protein 2 (RON2) receptor, makes significant contacts with the antibody. R31C2 inhibits binding of the Rhoptry Neck Protein 2 (RON2) receptor by steric blocking of the hydrophobic groove and by preventing the displacement of the D2 loop which is essential for exposing the complete binding site on AMA1. R31C2 recognizes a non-polymorphic epitope and should thus be cross-strain reactive. PkAMA1 is much less polymorphic than the P. falciparum and P. vivax orthologues. Unlike these two latter species, there are no polymorphic sites close to the RON2-binding site of PkAMA1, suggesting that P. knowlesi has not developed a mechanism of immune escape from the host’s humoral response to AMA1. PMID:25886591

  12. Low Levels of Polymorphisms and No Evidence for Diversifying Selection on the Plasmodium knowlesi Apical Membrane Antigen 1 Gene

    PubMed Central

    Faber, Bart W.; Abdul Kadir, Khamisah; Rodriguez-Garcia, Roberto; Remarque, Edmond J; Saul, Frederick A.; Vulliez-Le Normand, Brigitte; Bentley, Graham A.; Kocken, Clemens H. M.; Singh, Balbir

    2015-01-01

    Infection with Plasmodium knowlesi, a zoonotic primate malaria, is a growing human health problem in Southeast Asia. P. knowlesi is being used in malaria vaccine studies, and a number of proteins are being considered as candidate malaria vaccine antigens, including the Apical Membrane Antigen 1 (AMA1). In order to determine genetic diversity of the ama1 gene and to identify epitopes of AMA1 under strongest immune selection, the ama1 gene of 52 P. knowlesi isolates derived from human infections was sequenced. Sequence analysis of isolates from two geographically isolated regions in Sarawak showed that polymorphism in the protein is low compared to that of AMA1 of the major human malaria parasites, P. falciparum and P. vivax. Although the number of haplotypes was 27, the frequency of mutations at the majority of the polymorphic positions was low, and only six positions had a variance frequency higher than 10%. Only two positions had more than one alternative amino acid. Interestingly, three of the high-frequency polymorphic sites correspond to invariant sites in PfAMA1 or PvAMA1. Statistically significant differences in the quantity of three of the six high frequency mutations were observed between the two regions. These analyses suggest that the pkama1 gene is not under balancing selection, as observed for pfama1 and pvama1, and that the PkAMA1 protein is not a primary target for protective humoral immune responses in their reservoir macaque hosts, unlike PfAMA1 and PvAMA1 in humans. The low level of polymorphism justifies the development of a single allele PkAMA1-based vaccine. PMID:25881166

  13. Genetic detection of Babesia bigemina from Mongolian cattle using apical membrane antigen-1 gene-based PCR assay.

    PubMed

    Sivakumar, Thillaiampalam; Altangerel, Khukhuu; Battsetseg, Badgar; Battur, Banzragch; Aboulaila, Mahmoud; Munkhjargal, Tserendorj; Yoshinari, Takeshi; Yokoyama, Naoaki; Igarashi, Ikuo

    2012-06-01

    We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina. PMID:22284301

  14. Intestinal absorption mechanism of tebipenem pivoxil, a novel oral carbapenem: involvement of human OATP family in apical membrane transport.

    PubMed

    Kato, Kazuhiko; Shirasaka, Yoshiyuki; Kuraoka, Erika; Kikuchi, Akihiro; Iguchi, Maki; Suzuki, Hisashi; Shibasaki, Shigeki; Kurosawa, Tohru; Tamai, Ikumi

    2010-10-01

    Tebipenem pivoxil (TBPM-PI) is an oral carbapenem antibiotic for treating otolaryngologic and respiratory infections in pediatric patients. This agent is a prodrug to improve intestinal absorption of TBPM, an active form, and an absorption rate of TBPM-PI is higher than those of other prodrug-type β-lactam antibiotics. In the present study, we hypothesized that a certain mechanism other than simple diffusion is involved in the process of improved intestinal absorption of TBPM-PI and examined the mechanism. TBPM-PI uptake by Caco-2 cells was decreased by ATP-depletion and lowering the temperature to 4 °C, suggesting the contribution of carrier-mediated transport mechanisms. This uptake was partially decreased by ACE inhibitors, and the reduction of the absorption by captopril was observed by in vivo study and in situ single-pass intestinal perfusion study in rat, supporting the contribution of influx transporters. Since some ACE inhibitors and β-lactam antibiotics are reported to be substrates of PEPT and OATP families, we measured transporting activity of TBPM-PI by intestinally expressed transporters, PEPT1, OATP1A2, and OATP2B1. As a result, significant transport activities were observed by both OATP1A2 and OATP2B1 but not by PEPT1. Interestingly, pH dependence of TBPM-PI transports was different between OATP1A2 and OATP2B1, showing highest activity by OATP1A2 at pH 6.5, while OATP2B1-mediated uptake was higher at neutral and weak alkaline pH. OATP1A2 exhibited higher affinity for TBPM-PI (K(m) = 41.1 μM) than OATP2B1 (K(m) > 1 mM) for this agent. These results suggested that TBPM-PI has high intestinal apical membrane permeability due to plural intestinal transport routes, including the uptake transporters such as OATP1A2 and OATP2B1 as well as simple diffusion. PMID:20735088

  15. Overcoming Antigenic Diversity by Enhancing the Immunogenicity of Conserved Epitopes on the Malaria Vaccine Candidate Apical Membrane Antigen-1

    PubMed Central

    Dutta, Sheetij; Dlugosz, Lisa S.; Drew, Damien R.; Ge, Xiopeng; Ababacar, Diouf; Rovira, Yazmin I.; Moch, J. Kathleen; Shi, Meng; Long, Carole A.; Foley, Michael; Beeson, James G.; Anders, Robin F.; Miura, Kazutoyo; Haynes, J. David; Batchelor, Adrian H.

    2013-01-01

    Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal

  16. First report of Babesia divergens infection in an HIV patient.

    PubMed

    González, Luis M; Castro, Emma; Lobo, Cheryl A; Richart, Alberto; Ramiro, Raquel; González-Camacho, Fernando; Luque, Daniel; Velasco, Aurelio C; Montero, Estrella

    2015-04-01

    Human babesiosis is a zoonosis primarily transmitted through Ixodes ticks and alternatively by routes such as blood transfusions from asymptomatic donors. We report the first case of human babesiosis caused by Babesia divergens in a patient with HIV. This study also focuses on elucidating the possible transmission route of infection in this patient, who received numerous blood transfusions but showed patent symptoms only after splenectomy. A battery of detection tools along with a novel Western-Blot Assay and Enzyme Linked Immunosorbent Assay using the major surface protein of B. divergens (Bd37) as a target were used to evaluate the presence of B. divergens or antibodies against the parasite in samples from the patient and the blood donors involved in this case. A retrospective study of the humoral status against the parasite revealed B. divergens IgG antibodies in one of the implicated donors, but also showed that the patient had been already exposed to the parasite before any transfusion. Thus, this analysis of natural and transfusion transmission routes suggests a pre-existing subclinical babesiosis in the patient. PMID:25686807

  17. Apical Membrane Localization of the Adenomatous Polyposis Coli Tumor Suppressor Protein and Subcellular Distribution of the β-Catenin Destruction Complex in Polarized Epithelial Cells

    PubMed Central

    Reinacher-Schick, Anke; Gumbiner, Barry M.

    2001-01-01

    The adenomatous polyposis coli (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. APC is known to function as a tumor suppressor through downregulation of β-catenin as part of a high molecular weight complex known as the β-catenin destruction complex. The molecular composition of the intact complex and its site of action in the cell are still not well understood. Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton. To better understand the role of APC and the destruction complex in colorectal cancer, we have begun to characterize and isolate these complexes from confluent polarized human colon epithelial cell monolayers and other epithelial cell types. Subcellular fractionation and immunofluorescence microscopy reveal that a predominant fraction of APC associates tightly with the apical plasma membrane in a variety of epithelial cell types. This apical membrane association is not dependent on the mutational status of either APC or β-catenin. An additional pool of APC is cytosolic and fractionates into two distinct high molecular weight complexes, 20S and 60S in size. Only the 20S fraction contains an appreciable portion of the cellular axin and small but detectable amounts of glycogen synthase kinase 3β and β-catenin. Therefore, it is likely to correspond to the previously characterized β-catenin destruction complex. Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex. The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of β-catenin, or alternatively, whether they could be involved in other functions of the protein that

  18. Staphylococcus aureus enterotoxins A- and B: binding to the enterocyte brush border and uptake by perturbation of the apical endocytic membrane traffic.

    PubMed

    Danielsen, E Michael; Hansen, Gert H; Karlsdóttir, Edda

    2013-04-01

    Enterotoxins of Staphylococcus aureus are among the most common causes of food poisoning. Acting as superantigens they intoxicate the organism by causing a massive uncontrolled T cell activation that ultimately may lead to toxic shock and death. In contrast to our detailed knowledge regarding their interaction with the immune system, little is known about how they penetrate the epithelial barrier to gain access to their targets. We therefore studied the uptake of two staphylococcal enterotoxins (SEs), SEA and SEB, using organ cultured porcine jejunal explants as model system. Attachment of both toxins to the villus surface was scarce and patchy compared with that of cholera toxin B (CTB). SEA and SEB also bound to microvillus membrane vesicles in vitro, but less efficiently than CTB, and the binding was sensitive to treatment with endoglycoceramidase II, indicating that a glycolipid, possibly digalactosylceramide, acts as cell surface receptor at the brush border. Both SEs partitioned poorly with detergent resistant membranes (DRMs) of the microvillus, suggesting a weak association with lipid raft microdomains. Where attachment occurred, cellular uptake of SEA and SEB was also observed. In enterocytes, constitutive apical endocytosis normally proceeds only to subapical early endosomes present in the actomyosin-rich "terminal web" region. But, like CTB, both SEA and SEB penetrated deep into the cytoplasm. In conclusion, the data show that after binding to the enterocyte brush border SEA and SEB perturb the apical membrane trafficking, enabling them to engage in transcytosis to reach their target cells in the subepithelial lamina propria. PMID:23180309

  19. Commensal-Associated Molecular Patterns Induce Selective Toll-Like Receptor-Trafficking from Apical Membrane to Cytoplasmic Compartments in Polarized Intestinal Epithelium

    PubMed Central

    Cario, Elke; Brown, Dennis; McKee, Mary; Lynch-Devaney, Kathryn; Gerken, Guido; Podolsky, Daniel K.

    2002-01-01

    Commensal-associated molecular patterns, the major products of nonpathogenic bacteria, are present at high concentrations at the apical surface of the intestinal epithelium. However, the nature of the interaction of commensal-associated molecular patterns with the lumenal surface of the epithelium has not been defined. We have recently demonstrated that intestinal epithelial cells constitutively express several Toll-like receptors (TLRs) in vitro and in vivo that seem to be the key receptors responsible for immune cell activation in response to various bacterial products. In this study we characterize the subcellular distribution of two major TLRs, TLR2 and TLR4, and their ligand-specific dynamic regulation in the model human intestinal epithelial cell line T84. Immunocytochemical studies indicate that TLR2 and TLR4 are constitutively expressed at the apical pole of differentiated T84 cells. After stimulation with lipopolysaccharide or peptidoglycan, TLRs selectively traffic to cytoplasmic compartments near the basolateral membrane. Thus, we demonstrate that TLRs are positioned at the apical pole where they are poised to monitor the sensitive balance of the lumenal microbial array. The results of this dynamic epithelial surveillance can then be conveyed to the underlying cell populations of the lamina propria via these innate immune pattern recognition receptors. PMID:11786410

  20. TRPP2 and TRPV4 Form an EGF-Activated Calcium Permeable Channel at the Apical Membrane of Renal Collecting Duct Cells

    PubMed Central

    Song, Binlin; Gooz, Monika; Zhang, Jia-Ning; Yu, Chang-Jiang; Jiang, Shuai; Baldys, Aleksander; Gooz, Pal; Steele, Stacy; Owsianik, Grzegorz; Nilius, Bernd; Komlosi, Peter; Bell, P. Darwin

    2013-01-01

    Objective Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear. Methods and Results We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\\TRPV4 channel mediated increase in intracellular calcium. Conclusion We conclude that in the absence of cilia, an EGF activated TRPP2\\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis. PMID:23977387

  1. Identification and apical membrane localization of an electrogenic Na+/Ca2+ exchanger NCX2a likely to be involved in renal Ca2+ excretion by seawater fish

    PubMed Central

    Islam, Zinia; Romero, Michael F.; Hirose, Shigehisa

    2011-01-01

    Seawater (SW) contains ∼10 mM Ca2+, yet marine fish must drink seawater as their major water source. Thus marine teleosts fish need to excrete Ca2+ to maintain whole body Ca2+ homeostasis. In the intestine, seawater Ca2+ interreacts with epithelial-secreted HCO3− by the intestinal epithelium, and the resulting CaCO3 precipitates, which is rectally excreted. Recently the transporters involved in intestinal HCO3− secretion were identified. Ca2+ is also excreted by the kidney, but the protein(s) involved in renal Ca2+ excretion have not been identified. Here we identified a candidate transporter by using SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus), which are becoming useful animal models for studying molecular mechanisms of seawater adaptation. RT-PCR analyses of Na+/Ca2+ exchanger (NCX) family members in various torafugu tissues demonstrated that only NCX2a is highly expressed in the kidney. Renal expression of NCX2a was markedly elevated when mefugu were transferred from freshwater to seawater. In situ hybridization and immunohistochemical analyses indicated that NCX2a is expressed in the proximal tubule at the apical membrane. NCX2a, expressed in Xenopus oocytes, conferred [Ca2+]out- and Na+-dependent currents. These results suggest that NCX2a mediates renal Ca2+ secretion at the apical membrane of renal proximal tubules and has an important role in whole body Ca2+ homeostasis of marine teleosts. PMID:21880864

  2. In Vitro Evaluation of Drug Susceptibilities of Babesia divergens Isolates

    PubMed Central

    Brasseur, Philippe; Lecoublet, Sophie; Kapel, Nathalie; Favennec, Loic; Ballet, Jean J.

    1998-01-01

    The susceptibilities of three bovine and two human Babesia divergens isolates to antimicrobial agents were evaluated in vitro by a tritiated hypoxanthine incorporation assay. The MICs at which 50% of isolates are inhibited (MIC50s) for mefloquine (chlorhydrate), chloroquine (sulfate), quinine (chlorhydrate), clindamycin (phosphate), pentamidine (isethionate), phenamidine (isethionate) plus oxomemazine (chlorhydrate), lincomycin (chlorhydrate monohydrate), and imidocarb (dipropionate) were determined. Except for imidocarb, the MIC50s observed for the different isolates were close. Imidocarb and the combination of phenamidine plus oxomemazine exhibited the highest in vitro activity, while antimalarial agents such as mefloquine, choroquine, and quinine were inactive. Other drugs had intermediate activities. The data support further in vitro evaluation of antimicrobial agents active against B. divergens for the improvement of therapeutic strategies. PMID:9559789

  3. In vitro evaluation of drug susceptibilities of Babesia divergens isolates.

    PubMed

    Brasseur, P; Lecoublet, S; Kapel, N; Favennec, L; Ballet, J J

    1998-04-01

    The susceptibilities of three bovine and two human Babesia divergens isolates to antimicrobial agents were evaluated in vitro by a tritiated hypoxanthine incorporation assay. The MICs at which 50% of isolates are inhibited (MIC50s) for mefloquine (chlorhydrate), chloroquine (sulfate), quinine (chlorhydrate), clindamycin (phosphate), pentamidine (isethionate), phenamidine (isethionate) plus oxomemazine (chlorhydrate), lincomycin (chlorhydrate monohydrate), and imidocarb (dipropionate) were determined. Except for imidocarb, the MIC50s observed for the different isolates were close. Imidocarb and the combination of phenamidine plus oxomemazine exhibited the highest in vitro activity, while antimalarial agents such as mefloquine, choroquine, and quinine were inactive. Other drugs had intermediate activities. The data support further in vitro evaluation of antimicrobial agents active against B. divergens for the improvement of therapeutic strategies. PMID:9559789

  4. Uptake of 4-chloro-2-methylphenoxyacetic acid (MCPA) from the apical membrane of Caco-2 cells by the monocarboxylic acid transporter

    SciTech Connect

    Kimura, Osamu; Tsukagoshi, Kensuke; Endo, Tetsuya

    2008-03-15

    The cellular uptake mechanism of 4-chloro-2-methylphenoxyacetic acid (MCPA), a phenoxyacetic acid derivative, was investigated using Caco-2 epithelial cells. The cells were incubated with 50 {mu}M MCPA at pH 6.0 and 37 deg. C, and the uptake of MCPA from the apical membranes was measured. The uptake of MCPA was significantly decreased by incubation at low temperature (4 {sup o}C) and markedly increased by lowering the extracellular pH. Pretreatment with a protonophore, carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (25 {mu}M), or metabolic inhibitors, 2,4-dinitrophenol (1 mM) and sodium azide (10 mM), significantly decreased the uptake of MCPA by 53%, 45% and 48%, respectively. Coincubation of MCPA with 10 mM L-lactic acid or {alpha}-cyano-4-hydroxycinnamate, which is a substrate or an inhibitor of the monocarboxylic acid transporters (MCTs), significantly decreased the uptake of MCPA by 31% and 20%, respectively, and coincubation with benzoic acid profoundly decreased the uptake by 68%. In contrast, coincubation with succinic acid (a dicarboxylic acid) did not affect the uptake. Kinetic analysis of initial MCPA uptake suggested that MCPA is taken up via a carrier-mediated process [K{sub m} = 1.37 {+-} 0.15 mM, V{sub max} = 115 {+-} 6 nmol (mg protein){sup -1} (3 min){sup -1}]. Lineweaver-Burk plots show that benzoic acid competitively inhibits the uptake of MCPA with a K{sub i} value of 4.68 {+-} 1.76 mM. A trans-stimulation effect on MCPA uptake was found in cells preloaded with benzoic acid. These results suggest that the uptake of MCPA from the apical membrane of Caco-2 cells is mainly mediated by common MCTs along with benzoic acid but also in part by L-lactic acid.

  5. Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

    SciTech Connect

    Harvey, B.; Lacoste, I.; Ehrenfeld, J. )

    1991-04-01

    We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride, indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+).

  6. Trace Amine-Associated Receptor 1 Localization at the Apical Plasma Membrane Domain of Fisher Rat Thyroid Epithelial Cells Is Confined to Cilia

    PubMed Central

    Szumska, Joanna; Qatato, Maria; Rehders, Maren; Führer, Dagmar; Biebermann, Heike; Grandy, David K.; Köhrle, Josef; Brix, Klaudia

    2015-01-01

    Background The trace amine-associated receptor 1 (Taar1) is one member of the Taar family of G-protein-coupled receptors (GPCR) accepting various biogenic amines as ligands. It has been proposed that Taar1 mediates rapid, membrane-initiated effects of thyronamines, the endogenous decarboxylated and deiodinated relatives of the classical thyroid hormones T4 and T3. Objectives Although the physiological actions of thyronamines in general and 3-iodothyronamine (T1AM) in particular are incompletely understood, studies published to date suggest that synthetic T1AM-activated Taar1 signaling antagonizes thyromimetic effects exerted by T3. However, the location of Taar1 is currently unknown. Methods To fill this gap in our knowledge we employed immunofluorescence microscopy and a polyclonal antibody to detect Taar1 protein expression in thyroid tissue from Fisher rats, wild-type and taar1-deficient mice, and in the polarized FRT cells. Results With this approach we found that Taar1 is expressed in the membranes of subcellular compartments of the secretory pathway and on the apical plasma membrane of FRT cells. Three-dimensional analyses further revealed Taar1 immunoreactivity in cilial extensions of postconfluent FRT cell cultures that had formed follicle-like structures. Conclusions The results suggest Taar1 transport along the secretory pathway and its accumulation in the primary cilium of thyrocytes. These findings are of significance considering the increasing interest in the role of cilia in harboring functional GPCR. We hypothesize that thyronamines can reach and activate Taar1 in thyroid follicular epithelia by acting from within the thyroid follicle lumen, their potential site of synthesis, as part of a nonclassical mechanism of thyroid autoregulation. PMID:26601071

  7. Endodontic management of nonvital permanent teeth having immature roots with one step apexification, using mineral trioxide aggregate apical plug and autogenous platelet-rich fibrin membrane as an internal matrix: Case series.

    PubMed

    Sharma, Vivek; Sharma, Sarang; Dudeja, Pooja; Grover, Shibani

    2016-01-01

    A tooth with blunderbuss canal and open apex can be an endodontic challenge because of difficulty in obtaining an apical seal, and existing thin radicular walls which are susceptible to fracture. To overcome the limitations of traditional long-term calcium hydroxide apexification procedures, nonsurgical one step apexification using an array of materials such as mineral trioxide aggregate (MTA) has been suggested. However, adequate compaction of MTA in teeth with wide open apices can be an arduous task, and an internal matrix is required for controlled placement of MTA against which obturating material can be condensed. Platelet-rich fibrin (PRF), a second generation platelet concentrate containing several growth factors that promotes hard and soft-tissue healing, has been used as an internal matrix to create an apical plug of MTA and hence prevent extrusion of filling materials. This case series presents the endodontic management of immature permanent teeth with open apices using internal matrix of autologous PRF membrane and one step apical barrier placement of MTA. PMID:27041904

  8. Endodontic management of nonvital permanent teeth having immature roots with one step apexification, using mineral trioxide aggregate apical plug and autogenous platelet-rich fibrin membrane as an internal matrix: Case series

    PubMed Central

    Sharma, Vivek; Sharma, Sarang; Dudeja, Pooja; Grover, Shibani

    2016-01-01

    A tooth with blunderbuss canal and open apex can be an endodontic challenge because of difficulty in obtaining an apical seal, and existing thin radicular walls which are susceptible to fracture. To overcome the limitations of traditional long-term calcium hydroxide apexification procedures, nonsurgical one step apexification using an array of materials such as mineral trioxide aggregate (MTA) has been suggested. However, adequate compaction of MTA in teeth with wide open apices can be an arduous task, and an internal matrix is required for controlled placement of MTA against which obturating material can be condensed. Platelet-rich fibrin (PRF), a second generation platelet concentrate containing several growth factors that promotes hard and soft-tissue healing, has been used as an internal matrix to create an apical plug of MTA and hence prevent extrusion of filling materials. This case series presents the endodontic management of immature permanent teeth with open apices using internal matrix of autologous PRF membrane and one step apical barrier placement of MTA. PMID:27041904

  9. In Vitro Studies with Recombinant Plasmodium falciparum Apical Membrane Antigen 1 (AMA1): Production and Activity of an AMA1 Vaccine and Generation of a Multiallelic Response

    PubMed Central

    Kennedy, Michael C.; Wang, Jin; Zhang, Yanling; Miles, Aaron P.; Chitsaz, Farideh; Saul, Allan; Long, Carole A.; Miller, Louis H.; Stowers, Anthony W.

    2002-01-01

    Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage. PMID:12438374

  10. Multilevel Precision-Based Rational Design of Chemical Inhibitors Targeting the Hydrophobic Cleft of Toxoplasma gondii Apical Membrane Antigen 1 (AMA1)

    PubMed Central

    Muralikumar, Shalini; Mahalakshmi, B; Lily Therese, K; Madhavan, HN; Alameen, Mohamed; Thirumudi, Indhuja

    2016-01-01

    Toxoplasma gondii is an intracellular Apicomplexan parasite and a causative agent of toxoplasmosis in human. It causes encephalitis, uveitis, chorioretinitis, and congenital infection. T. gondii invades the host cell by forming a moving junction (MJ) complex. This complex formation is initiated by intermolecular interactions between the two secretory parasitic proteins—namely, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) and is critically essential for the host invasion process. By this study, we propose two potential leads, NSC95522 and NSC179676 that can efficiently target the AMA1 hydrophobic cleft, which is a hotspot for targeting MJ complex formation. The proposed leads are the result of an exhaustive conformational search-based virtual screen with multilevel precision scoring of the docking affinities. These two compounds surpassed all the precision levels of docking and also the stringent post docking and cumulative molecular dynamics evaluations. Moreover, the backbone flexibility of hotspot residues in the hydrophobic cleft, which has been previously reported to be essential for accommodative binding of RON2 to AMA1, was also highly perturbed by these compounds. Furthermore, binding free energy calculations of these two compounds also revealed a significant affinity to AMA1. Machine learning approaches also predicted these two compounds to possess more relevant activities. Hence, these two leads, NSC95522 and NSC179676, may prove to be potential inhibitors targeting AMA1-RON2 complex formation towards combating toxoplasmosis. PMID:27445648

  11. Multilevel Precision-Based Rational Design of Chemical Inhibitors Targeting the Hydrophobic Cleft of Toxoplasma gondii Apical Membrane Antigen 1 (AMA1).

    PubMed

    Vetrivel, Umashankar; Muralikumar, Shalini; Mahalakshmi, B; Lily Therese, K; Madhavan, H N; Alameen, Mohamed; Thirumudi, Indhuja

    2016-06-01

    Toxoplasma gondii is an intracellular Apicomplexan parasite and a causative agent of toxoplasmosis in human. It causes encephalitis, uveitis, chorioretinitis, and congenital infection. T. gondii invades the host cell by forming a moving junction (MJ) complex. This complex formation is initiated by intermolecular interactions between the two secretory parasitic proteins-namely, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) and is critically essential for the host invasion process. By this study, we propose two potential leads, NSC95522 and NSC179676 that can efficiently target the AMA1 hydrophobic cleft, which is a hotspot for targeting MJ complex formation. The proposed leads are the result of an exhaustive conformational search-based virtual screen with multilevel precision scoring of the docking affinities. These two compounds surpassed all the precision levels of docking and also the stringent post docking and cumulative molecular dynamics evaluations. Moreover, the backbone flexibility of hotspot residues in the hydrophobic cleft, which has been previously reported to be essential for accommodative binding of RON2 to AMA1, was also highly perturbed by these compounds. Furthermore, binding free energy calculations of these two compounds also revealed a significant affinity to AMA1. Machine learning approaches also predicted these two compounds to possess more relevant activities. Hence, these two leads, NSC95522 and NSC179676, may prove to be potential inhibitors targeting AMA1-RON2 complex formation towards combating toxoplasmosis. PMID:27445648

  12. Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule.

    PubMed

    Sabolić, I; Shi, L B; Brown, D; Ausiello, D A; Verkman, A S

    1992-01-10

    A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis. PMID:1309658

  13. Two water-specific aquaporins at the apical and basal plasma membranes of insect epithelia: molecular basis for water recycling through the cryptonephric rectal complex of lepidopteran larvae.

    PubMed

    Azuma, Masaaki; Nagae, Tomone; Maruyama, Mariya; Kataoka, Naoya; Miyake, Seiji

    2012-04-01

    Larval lepidopteran and coleopteran insects have evolved a specialised cryptonephric system in the hindgut in which water is constantly and rapidly taken up before defecation. In the silkworm, Bombyx mori, the movement of water through the epithelia within the cryptonephric rectal complex is likely facilitated by the two aquaporins, AQP-Bom1 and AQP-Bom3. Both are functionally water-specific and are predominantly expressed in the hindgut (colon and rectum). Phylogenetically, AQP-Bom1 and AQP-Bom3 belong to the DRIP (Drosophila integral protein) and PRIP (Pyrocoelia rufa integral protein) subfamilies, respectively, of the insect AQP clade. In immunoblot analyses using antipeptide antibodies for each Bombyx AQP, the predicted molecular mass for the respective AQPs were around 25 kDa, and further indicated that both tended to be oligomerised as a homotetramer (∼110 kDa). AQP-Bom1 [DRIP] was exclusively expressed at the apical plasma membrane of colonic and rectal epithelial cells, whereas AQP-Bom3 [PRIP] was expressed at the basal plasma membrane of these cells. This polarised localisation of DRIP/PRIP was also observed in the outer cryptonephric Malpighian tubules (outer cMT) and in the six tubules just outside the cryptonephric rectal complex (rectal lead MT). In the rectal epithelia, water is transported from the rectal lumen to the perinephric space and then deposited into the lumen of the outer cMT; the water then goes through the tubular lumen to exit the complex and is finally transported across the rectal lead MT. We conclude that rectal water retrieval into the haemocoele occurs at the very limited region of the water-permeable sites in MT epithelia after passing the rectal and cMT epithelia and that the high osmotic permeability is due to the presence of two distinct water-specific AQPs (DRIP and PRIP) in the epithelial cells of lepidopteran hindgut. PMID:22285686

  14. Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti.

    PubMed

    Ord, Rosalynn L; Rodriguez, Marilis; Cursino-Santos, Jeny R; Hong, Hyunryung; Singh, Manpreet; Gray, Jeremy; Lobo, Cheryl A

    2016-05-01

    Apicomplexan parasites include those of the genera Plasmodium, Cryptosporidium, and Toxoplasma and those of the relatively understudied zoonotic genus Babesia In humans, babesiosis, particularly transfusion-transmitted babesiosis, has been emerging as a major threat to public health. Like malaria, the disease pathology is a consequence of the parasitemia which develops through cyclical replication of Babesia parasites in host erythrocytes. However, there are no exoerythrocytic stages in Babesia, so targeting of the blood stage and associated proteins to directly prevent parasite invasion is the most desirable option for effective disease control. Especially promising among such molecules are the rhoptry neck proteins (RONs), whose homologs have been identified in many apicomplexan parasites. RONs are involved in the formation of the moving junction, along with AMA1, but no RON has been identified and characterized in any Babesia spp. Here we identify the RON2 proteins of Babesia divergens (BdRON2) and B. microti (BmRON2) and show that they are localized apically and that anti-BdRON2 antibodies are significant inhibitors of parasite invasion in vitro Neither protein is immunodominant, as both proteins react only marginally with sera from infected animals. Further characterization of the direct role of both BdRON2 and BmRON2 in parasite invasion is required, but knowledge of the level of conformity of RON2 proteins within the apicomplexan phylum, particularly that of the AMA1-RON2 complex at the moving junction, along with the availability of an animal model for B. microti studies, provides a key to target this complex with a goal of preventing the erythrocytic invasion of these parasites and to further our understanding of the role of these conserved ligands in invasion. PMID:26953328

  15. A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens.

    PubMed Central

    Worobo, R W; Van Belkum, M J; Sailer, M; Roy, K L; Vederas, J C; Stiles, M E

    1995-01-01

    Divergicin A is a strongly hydrophobic, narrow-spectrum, nonlantibiotic bacteriocin produced by Carnobacterium divergens LV13. This strain of C. divergens contains a 3.4-kb plasmid that mediates production of, and immunity to, the bacteriocin. N-terminal amino acid sequencing of the purified divergicin A was used to locate the structural gene (dvnA). The structural gene encodes a prepeptide of 75 amino acids consisting of a 29-amino-acid N-terminal extension and a mature peptide of 46 amino acids. Directly downstream of dvnA there is a second open reading frame that encodes the immunity protein for divergicin A. Divergicin A has a calculated molecular mass of 4,223.89 Da. The molecular mass determined by mass spectrometry is 4,223.9 Da, indicating that there is no posttranslational modification of the peptide. The N-terminal extension of divergicin A has an Ala-Ser-Ala (positions -3 to -1) cleavage site and acts as a signal peptide that accesses the general export system of the cell (such as the sec pathway in Escherichia coli). This is the first bacteriocin of lactic acid bacteria to be reported that does not have dedicated maturation and secretion genes. Production of divergicin A was observed in heterologous hosts containing only the two genes associated with divergicin A production and immunity. Fusing alkaline phosphatase behind the signal peptide for divergicin resulted in the secretion of this enzyme in the periplasmic space and supernatant of E. coli. PMID:7768812

  16. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area.

    PubMed

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-04-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs. PMID:26825252

  17. Toxin pharmacology of the large-conductance Ca(2+)-activated K+ channel in the apical membrane of rabbit proximal convoluted tubule in primary culture.

    PubMed

    Tauc, M; Congar, P; Poncet, V; Merot, J; Vita, C; Poujeol, P

    1993-10-01

    The patch-clamp technique was used to study the toxin pharmacology of the large-conductance Ca(2+)-activated K+ channel (BKCa) present in the apical membrane of rabbit proximal convoluted tubules (PCT) in primary culture. Experiments were performed with the inside-out configuration. This channel was very selective for K+ against Na+ and had a conductance of 180 pS with 140 mmol/l in the pipette and the bath. The action of toxins was studied on the extracellular side of the channel by using the pipette perfusion technique. Experimental conditions were 140 mmol/l KCl in the pipette and 140 mmol/l NaCl in the bath. Pipette potential was maintained at 0 mV. Perfusion of crude venom from Leiurus quinquestriatus hebraeus inhibited reversibly the open probability (Po) in a concentration-dependent fashion (IC50 = 0.8 mg/l; n = 3). The following synthetic or purified toxins were tested: synthetic charybdotoxin (ChTX) IC50 = 7.3 x 10(-9) M (n = 5); iberiotoxin (IbTX) IC50 = 5.5 x 10(-7) mol/l (n = 3); and kaliotoxin (KTX) IC50 = 4.8 x 10(-7) mol/l (n = 3). The suppression of the six first N-terminal amino-acids slightly reduced the affinity of ChTX (IC50 = 1.2 x 10(-8) mol/l, n = 4). Neither Dendroaspis polylepis venom nor purified alpha dendrotoxin modified Po even at high concentrations (20 mg/l and 10(-6) mol/l respectively). Apamin, which blocked the small-conductance K+ channel in cultured PCT, did not act on BKCa. These results indicate that ChTX is the most efficient known toxin against the epithelial BKCa in primary cultures of PCT.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7505914

  18. High-Level Expression, Purification and Characterization of A Recombinant Plasmodium vivax Apical Membrane Antigen 1: Implication for vivax Malaria Vaccine Development

    PubMed Central

    Salavatifar, Maryam; Zakeri, Sedigheh; Hayati Roodbari, Nasim; Djadid, Navid Dinparast

    2015-01-01

    Objective The apical membrane antigen-1 (AMA-1) is considered as a promising candidate for development of a malaria vaccine against Plasmodium parasites. The correct conformation of this protein appears to be necessary for the stimulation of parasite-inhibitory responses, and these responses, in turn, seem to be antibody-mediated. Therefore, in the present investigation, we expressed the Plasmodium vivax AMA-1 (PvAMA-1) ectodomain in Escherichia coli (E. coli), purified it using standard procedures and characterized it to determine its biological activities for it to be used as a potential target for developing a protective and safe vivax malaria vaccine. Materials and Methods In this experimental investigation, the ectodomain of PvAMA-1 antigen (GenBank accession no. JX624741) was expressed in the E. coli M15pQE30 expression system and purified with immobilized-metal affinity chromatography. The correct conformation of the recombinant protein was evaluated by Western blotting and indirect immunofluorescence antibody (IFA) test. In addition, the immunogenic properties of PvAMA-1 were evaluated in BALB/c mice with the purified protein emulsified in Freund’s adjuvant. Results In the present study, the PvAMA-1 ectodomain was expressed at a high-level (65 mg/L) using a bacterial system. Reduced and non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as Western blot analysis confirmed the appropriate conformation and folding of PvAMA-1. The evaluation of immunogenic properties of PvAMA-1 showed that both T helper-1 and 2 cells (Th1 and Th2) responses were present in mice after three immunizations and persisted up to one year after the first immunization. Moreover, the antibodies raised against the recombinant PvAMA-1 in injected mice could recognize the native protein localized on P. vivax parasites. Conclusion We demonstrate that our recombinant protein had proper conformation and folding. Also, there were common epitopes in the

  19. Coccidioidomycosis in a Pacific walrus (Odobenus rosmarus divergens).

    PubMed

    Schmitt, Todd L; Procter, Diana G

    2014-03-01

    An 11 yr-old female Pacific walrus (Odobenus rosmarus divergens) demonstrated decreased appetite and weight loss approximately 4 wk after truck transport from a northern California facility to a southern California facility. An initial blood analysis revealed a leukocytosis of 22,800 white blood cells (WBC)/microl, with a left shift, low iron (58 microg/dl), and mild hyperglobulinemia (4.3 g/dl). Empiric antibiotic therapy was started with amoxicillin and clavulanic acid (14 mg/kg p.o. b.i.d.). Clinical improvement was observed initially; however, follow-up blood analysis demonstrated a persistent leukocytosis (24,000 WBC/microl), with left shift and progressive hyperglobulinemia (6.7 mg/dl). As a result of the relapse of clinical signs on antibiotic therapy, aggressive antifungal therapy was initiated with voriconazole (1.8 mg/kg p.o. s.i.d.). Concurrent fungal immunodiffusion antibody assays and complement fixation were repetitively positive for coccidioidomycosis. The walrus improved clinically over the next 3 mo and is currently stable on antifungal therapy at its originating facility in northern California. PMID:24712180

  20. The apical membrane of intestinal brush cells possesses a specialised, but species-specific, composition of glycoconjugates--on-section and in vivo lectin labelling in rats, guinea-pigs and mice.

    PubMed

    Gebert, A; al-Samir, K; Werner, K; Fassbender, S; Gebhard, A

    2000-05-01

    Brush cells are specialised epithelial cells that are assumed to represent chemoreceptors of the digestive tract. They comprise a small population of the epithelial cells lining the intestine, possess a unique ultrastructure and, in many aspects, resemble the receptor cells of taste buds. To characterise glycoconjugates possibly involved in a sensory function, we investigated brush cells in the small intestine of three species using lectin histochemistry in confocal light and thin-section electron microscopy. Brush cells of rats were selectively labelled by the sialic acid-specific lectin Maackia amurensis agglutinin, those of guinea-pigs by the D-galactose-specific lectin Bandeiraea simplicifolia agglutinin, isolectin B4 and those of mice by the L-fucose-specific lectin Ulex europaeus agglutinin lectin I. Lectin binding sites were consistently located in the glycocalyx of the apical membrane and in that of cytoplasmic vesicles. In vivo lectin labelling revealed that the glycoconjugates of the apical membrane are accessible under physiological conditions, that brush cells do not endocytose and that they probably possess a high membrane turnover rate. The results show that specialisations exist in the composition of glycoconjugates forming the glycocalyx of brush cells in all species investigated. The presence of brush cell-specific glycoconjugates would be in accordance with the current hypothesis of a receptive function of brush cells. Differences in the specific glycosylation patterns among rats, guinea-pigs and mice indicate that species-specific adaptations exist. PMID:10883398

  1. Identification of common antigens in Babesia bovis, B. bigemina, and B. divergens.

    PubMed

    Figueroa, Julio V; Precigout, Eric; Carcy, Bernard; Gorenflot, André

    2006-10-01

    Bovine babesiosis, caused by Babesia bovis, B. bigemina, and B. divergens, is a significant impediment to livestock production in countries with tropical/subtropical and temperate climates. Previous studies conducted on the immunoprophylaxis against the disease and diagnosis of these parasites has demonstrated the presence of similar antigens. The objective of this article was to identify and partially characterize antigens conserved among these three species. Immunochemical analysis using sera from cattle immunized individually with antigens from these three Babesia species revealed a number of antigens recognized by heterologous antisera. Cross-reactions were more evident in sera from cattle immunized with B. bovis/B. bigemina which recognized several antigens (15 kDa to >200 kDa) in B. divergens. Immunoscreening of a B. divergens cDNA library with bovine serum to B. bigemina allowed the isolation of five clones and DNA sequencing of plasmid BdJF5 showed a 680 bp cDNA insert. Basic Local Alignment Search Tool (BLAST) analysis of the predicted amino acid sequence revealed 47% identity with a protein identified as alphaNAC. Serum from mice immunized with a recombinant Glutathione S-Transferase-BdJF5 fusion protein immunoprecipitated a 20 kDa B. bovis antigen. However, 30 kDa and 18 kDa antigens were immunoprecipitated from B. divergens and immunoblotting analysis revealed the recognition of a 35 kDa B. bigemina antigen. An indirect fluorescence antibody assay on merozoites showed strong reaction with B. divergens and weak recognition of B. bovis and B. bigemina. Despite the existent antigenic polymorphism among the Babesia spp., these results demonstrated that common antigens occur between European B. divergens and Mexican B. bovis/B. bigemina. PMID:17135542

  2. Bicarbonate exchangers SLC26A3 and SLC26A6 are localized at the apical membrane of porcine vas deferens epithelium

    PubMed Central

    Pierucci-Alves, Fernando; Akoyev, Vladimir; Schultz, Bruce D

    2015-01-01

    The goal of this study was to test for expression of HCO3− exchangers SLC26A3 and SLC26A6 in primary cultures of porcine vas deferens epithelial cells (1°PVD) and native porcine vas deferens. Quantitative RT-PCR revealed that mRNA coding for SLC26A6 was six times more abundant than mRNA coding for SLC26A3 in 1°PVD cells. Western blot analyses combined with surface biotinylation of 1°PVD demonstrated SLC26A3 and SLC26A6 immunoreactivities in whole-cell lysates and apical surfaces of monolayers. Laser scanning confocal microscopy (LSCM) of the 1°PVD cell monolayers demonstrated that SLC26A3 immunoreactivity was primarily in the apical region but present throughout the basal-apical cellular axis, whereas SLC26A6 immunoreactivity was present in the apical region and sometimes accumulated in the nuclear region. LSCM also demonstrated SLC26A3 and SLC26A6 immunoreactivities present along the entire apical lining of the native porcine vas deferens epithelium and in basal cells. The patterns and apparent abundance of SLC26A3 and SLC26A6 immunoreactivities in the proximal vas deferens were not different from the corresponding immunoreactivities in the distal region. There is no evidence of preferential expression of SLC26A3 or SLC26A6 in any portion of the vas deferens, as has been proposed for epithelia that secrete HCO3− in other duct systems. Thus, vas deferens epithelia express transporters throughout the duct that can contribute to rapid alkalinization of the luminal contents as it has been demonstrated in vivo. PMID:25907791

  3. Uptake of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) and dicamba (3,6-dichloro-2-methoxybenzoic acid) from the apical membranes of the human intestinal Caco-2 cells.

    PubMed

    Kimura, Osamu; Tsukagoshi, Kensuke; Hayasaka, Moriaki; Endo, Tetsuya

    2012-01-01

    We investigated whether the uptake of triclopyr (3, 5, 6-trichloro-2-pyridinyloxyacetic acid) and dicamba (3,6-dichloro-2-methoxybenzoic acid) across the apical membrane of Caco-2 cells was mediated via proton-linked monocarboxylic acid transporters (MCTs). The uptake of triclopyr from the apical membranes was fast, pH-, temperature-, and concentration dependent, required metabolic energy to proceed, and was competitively inhibited by monocarboxylic acids such as benzoic acid and ferulic acid (substrates of L-lactic acid-insensitive MCTs), but not by L-lactic acid. Thus, the uptake of triclopyr in Caco-2 cells appears to be mediated mainly via L-lactic acid-insensitive MCTs. In contrast, the uptake of dicamba (a benzoic acid derivative) was slow, and it was both pH- and temperature dependent. Coincubation with ferulic acid did not decrease the uptake of dicamba, although coincubation with benzoic acid moderately decreased it. The uptake of dicamba appears to be mediated mainly via passive diffusion, which is in contrast to the uptake of benzoic acid via MCTs. We speculate that the substituted groups in dicamba may inhibit uptake via MCTs. PMID:21766207

  4. Morphological and Molecular Descriptors of the Developmental Cycle of Babesia divergens Parasites in Human Erythrocytes

    PubMed Central

    Rossouw, Ingrid; Maritz-Olivier, Christine; Niemand, Jandeli; van Biljon, Riette; Smit, Annel; Olivier, Nicholas A.; Birkholtz, Lyn-Marie

    2015-01-01

    Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is on the increase, resulting in renewed attentiveness to this potentially life threatening emerging zoonotic disease. The molecular mechanisms underlying the pathophysiology and intra-erythrocytic development of these parasites are poorly understood. This impedes concerted efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell biological and molecular functional genomics tools, we describe the intra-erythrocytic development cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic Babesia spp. This is further correlated for the first time to nuclear content increases during intra-erythrocytic development progression, providing insight into the part of the life cycle that occurs during human infection. High-content temporal evaluation elucidated the contribution of the different stages to life cycle progression. Moreover, molecular descriptors indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation. Additionally, differential expression is observed as the parasite equilibrates its developmental stages during its life cycle. Together, this information provides the first temporal evaluation of the functional transcriptome of B. divergens parasites, information that could be useful in identifying biological processes essential to parasite survival for future anti-babesiacidal discoveries. PMID:25955414

  5. Morphological and Molecular Descriptors of the Developmental Cycle of Babesia divergens Parasites in Human Erythrocytes.

    PubMed

    Rossouw, Ingrid; Maritz-Olivier, Christine; Niemand, Jandeli; van Biljon, Riette; Smit, Annel; Olivier, Nicholas A; Birkholtz, Lyn-Marie

    2015-05-01

    Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is on the increase, resulting in renewed attentiveness to this potentially life threatening emerging zoonotic disease. The molecular mechanisms underlying the pathophysiology and intra-erythrocytic development of these parasites are poorly understood. This impedes concerted efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell biological and molecular functional genomics tools, we describe the intra-erythrocytic development cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic Babesia spp. This is further correlated for the first time to nuclear content increases during intra-erythrocytic development progression, providing insight into the part of the life cycle that occurs during human infection. High-content temporal evaluation elucidated the contribution of the different stages to life cycle progression. Moreover, molecular descriptors indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation. Additionally, differential expression is observed as the parasite equilibrates its developmental stages during its life cycle. Together, this information provides the first temporal evaluation of the functional transcriptome of B. divergens parasites, information that could be useful in identifying biological processes essential to parasite survival for future anti-babesiacidal discoveries. PMID:25955414

  6. Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1.

    PubMed

    Norden, Pieter R; Kim, Dae Joong; Barry, David M; Cleaver, Ondine B; Davis, George E

    2016-01-01

    A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network

  7. Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1

    PubMed Central

    Norden, Pieter R.; Kim, Dae Joong; Barry, David M.; Cleaver, Ondine B.; Davis, George E.

    2016-01-01

    A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network

  8. Babesia divergens, a Bovine Blood Parasite of Veterinary and Zoonotic Importance

    PubMed Central

    Zintl, Annetta; Mulcahy, Grace; Skerrett, Helen E.; Taylor, Stuart M.; Gray, Jeremy S.

    2003-01-01

    Babesia divergens is an intraerythrocytic protozoan parasite, transmitted by the tick Ixodes ricinus, and is the main agent of bovine babesiosis in Europe. It is not only a cause of significant loss to the cattle industry; it can also infect immunocompromised humans, causing medical emergencies characterized by rapid fulmination and parasitemias that may exceed 70%. The current emphasis in Europe on sustainable agriculture and extensification is likely to lead to an increase in vector tick populations with increased risk of infection. Despite the veterinary and zoonotic importance of this parasite, relatively little research has been carried out on B. divergens, and many questions regarding the parasite's epidemiology and the host's response remain unanswered. A better understanding of the species' biology and host-parasite interactions may lead to improved control mechanisms and new trends in vaccine and antibabesial drug development. This review provides the first comprehensive summary of B. divergens biology, including its morphology, life cycle, and host specificity, and the current state of knowledge of both human and bovine infections. PMID:14557289

  9. Apical sterol-rich membranes are essential for localizing cell end markers that determine growth directionality in the filamentous fungus Aspergillus nidulans.

    PubMed

    Takeshita, Norio; Higashitsuji, Yuhei; Konzack, Sven; Fischer, Reinhard

    2008-01-01

    In filamentous fungi, hyphal extension depends on the continuous delivery of vesicles to the growing tip. Here, we describe the identification of two cell end marker proteins, TeaA and TeaR, in Aspergillus nidulans, corresponding to Tea1 and Mod5 in Schizosaccharomyces pombe. Deletion of teaA or teaR caused zig-zag-growing and meandering hyphae, respectively. The Kelch-repeat protein TeaA, the putatively prenylated TeaR protein, and the formin SepA were highly concentrated in the Spitzenkörper, a vesicle transit station at the tip, and localized along the tip membrane. TeaA localization at tips depended on microtubules, and TeaA was required for microtuble convergence in the hyphal apex. The CENP-E family kinesin KipA was necessary for proper localization of TeaA and TeaR, but not for their transportation. TeaA and TeaR localization were interdependent. TeaA interacted in vivo with TeaR, and TeaA colocalized with SepA. Sterol-rich membrane domains localized at the tip in teaA and teaR mutants like in wild type, and filipin treatment caused mislocalization of both proteins. This suggests that sterol-rich membrane domains determine cell end factor destinations and thereby polarized growth. PMID:18003978

  10. Development of culture-based serological assays to diagnose Babesia divergens infections.

    PubMed

    Gabrielli, Simona; Galuppi, Roberta; Marcer, Federica; Marini, Carla; Tampieri, Maria Paola; Moretti, Annabella; Pietrobelli, Mario; Cancrini, Gabriella

    2012-02-01

    Babesioses are hematic tick-borne diseases that induce malaria-like disorders in domestic, wild animals, and humans. Although indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) commercial kits are available to test the presence of antibodies against most Babesia species, no kit exists to serologically diagnose the infections due to Babesia divergens, one of the most important zoonotic species. To fill this gap and to develop assays to detect animal and human infections, in vitro cultures (microaerophilous stationary phase system) of B. divergens were organized. Infected erythrocytes were adsorbed as corpuscular antigen (CA) on IFAT slides and ELISA microwells. The supernatant medium of the cultures (metabolic antigen, MA) was collected and employed in ELISA and western blot (WB) assays. B. divergens was also used to produce positive sera in Meriones unguiculatus and to infect a calf. Serological tests were set up with sera from experimentally/naturally infected animals, and possible cross-reactions were evaluated using heterologous sera from cattle positive to other piroplasms. Sera from clinically healthy people at risk of infection were also tested. As expected, assays based on the purified MAs from in vitro cultures proved more sensitive and specific than CA-IFAT and CA-ELISA. In fact, MA-ELISA provided satisfactory performances (even if 8.4%-15.7% cross-reactions were evidenced), and the WB developed proved totally sensitive and specific. WB indicated as immunodominant antigens two major protein bands at 33 and 37 kDa, which were also evidenced in 2.2% of the human sera tested, proving the parasite transmission to humans also in Italy. PMID:21995263

  11. Apical Functionalization of Tribenzotriquinacenes.

    PubMed

    Dhara, Ayan; Weinmann, Joshua; Krause, Ana-Maria; Beuerle, Florian

    2016-08-22

    The introduction of one alkyne moiety at the central carbon atom of the tripodal tribenzotriquinacene scaffold allows easy access to a great variety of apically functionalized derivatives. The spatially well-separated arrangement of different functional units on the convex face and outer rim was further proven by single-crystal X-ray studies. Subsequent modifications that feature a general protecting group-free strategy for the demethylation of protected catechols in the presence of a terminal alkyne group, an azide-alkyne Huisgen cycloaddition, and Sonogashira cross-coupling reactions showcase the high synthetic potential of this modular approach for tribenzotriquinacene derivatization. PMID:27444414

  12. Babesia divergens infections in the Mongolian gerbil: characteristics of a human strain.

    PubMed

    Liddell, K G; Lucas, S B; Williams, H

    1981-04-01

    A strain of the cattle piroplasm Babesia divergens isolated from a fatal human infection was propagated in the Mongolian gerbil through 150 semi-continuous intraperitoneal passages. The infection was normally fatal; death, accompanied by profuse haemoglobinuria and debilitation, occurred as early as 44 h after intraperitoneal inoculation of heavily parasitized blood with precipitous drops in red blood cell and platelet counts. The average maximum parasitaemia achieved increased on continuous passage reaching 80% by the 150th stage. Twenty-four hours after infection erythrophagocytosis and splenic congestion were apparent by light and electron-microscopical examination and by 48 h hepatic necrosis, renal tubular damage with haemoglobin cast accumulation and ischaemic necrosis of ileal mucosa had developed. Gerbils were highly susceptible to small numbers of parasites when the inoculum was either fresh parasitized blood in high dilution or erythrocytes concentrated from animals showing minimal parasitaemia. Animals inoculated with parasites preserved in dimethyl sulphoxide at low temperatures usually developed fatal infections. However, occasionally animals suffered at most a low grade parasitaemia subsequent to recovery with parasite elimination. These animals were immune to further challenge, and no chronic infections developed. A field strain of B. divergens isolated locally from a case of bovine redwater behaved similarly to the human strain on continuous passage in gerbils. PMID:7220085

  13. Identification and Characterization of the RouenBd1987 Babesia divergens Rhopty-Associated Protein 1

    PubMed Central

    Rodriguez, Marilis; Alhassan, Andy; Ord, Rosalynn L.; Cursino-Santos, Jeny R.; Singh, Manpreet; Gray, Jeremy; Lobo, Cheryl A.

    2014-01-01

    Human babesiosis is caused by one of several babesial species transmitted by ixodid ticks that have distinct geographical distributions based on the presence of competent animal hosts. The pathology of babesiosis, like malaria, is a consequence of the parasitaemia which develops through the cyclical replication of Babesia parasites in a patient's red blood cells, though symptoms typically are nonspecific. We have identified the gene encoding Rhoptry-Associated Protein −1 (RAP-1) from a human isolate of B. divergens, Rouen1987 and characterized its protein product at the molecular and cellular level. Consistent with other Babesia RAP-1 homologues, BdRAP-1 is expressed as a 46 kDa protein in the parasite rhoptries, suggesting a possible role in red cell invasion. Native BdRAP-1 binds to an unidentified red cell receptor(s) that appears to be non-sialylated and non-proteinacious in nature, but we do not find significant reduction in growth with anti-rRAP1 antibodies in vitro, highlighting the possibility the B. divergens is able to use alternative pathways for invasion, or there is an alternative, complementary, role for BdRAP-1 during the invasion process. As it is the parasite's ability to recognize and then invade host cells which is central to clinical disease, characterising and understanding the role of Babesia-derived proteins involved in these steps are of great interest for the development of an effective prophylaxis. PMID:25226276

  14. Pulmonary Mast Cell Tumor and Possible Paraganglioma in a Free-ranging Pacific Walrus ( Odobenus rosmarus divergens), Barrow, Alaska, USA.

    PubMed

    Seguel, Mauricio; Stimmelmayr, Raphaela; Howerth, Elizabeth; Gottdenker, Nicole

    2016-04-28

    We describe a pulmonary mast cell tumor in a subsistence-harvested free-ranging Pacific walrus (Odobenus rosmarus divergens). Neoplastic cells effacing a focal area of pulmonary parenchyma were characterized by rare metachromatic granules and positive staining for C-kit. We also report co-occurrence of a peribronchial mass with a morphologic and immunohistochemical profile compatible with paraganglioma. PMID:27054472

  15. Apical potassium channels in the rat connecting tubule.

    PubMed

    Frindt, Gustavo; Palmer, Lawrence G

    2004-11-01

    Apical membrane K channels in the rat connecting tubule (CNT) were studied using the patch-clamp technique. Tubules were isolated from the cortical labyrinth of the kidney and split open to provide access to the apical membrane. Cell-attached patches were formed on presumed principal and/or connecting tubule cells. The major channel type observed had a single-channel conductance of 52 pS, high open probability and kinetics that were only weakly dependent on voltage. These correspond closely to the "SK"-type channels in the cortical collecting duct, identified with the ROMK (Kir1.1) gene product. A second channel type, which was less frequently observed, mediated larger currents and was strongly activated by depolarization of the apical membrane voltage. These were identified as BK or maxi-K channels. The density of active SK channels revealed a high degree of clustering. Although heterogeneity of tubules or of cell types within a tubule could not be excluded, the major factor underlying the distribution appeared to be the presence of channel clusters on the membrane of individual cells. The overall density of channels was higher than that previously found in the cortical collecting tubule (CCT). In contrast to results in the CCT, we did not detect an increase in the overall density of SK channels in the apical membrane after feeding the animals a high-K diet. However, the activity of amiloride-sensitive Na channels was undetectable under control conditions but was increased after both 1 day (90 +/- 24 pA/cell) or 7 days (385 +/- 82 pA/cell) of K loading. Thus one important factor leading to an increased K secretion in the CNT in response to increased dietary K is an increased apical Na conductance, leading to depolarization of the apical membrane voltage and an increased driving force for K movement out into the tubular lumen. PMID:15280155

  16. Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures.

    PubMed

    Cholon, Deborah M; O'Neal, Wanda K; Randell, Scott H; Riordan, John R; Gentzsch, Martina

    2010-03-01

    CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, DeltaF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in DeltaF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of DeltaF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of DeltaF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking. PMID:20008117

  17. Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures

    PubMed Central

    Cholon, Deborah M.; O'Neal, Wanda K.; Randell, Scott H.; Riordan, John R.

    2010-01-01

    CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, ΔF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in ΔF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of ΔF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of ΔF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking. PMID:20008117

  18. Novel Diamidines with Activity against Babesia divergens In Vitro and Babesia microti In Vivo ▿

    PubMed Central

    Nehrbass-Stuedli, Angela; Boykin, David; Tidwell, Richard R.; Brun, Reto

    2011-01-01

    Dicationic diamidines, such as diminazene and pentamidine, are well-studied chemotherapeutic agents with significant activity against parasitic diseases. The in vitro activities of novel diamidine compounds against the Babesia divergens strains 1903B and 4201 were investigated. The most potent compound, a diphenyl furan, had a 50% inhibitory concentration (IC50) of 1.5 ng/ml. In a murine model, several test compounds were effective enough to cure mice infected with Babesia microti at a dose of 12.5 and/or 25 mg/kg of body weight given by the subcutaneous route for 4 days. The best antibabesial properties were exhibited by terphenyls, benzimidazoles, diphenyl furans, pentamidine, and pentamidine analogues. PMID:21537025

  19. [Tick infestation and the prevalence of Borrelia burgdorferi and Babesia divergens in cattle in Bavaria].

    PubMed

    Lengauer, Heidi; Just, Frank Thomas; Edelhofer, Renate; Pfister, Kurt

    2006-01-01

    During the grazing period 2002 319 cattle from 31 farms located in 6 districts of southern Bavaria were examined for the presence of ticks in 4- to 5-week intervals, and 287 serum samples were tested for the presence of antibodies against Borrelia burgdorferi and Babesia divergens. Ticks were detected in all 31 farms with a mean prevalence of 69%. 3218 out of 3453 collected ticks were Ixodes ricinus; 139 nymphs, 19 larvae and 77 damaged adult specimens could only be determined to the Genus level (Ixodes). The seasonal pattern revealed the highest frequencies of ticks in May/June and September. The intensity of tick infestation of positive animals was generally low. 76.5% of parasitized cattle had 1-6 ticks per day of investigation. Individual cattle showed up to 250 ticks per day. The percentage of infested animals in each herd varied within the period between 0-100%. The examination of serum samples by immunofluorescence technique (IFAT) revealed positive anti-Borrelia antibody titers (> or = 1:64) for 45.6% of the animals. The within-farm seroprevalence of borreliosis ranged from 20 to 100% in 27 of the 31 farms. A significant correlation could be detected between the number of ticks/cattle and the anti-Borrelia burgdorferi IgG-titer. By contrast, there was no significant correlation between the age of the animals and anti-Borrelia serum titers. For comparative reasons, 64 IFAT-positive serum samples were tested by Western blot techniques for the presence of antibodies cross-reacting with Borrelia garinii antigen. These analyses revealed that 69% of the samples reacted positively, 28% were unclear and 3% were negative. Examinations of the 287 serum samples for the presence of anti-Babesia divergens antibodies revealed one positive animal with a titer of 1:16. PMID:17009719

  20. Roles of external and cellular Cl- ions on the activation of an apical electrodiffusional Cl- pathway in toad skin.

    PubMed

    Procopio, J; Lacaz-Vieira, F

    1990-07-01

    This study is concerned with the short-circuit current, Isc, responses of the Cl(-)-transporting cells of toad skin submitted to sudden changes of the external Cl- concentration, [Cl]o. Sudden changes of [Cl]o, carried out under apical membrane depolarization, allowed comparison of the roles of [Cl]o and [Cl]cell on the activation of the apical Cl- pathways. Equilibration of short-circuited skins symmetrically in K-Ringer's solutions of different Cl- concentrations permitted adjustment of [Cl]cell to different levels. For a given Cl- concentration (in the range of 11.7 to 117 mM) on both sides of a depolarized apical membrane, this structure exhibits a high Cl- permeability, P(Cl)apical. On the other hand, for the same range of [Cl]cell but with [Cl]o = 0, P(Cl)apical is reduced to negligible values. These observations indicate that when the apical membrane is depolarized P(Cl)apical is modulated by [Cl]o; in the absence of external Cl- ions, intracellular Cl- is not sufficient to activate P(Cl)apical. Computer simulation shows that the fast Cl- currents induced across the apical membrane by sudden shifts of [Cl]o from a control equilibrium value strictly follow the laws of electrodiffusion. For each experimental group, the computer-generated Isc versus [( Cl]cell - [Cl]o) curve which best fits the experimental data can only be obtained by a unique pair of P(Cl)apical and Rb (resistance of the basolateral membrane), thus allowing the calculation of these parameters. The electrodiffusional behavior of the net Cl- flux across the apical membrane supports the channel nature of the apical Cl- pathways in the Cl(-)-transporting cells. Cl- ions contribute significantly to the overall conductance of the basolateral membrane even in the presence of a high K concentration in the internal solution. PMID:1698229

  1. Large-scale drug screening against Babesia divergens parasite using a fluorescence-based high-throughput screening assay.

    PubMed

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; AbouLaila, Mahmoud; Tuvshintulga, Bumduuren; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-08-30

    The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the efficacies of large chemical libraries against Babesia divergens (bovine strain) in in vitro cultures was evaluated in this study. Hematocrits (HCTs) of 2.5%, 5%, and 10% were used for the in vitro culture at 1% parasitemia without daily replacement of the medium. Linearity and HTS assay results revealed that the best HCTs were 5% and 10%. The obtained IC50 values of diminazene aceturate, either by fluorescence-based HTS assay with and without daily replacement of medium or by fluorescence- and microscopy-based methods, did not differ significantly at 5% HCT. Actinonin and chloroquine diphosphate were the most effective drugs against the in vitro growth of B. divergens, followed by pyronaridine tetraphosphate- and luteolin-treated cultures. On contrary, tetracycline hydrochloride and (-)-epigallocatechin-3-gallate from green tea exhibited poor activity as compared with diminazene aceturate (positive control drug). The data indicated that 5% HCT without daily replacement of the culture medium mixed with bovine serum in vitro using a fluorescence-based HTS assay creates the best conditions for large-scale drug screening against B. divergens that infect cattle. PMID:27523944

  2. Clathrin and AP-1 regulate apical polarity and lumen formation during C. elegans tubulogenesis

    PubMed Central

    Zhang, Hongjie; Kim, Ahlee; Abraham, Nessy; Khan, Liakot A.; Hall, David H.; Fleming, John T.; Gobel, Verena

    2012-01-01

    Clathrin coats vesicles in all eukaryotic cells and has a well-defined role in endocytosis, moving molecules away from the plasma membrane. Its function on routes towards the plasma membrane was only recently appreciated and is thought to be limited to basolateral transport. Here, an unbiased RNAi-based tubulogenesis screen identifies a role of clathrin (CHC-1) and its AP-1 adaptor in apical polarity during de novo lumenal membrane biogenesis in the C. elegans intestine. We show that CHC-1/AP-1-mediated polarized transport intersects with a sphingolipid-dependent apical sorting process. Depleting each presumed trafficking component mislocalizes the same set of apical membrane molecules basolaterally, including the polarity regulator PAR-6, and generates ectopic lateral lumens. GFP::CHC-1 and BODIPY-ceramide vesicles associate perinuclearly and assemble asymmetrically at polarized plasma membrane domains in a co-dependent and AP-1-dependent manner. Based on these findings, we propose a trafficking pathway for apical membrane polarity and lumen morphogenesis that implies: (1) a clathrin/AP-1 function on an apically directed transport route; and (2) the convergence of this route with a sphingolipid-dependent apical trafficking path. PMID:22535410

  3. In vitro host erythrocyte specificity and differential morphology of Babesia divergens and a zoonotic Babesia sp. from eastern cottontail rabbits (Sylvilagus floridanus).

    PubMed

    Spencer, Angela M; Goethert, Heidi K; Telford, Samuel R; Holman, Patricia J

    2006-04-01

    A Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus floridanus) is morphologically similar and genetically identical, based on SSU rRNA gene comparisons, to 2 agents responsible for human babesiosis in the United States. This zoonotic agent is closely related to the European parasite, Babesia divergens. The 2 organisms were characterized by in vitro comparisons. In vitro growth of the rabbit Babesia sp. was supported in human and cottontail rabbit erythrocytes, but not in bovine cells. Babesia divergens was supported in vitro in bovine and human erythrocytes, but not in cottontail rabbit cells. Morphometric analysis classifies B. divergens as a small babesia in bovine erythrocytes, but the parasite exceeds this size in human erythrocytes. The rabbit Babesia sp. is large, the same size in both human or rabbit erythrocytes, and is significantly larger than B. divergens. Eight or more rabbit Babesia sp. parasites may occur within a single erythrocyte, sometimes in a floret array, unlike B. divergens. The erythrocyte specificity and morphological differences reported in this study agree with previous in vivo results and validate the use of in vitro methods for characterization of Babesia species. PMID:16729690

  4. Apical infection spreading to adjacent teeth: a case report.

    PubMed

    Komabayashi, Takashi; Jiang, Jin; Zhu, Qiang

    2011-06-01

    This case report describes apical infection on tooth number 24 that spread to adjacent teeth, resulting in devitalized teeth numbers 23 and 25. The 25-year-old Caucasian female patient was referred to the endodontic resident clinic because of uncontrolled apical infection. Root-end surgery and root-end filling of teeth numbers 23, 24, and 25 were performed. The histopathological diagnosis was a periapical cyst; however, the clinical surgical finding of a purulence-filled bone cavity also revealed a periapical abscess. After root-end surgery and regenerative therapy using Mineral Trioxide Aggregate, Bio-Oss xenograft material, and Bio-Gide resorbable collagen membrane, the patient had no symptoms. Radiographs showed the apical lesion had healed satisfactorily at the 6-month, 1-year, and 2-year follow-ups. The clinical implication of this rare case suggests the importance of standard endodontic diagnostic procedures for pulpal and apical diagnosis, prevention of apical periodontitis exacerbation by reducing bacterial factors, and the effectiveness of healing large bone defects using regenerative materials. PMID:21458327

  5. Tubular endocytosis drives remodelling of the apical surface during epithelial morphogenesis in Drosophila.

    PubMed

    Fabrowski, Piotr; Necakov, Aleksandar S; Mumbauer, Simone; Loeser, Eva; Reversi, Alessandra; Streichan, Sebastian; Briggs, John A G; De Renzis, Stefano

    2013-01-01

    During morphogenesis, remodelling of cell shape requires the expansion or contraction of plasma membrane domains. Here we identify a mechanism underlying the restructuring of the apical surface during epithelial morphogenesis in Drosophila. We show that the retraction of villous protrusions and subsequent apical plasma membrane flattening is an endocytosis-driven morphogenetic process. Quantitation of endogenously tagged GFP::Rab5 dynamics reveals a massive increase in apical endocytosis that correlates with changes in apical morphology. This increase is accompanied by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of Rab5-positive endosomes. We identify the Rab5-effector Rabankyrin-5 as a regulator of this pathway and demonstrate that blocking dynamin activity results in the complete inhibition of tubular endocytosis, in the disappearance of Rab5 endosomes, and in the inhibition of surface flattening. These data collectively demonstrate a requirement for endocytosis in morphogenetic remodelling during epithelial development. PMID:23921440

  6. Performance of a satellite-linked GPS on Pacific walruses (Odobenus rosmarus divergens)

    USGS Publications Warehouse

    Jay, C.V.; Garner, G.W.

    2002-01-01

    We evaluated the utility of a satellite-linked GPS in obtaining location data from Pacific walruses (Odobenus rosmarus divergens). A unit was attached to one of the tusks of each of three adult male walruses in Bristol Bay, Alaska. The units were designed to relay GPS positions through the Argos Data Collection and Location System. The GPS was only minimally effective in obtaining location data. An average of only 5% of the attempts yielded a position, and only a small number of these were locations at sea. The paucity of successful attempts was probably due to infrequent and brief surfacings of the GPS, the proximity of cliffs to predominant haul-out sites in the study region, and the packing of animals when they were hauled out in herds. Argos was effective in relaying GPS positions in this study, but as GPS technology advances, and its application to marine mammal studies becomes more prevalent, it seems that the greatest challenge to the study of many species will be in data retrieval.

  7. Copper directs ATP7B to the apical domain of hepatic cells via basolateral endosomes.

    PubMed

    Nyasae, Lydia K; Schell, Michael J; Hubbard, Ann L

    2014-12-01

    Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu-directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF-B and in the liver in vivo. Copper (10 µm) caused ATP7B to exit the trans-Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton-pump inhibitor bafilomycin-A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu-stimulated) case. Overall, loss of acidification-impaired Cu-regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane. PMID:25243755

  8. Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells

    PubMed Central

    Klingner, Christoph; Cherian, Anoop V.; Fels, Johannes; Diesinger, Philipp M.; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M.; Bathe, Mark

    2014-01-01

    Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin–Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. PMID:25313407

  9. Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent.

    PubMed

    Worrell, Roger T; Best, Alison; Crawford, Oscar R; Xu, Jie; Soleimani, Manoocher; Matthews, Jeffrey B

    2005-10-01

    Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). Current inhibition was not simply due to an increase in extracellular K(+)-like cations, in that the current magnitude was 95 +/- 5% with 10 mM apical K(+) and 46 +/- 3% with 10 mM apical NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is

  10. Sex-specific energetics of Pacific walruses (Odobenus rosmarus divergens) during the nursing interval

    USGS Publications Warehouse

    Noren, Shawn R.; Udevitz, Mark S.; Jay, Chadwick V.

    2016-01-01

    Habitat use and activity patterns of Pacific walruses (Odobenus rosmarus divergens) have changed with climate-induced reductions in sea ice. Increases in the time active in water could result in negative energy balance, precluding females from sustaining lactation, which could impact population demographics. Little is known about lactation costs in walruses. We examined the energetics of 0–2-yr-old walrus calves by using Bayesian hierarchical models based on longitudinal husbandry records of growth (n = 6 females and 7 males) and caloric intake (n = 5 females and 6 males) as a proxy for maternal lactation costs. Males and females had similar growth patterns; mean mass increased from 68 kg at birth to 301 kg by 2 yr. Females had a 2,000 kcal kg−1 higher mass storage (growth) cost than males; females typically synthesize and deposit greater amounts of adipose, which is more energy dense than lean tissue. In contrast, males had higher metabolic (basal and activity) costs, ranging from 600 to 1,800 kcal d−1 greater than similarly sized females; males are typically leaner, and muscle is more metabolically active than adipose. Yet total daily energy requirements (storage plus metabolic components) were similar across sexes, summing to approximately 190,000 kcal over the first month postpartum. Based on these estimates and assuming that 8,103 kcal is recovered from 1 kg of mass loss in adult female walruses, suckling calves could deplete 23 kg of their mother’s body mass over the first month after parturition if none of the lactation costs is met through ingested prey.

  11. Sex-Specific Energetics of Pacific Walruses (Odobenus rosmarus divergens) during the Nursing Interval.

    PubMed

    Noren, Shawn R; Udevitz, Mark S; Jay, Chadwick V

    2016-01-01

    Habitat use and activity patterns of Pacific walruses (Odobenus rosmarus divergens) have changed with climate-induced reductions in sea ice. Increases in the time active in water could result in negative energy balance, precluding females from sustaining lactation, which could impact population demographics. Little is known about lactation costs in walruses. We examined the energetics of 0-2-yr-old walrus calves by using Bayesian hierarchical models based on longitudinal husbandry records of growth (n = 6 females and 7 males) and caloric intake (n = 5 females and 6 males) as a proxy for maternal lactation costs. Males and females had similar growth patterns; mean mass increased from 68 kg at birth to 301 kg by 2 yr. Females had a 2,000 kcal kg(-1) higher mass storage (growth) cost than males; females typically synthesize and deposit greater amounts of adipose, which is more energy dense than lean tissue. In contrast, males had higher metabolic (basal and activity) costs, ranging from 600 to 1,800 kcal d(-1) greater than similarly sized females; males are typically leaner, and muscle is more metabolically active than adipose. Yet total daily energy requirements (storage plus metabolic components) were similar across sexes, summing to approximately 190,000 kcal over the first month postpartum. Based on these estimates and assuming that 8,103 kcal is recovered from 1 kg of mass loss in adult female walruses, suckling calves could deplete 23 kg of their mother's body mass over the first month after parturition if none of the lactation costs is met through ingested prey. PMID:27082720

  12. Energy demands for maintenance, growth, pregnancy, and lactation of female Pacific walruses (Odobenus rosmarus divergens).

    PubMed

    Noren, Shawn R; Udevitz, Mark S; Jay, Chadwick V

    2014-01-01

    Decreases in sea ice have altered habitat use and activity patterns of female Pacific walruses Odobenus rosmarus divergens and could affect their energetic demands, reproductive success, and population status. However, a lack of physiological data from walruses has hampered efforts to develop the bioenergetics models required for fully understanding potential population-level impacts. We analyzed long-term longitudinal data sets of caloric consumption and body mass from nine female Pacific walruses housed at six aquaria using a hierarchical Bayesian approach to quantify relative energetic demands for maintenance, growth, pregnancy, and lactation. By examining body mass fluctuations in response to food consumption, the model explicitly uncoupled caloric demand from caloric intake. This is important for pinnipeds because they sequester and deplete large quantities of lipids throughout their lifetimes. Model outputs were scaled to account for activity levels typical of free-ranging Pacific walruses, averaging 83% of the time active in water and 17% of the time hauled-out resting. Estimated caloric requirements ranged from 26,900 kcal d(-1) for 2-yr-olds to 93,370 kcal d(-1) for simultaneously lactating and pregnant walruses. Daily consumption requirements were higher for pregnancy than lactation, reflecting energetic demands of increasing body size and lipid deposition during pregnancy. Although walruses forage during lactation, fat sequestered during pregnancy sustained 27% of caloric requirements during the first month of lactation, suggesting that walruses use a mixed strategy of capital and income breeding. Ultimately, this model will aid in our understanding of the energetic and population consequences of sea ice loss. PMID:25461648

  13. Energy demands for maintenance, growth, pregnancy, and lactation of female Pacific walruses (Odobenus rosmarus divergens)

    USGS Publications Warehouse

    Noren, Shawn R.; Udevitz, Mark S.; Jay, Chadwick V.

    2014-01-01

    Decreases in sea ice have altered habitat use and activity patterns of female Pacific walruses Odobenus rosmarus divergens and could affect their energetic demands, reproductive success, and population status. However, a lack of physiological data from walruses has hampered efforts to develop the bioenergetics models required for fully understanding potential population-level impacts. We analyzed long-term longitudinal data sets of caloric consumption and body mass from nine female Pacific walruses housed at six aquaria using a hierarchical Bayesian approach to quantify relative energetic demands for maintenance, growth, pregnancy, and lactation. By examining body mass fluctuations in response to food consumption, the model explicitly uncoupled caloric demand from caloric intake. This is important for pinnipeds because they sequester and deplete large quantities of lipids throughout their lifetimes. Model outputs were scaled to account for activity levels typical of free-ranging Pacific walruses, averaging 83% of the time active in water and 17% of the time hauled-out resting. Estimated caloric requirements ranged from 26,900 kcal d−1 for 2-yr-olds to 93,370 kcal d−1 for simultaneously lactating and pregnant walruses. Daily consumption requirements were higher for pregnancy than lactation, reflecting energetic demands of increasing body size and lipid deposition during pregnancy. Although walruses forage during lactation, fat sequestered during pregnancy sustained 27% of caloric requirements during the first month of lactation, suggesting that walruses use a mixed strategy of capital and income breeding. Ultimately, this model will aid in our understanding of the energetic and population consequences of sea ice loss.

  14. Bioenergetics model for estimating food requirements of female Pacific walruses (Odobenus rosmarus divergens)

    USGS Publications Warehouse

    Noren, S.R.; Udevitz, M.S.; Jay, C.V.

    2012-01-01

    Pacific walruses Odobenus rosmarus divergens use sea ice as a platform for resting, nursing, and accessing extensive benthic foraging grounds. The extent of summer sea ice in the Chukchi Sea has decreased substantially in recent decades, causing walruses to alter habitat use and activity patterns which could affect their energy requirements. We developed a bioenergetics model to estimate caloric demand of female walruses, accounting for maintenance, growth, activity (active in-water and hauled-out resting), molt, and reproductive costs. Estimates for non-reproductive females 0–12 yr old (65−810 kg) ranged from 16359 to 68960 kcal d−1 (74−257 kcal d−1 kg−1) for years with readily available sea ice for which we assumed animals spent 83% of their time in water. This translated into the energy content of 3200–5960 clams per day, equivalent to 7–8% and 14–9% of body mass per day for 5–12 and 2–4 yr olds, respectively. Estimated consumption rates of 12 yr old females were minimally affected by pregnancy, but lactation had a large impact, increasing consumption rates to 15% of body mass per day. Increasing the proportion of time in water to 93%, as might happen if walruses were required to spend more time foraging during ice-free periods, increased daily caloric demand by 6–7% for non-lactating females. We provide the first bioenergetics-based estimates of energy requirements for walruses and a first step towards establishing bioenergetic linkages between demography and prey requirements that can ultimately be used in predicting this population’s response to environmental change.

  15. Projected status of the Pacific walrus (Odobenus rosmarus divergens) in the twenty-first century

    USGS Publications Warehouse

    Jay, Chadwick V.; Marcot, Bruce G.; Douglas, David C.

    2011-01-01

    Extensive and rapid losses of sea ice in the Arctic have raised conservation concerns for the Pacific walrus (Odobenus rosmarus divergens), a large pinniped inhabiting arctic and subarctic continental shelf waters of the Chukchi and Bering seas. We developed a Bayesian network model to integrate potential effects of changing environmental conditions and anthropogenic stressors on the future status of the Pacific walrus population at four periods through the twenty-first century. The model framework allowed for inclusion of various sources and levels of knowledge, and representation of structural and parameter uncertainties. Walrus outcome probabilities through the century reflected a clear trend of worsening conditions for the subspecies. From the current observation period to the end of century, the greatest change in walrus outcome probabilities was a progressive decrease in the outcome state of robust and a concomitant increase in the outcome state of vulnerable. The probabilities of rare and extirpated states each progressively increased but remained <10% through the end of the century. The summed probabilities of vulnerable, rare, and extirpated (P(v,r,e)) increased from a current level of 10% in 2004 to 22% by 2050 and 40% by 2095. The degree of uncertainty in walrus outcomes increased monotonically over future periods. In the model, sea ice habitat (particularly for summer/fall) and harvest levels had the greatest influence on future population outcomes. Other potential stressors had much smaller influences on walrus outcomes, mostly because of uncertainty in their future states and our current poor understanding of their mechanistic influence on walrus abundance.

  16. Projected status of the Pacific walrus (Odobenus rosmarus divergens) in the twenty-first century

    USGS Publications Warehouse

    Jay, C.V.; Marcot, B.G.; Douglas, D.C.

    2011-01-01

    Extensive and rapid losses of sea ice in the Arctic have raised conservation concerns for the Pacific walrus (Odobenus rosmarus divergens), a large pinniped inhabiting arctic and subarctic continental shelf waters of the Chukchi and Bering seas. We developed a Bayesian network model to integrate potential effects of changing environmental conditions and anthropogenic stressors on the future status of the Pacific walrus population at four periods through the twenty-first century. The model framework allowed for inclusion of various sources and levels of knowledge, and representation of structural and parameter uncertainties. Walrus outcome probabilities through the century reflected a clear trend of worsening conditions for the subspecies. From the current observation period to the end of century, the greatest change in walrus outcome probabilities was a progressive decrease in the outcome state of robust and a concomitant increase in the outcome state of vulnerable. The probabilities of rare and extirpated states each progressively increased but remained >10% through the end of the century. The summed probabilities of vulnerable, rare, and extirpated (P(v,r,e)) increased from a current level of 10% in 2004 to 22% by 2050 and 40% by 2095. The degree of uncertainty in walrus outcomes increased monotonically over future periods. In the model, sea ice habitat (particularly for summer/fall) and harvest levels had the greatest influence on future population outcomes. Other potential stressors had much smaller influences on walrus outcomes, mostly because of uncertainty in their future states and our current poor understanding of their mechanistic influence on walrus abundance. ?? 2011 US Government.

  17. The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells.

    PubMed

    Perez Bay, Andres E; Schreiner, Ryan; Benedicto, Ignacio; Paz Marzolo, Maria; Banfelder, Jason; Weinstein, Alan M; Rodriguez-Boulan, Enrique J

    2016-01-01

    The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station. PMID:27180806

  18. The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells

    PubMed Central

    Perez Bay, Andres E.; Schreiner, Ryan; Benedicto, Ignacio; Paz Marzolo, Maria; Banfelder, Jason; Weinstein, Alan M.; Rodriguez-Boulan, Enrique J.

    2016-01-01

    The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station. PMID:27180806

  19. aPKC regulates apical localization of Lgl to restrict elongation of microridges in developing zebrafish epidermis

    PubMed Central

    Raman, Renuka; Damle, Indraneel; Rote, Rahul; Banerjee, Shamik; Dingare, Chaitanya; Sonawane, Mahendra

    2016-01-01

    Epithelial cells exhibit apical membrane protrusions, which confer specific functions to epithelial tissues. Microridges are short actin protrusions that are laterally long and form a maze-like pattern in the apical domain. They are widely found on vertebrate squamous epithelia including epidermis and have functions in mucous retention, membrane storage and abrasion resistance. It is largely unknown how the formation of these laterally long actin projections is regulated. Here, we show that antagonistic interactions between aPKC and Lgl–regulators of apical and basolateral domain identity, respectively,–control the length of microridges in the zebrafish periderm, the outermost layer of the epidermis. aPKC regulates the levels of Lgl and the active form of non-muscle myosinII at the apical cortex to prevent actin polymerization-dependent precocious fusion and elongation of microridges. Our data unravels the functional significance of exclusion of Lgl from the apical domain in epithelial cells. PMID:27249668

  20. aPKC regulates apical localization of Lgl to restrict elongation of microridges in developing zebrafish epidermis.

    PubMed

    Raman, Renuka; Damle, Indraneel; Rote, Rahul; Banerjee, Shamik; Dingare, Chaitanya; Sonawane, Mahendra

    2016-01-01

    Epithelial cells exhibit apical membrane protrusions, which confer specific functions to epithelial tissues. Microridges are short actin protrusions that are laterally long and form a maze-like pattern in the apical domain. They are widely found on vertebrate squamous epithelia including epidermis and have functions in mucous retention, membrane storage and abrasion resistance. It is largely unknown how the formation of these laterally long actin projections is regulated. Here, we show that antagonistic interactions between aPKC and Lgl-regulators of apical and basolateral domain identity, respectively,-control the length of microridges in the zebrafish periderm, the outermost layer of the epidermis. aPKC regulates the levels of Lgl and the active form of non-muscle myosinII at the apical cortex to prevent actin polymerization-dependent precocious fusion and elongation of microridges. Our data unravels the functional significance of exclusion of Lgl from the apical domain in epithelial cells. PMID:27249668

  1. Apical Ca2+-activated potassium channels in mouse parotid acinar cells

    PubMed Central

    Almassy, Janos; Won, Jong Hak; Begenisich, Ted B.

    2012-01-01

    Ca2+ activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca2+] was used to investigate if Ca2+-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca2+-buffering conditions that produced brief, localized increases in [Ca2+] after focal laser photolysis of caged Ca2+. Conditions were used to isolate K+ and Cl− conductances. Photolysis at the apical PM resulted in a robust increase in K+ and Cl− currents. A localized reduction in [Ca2+] at the apical PM after photolysis of Diazo-2, a caged Ca2+ chelator, resulted in a decrease in both K+ and Cl− currents. The K+ currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance “maxi-K” (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34–sensitive K+ currents were also observed in BK-null parotid acini. In contrast, when the [Ca2+] was increased at the basal or lateral PM, no increase in either K+ or Cl− currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM. PMID:22291145

  2. Annexin XIIIb Associates with Lipid Microdomains to Function in Apical Delivery

    PubMed Central

    Lafont, Frank; Lecat, Sandra; Verkade, Paul; Simons, Kai

    1998-01-01

    A member of the annexin XIII sub-family, annexin XIIIb, has been implicated in the apical exocytosis of epithelial kidney cells. Annexins are phospholipid-binding proteins that have been suggested to be involved in membrane trafficking events although their actual physiological function remains open. Unlike the other annexins, annexin XIIIs are myristoylated. Here, we show by immunoelectron microscopy that annexin XIIIb is localized to the trans-Golgi network (TGN), vesicular carriers and the apical cell surface. Polarized apical sorting involves clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We now provide evidence for the raft association of annexin XIIIb. Using in vitro assays and either myristoylated or unmyristoylated recombinant annexin XIIIb, we demonstrate that annexin XIIIb in its native myristoylated form stimulates specifically apical transport whereas the unmyristoylated form inhibits this route. Moreover, we show that formation of apical carriers from the TGN is inhibited by an anti-annexin XIIIb antibody whereas it is stimulated by myristoylated recombinant annexin XIIIb. These results suggest that annexin XIIIb directly participates in apical delivery. PMID:9744874

  3. Pak1 Regulates the Orientation of Apical Polarization and Lumen Formation by Distinct Pathways

    PubMed Central

    Smits, Jos; ter Beest, Martin B.; Zegers, Mirjam M.

    2012-01-01

    The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways. PMID:22815903

  4. Apical localization of the coxsackie-adenovirus receptor by glycosyl-phosphatidylinositol modification is sufficient for adenovirus-mediated gene transfer through the apical surface of human airway epithelia.

    PubMed

    Walters, R W; van't Hof, W; Yi, S M; Schroth, M K; Zabner, J; Crystal, R G; Welsh, M J

    2001-08-01

    In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl(-) transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis. PMID:11462042

  5. Apical Localization of the Coxsackie-Adenovirus Receptor by Glycosyl-Phosphatidylinositol Modification Is Sufficient for Adenovirus-Mediated Gene Transfer through the Apical Surface of Human Airway Epithelia

    PubMed Central

    Walters, Robert W.; van't Hof, Wouter; Yi, Su Min P.; Schroth, Mary K.; Zabner, Joseph; Crystal, Ronald G.; Welsh, Michael J.

    2001-01-01

    In well-differentiated human airway epithelia, the coxsackie B and adenovirus type 2 and 5 receptor (CAR) resides primarily on the basolateral membrane. This location may explain the observation that gene transfer is inefficient when adenovirus vectors are applied to the apical surface. To further test this hypothesis and to investigate requirements and barriers to apical gene transfer to differentiated human airway epithelia, we expressed CAR in which the transmembrane and cytoplasmic tail were replaced by a glycosyl-phosphatidylinositol (GPI) anchor (GPI-CAR). As controls, we expressed wild-type CAR and CAR lacking the cytoplasmic domain (Tailless-CAR). All three constructs enhanced gene transfer with similar efficiencies in fibroblasts. In airway epithelia, GPI-CAR localized specifically to the apical membrane, where it bound adenovirus and enhanced gene transfer to levels obtained when vector was applied to the basolateral membrane. Moreover, GPI-CAR facilitated gene transfer of the cystic fibrosis transmembrane conductance regulator to cystic fibrosis airway epithelia, correcting the Cl− transport defect. In contrast, when we expressed wild-type CAR it localized to the basolateral membrane and failed to increase apical gene transfer. Only a small amount of Tailless-CAR resided in the apical membrane, and the effects on apical virus binding and gene transfer were minimal. These data indicate that binding of adenovirus to an apical membrane receptor is sufficient to mediate effective gene transfer to human airway epithelia and that the cytoplasmic domain of CAR is not required for this process. The results suggest that targeting apical receptors in differentiated airway epithelia may be sufficient for gene transfer in the genetic disease cystic fibrosis. PMID:11462042

  6. Fast and slow voltage modulation of apical Cl- permeability in toad skin at high [K+].

    PubMed

    Procopio, J

    1997-08-01

    The influence of voltage on the conductance of toad skin was studied to identify the time course of the activation/deactivation dynamics of voltage-dependent Cl- channels located in the apical membrane of mitochondrion-rich cells in this tissue. Positive apical voltage induced an important conductance inhibition which took a few seconds to fully develop and was instantaneously released by pulse inversion to negative voltage, indicating a short-duration memory of the inhibiting factors. Sinusoidal stimulation at 23.4 mM [Cl-] showed hysteresis in the current versus voltage curves, even at very low frequency, suggesting that the rate of voltage application was also relevant for the inhibition/releasing effect to develop. We conclude that the voltage modulation of apical Cl- permeability is essentially a fast process and the apparent slow components of activation/deactivation obtained in the whole skin are a consequence of a gradual voltage build-up across the apical membrane due to voltage sharing between apical and basolateral membranes. PMID:9361735

  7. Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells

    PubMed Central

    Wankel, Bret; Ouyang, Jiangyong; Guo, Xuemei; Hadjiolova, Krassimira; Miller, Jeremy; Liao, Yi; Tham, Daniel Kai Long; Romih, Rok; Andrade, Leonardo R.; Gumper, Iwona; Simon, Jean-Pierre; Sachdeva, Rakhee; Tolmachova, Tanya; Seabra, Miguel C.; Fukuda, Mitsunori; Schaeren-Wiemers, Nicole; Hong, Wan Jin; Sabatini, David D.; Wu, Xue-Ru; Kong, Xiangpeng; Kreibich, Gert; Rindler, Michael J.; Sun, Tung-Tien

    2016-01-01

    Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV–membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking. PMID:27009205

  8. Apical ABC transporters and cancer chemotherapeutic drug disposition.

    PubMed

    Durmus, Selvi; Hendrikx, Jeroen J M A; Schinkel, Alfred H

    2015-01-01

    ATP-binding cassette (ABC) transporters are transmembrane efflux transporters that mediate cellular extrusion of a broad range of substrates ranging from amino acids, lipids, and ions to xenobiotics including many anticancer drugs. ABCB1 (P-GP) and ABCG2 (BCRP) are the most extensively studied apical ABC drug efflux transporters. They are highly expressed in apical membranes of many pharmacokinetically relevant tissues such as epithelial cells of the small intestine and endothelial cells of the blood capillaries in brain and testis, and in the placental maternal-fetal barrier. In these tissues, they have a protective function as they efflux their substrates back to the intestinal lumen or blood and thus restrict the intestinal uptake and tissue disposition of many compounds. This presents a major challenge for the use of many (anticancer) drugs, as most currently used anticancer drugs are substrates of these transporters. Herein, we review the latest findings on the role of apical ABC transporters in the disposition of anticancer drugs. We discuss that many new, rationally designed anticancer drugs are substrates of these transporters and that their oral availability and/or brain disposition are affected by this interaction. We also summarize studies that investigate the improvement of oral availability and brain disposition of many cytotoxic (e.g., taxanes) and rationally designed (e.g., tyrosine kinase inhibitor) anticancer drugs, using chemical inhibitors of these transporters. These findings provide a better understanding of the importance of apical ABC transporters in chemotherapy and may therefore advance translation of promising preclinical insights and approaches to clinical studies. PMID:25640265

  9. Surgical treatments for vaginal apical prolapse.

    PubMed

    Kong, Mi Kyung; Bai, Sang Wook

    2016-07-01

    Pelvic organ prolapse is a common condition, occurring in up to 11% of women in the United States. Often, pelvic organ prolapse recurs after surgery; when it recurs after hysterectomy, it frequently presents as vaginal apical prolapse. There are many different surgical treatments for vaginal apical prolapse; among them, abdominal sacral colpopexy is considered the gold standard. However, recent data reveal that other surgical procedures also result in good outcome. This review discusses the various surgical treatments for vaginal apical prolapse including their risks and benefits. PMID:27462591

  10. Surgical treatments for vaginal apical prolapse

    PubMed Central

    Kong, Mi Kyung

    2016-01-01

    Pelvic organ prolapse is a common condition, occurring in up to 11% of women in the United States. Often, pelvic organ prolapse recurs after surgery; when it recurs after hysterectomy, it frequently presents as vaginal apical prolapse. There are many different surgical treatments for vaginal apical prolapse; among them, abdominal sacral colpopexy is considered the gold standard. However, recent data reveal that other surgical procedures also result in good outcome. This review discusses the various surgical treatments for vaginal apical prolapse including their risks and benefits. PMID:27462591

  11. Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

    PubMed

    Seiler, C; Nicolson, T

    1999-11-15

    Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. PMID:10526320

  12. Apical Na+ permeability of frog skin during serosal Cl- replacement.

    PubMed

    Leibowich, S; DeLong, J; Civan, M M

    1988-05-01

    Gluconate substitution for serosal Cl- reduces the transepithelial short-circuit current (Isc) and depolarizes short-circuited frog skins. These effects could result either from inhibition of basolateral K+ conductance, or from two actions to inhibit both apical Na+ permeability (PapNa) and basolateral pump activity. We have addressed this question by studying whole-and split-thickness frog skins. Intracellular Na+ concentration (CcNa) and PapNa have been monitored by measuring the current-voltage relationship for apical Na+ entry. This analysis was conducted by applying trains of voltage pulses, with pulse durations of 16 to 32 msec. Estimates of PapNa and CcNa were not detectably dependent on pulse duration over the range 16 to 80 msec. Serosal Cl- replacement uniformly depolarized short-circuited tissues. The depolarization was associated with inhibition of Isc across each split skin, but only occasionally across the whole-thickness preparations. This difference may reflect the better ionic exchange between the bulk medium and the extracellular fluid in contact with the basolateral membranes, following removal of the underlying dermis in the split-skin preparations. PapNa was either unchanged or increased, and CcNa either unchanged or reduced after the anionic replacement. These data are incompatible with the concept that serosal Cl- replacement inhibits PapNa and Na,K-pump activity. Gluconate substitution likely reduces cell volume, triggering inhibition of the basolateral K+ channels, consistent with the data and conclusions of S.A. Lewis, A.G. Butt, M.J. Bowler, J.P. Leader and A.D.C. Macknight (J. Membrane Biol. 83:119-137, 1985) for toad bladder. The resulting depolarization reduces the electrical force favoring apical Na+ entry. The volume-conductance coupling serves to conserve volume by reducing K+ solute loss. Its molecular basis remains to be identified. PMID:2458472

  13. EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells.

    PubMed

    Nakajo, Atsuhiro; Yoshimura, Shin-ichiro; Togawa, Hiroko; Kunii, Masataka; Iwano, Tomohiko; Izumi, Ayaka; Noguchi, Yuria; Watanabe, Ayako; Goto, Ayako; Sato, Toshiro; Harada, Akihiro

    2016-02-01

    The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport. PMID:26833786

  14. EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

    PubMed Central

    Nakajo, Atsuhiro; Togawa, Hiroko; Kunii, Masataka; Iwano, Tomohiko; Izumi, Ayaka; Noguchi, Yuria; Watanabe, Ayako; Goto, Ayako; Sato, Toshiro

    2016-01-01

    The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain–binding protein 1–like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1–dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport. PMID:26833786

  15. Activation of an apical Cl- conductance by Ca2+ ionophores in cystic fibrosis airway epithelia.

    PubMed

    Willumsen, N J; Boucher, R C

    1989-02-01

    Cystic fibrosis (CF) airway epithelia express a defect in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of apical membrane Cl- channels. Recent patch-clamp studies have raised the possibility that Ca2+ -dependent mechanisms for the activation of Cl- secretion may be preserved in CF airway epithelia. To determine 1) whether intact normal (N1) and CF airway epithelia exhibit a Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanisms initiate Cl- secretion via activation of an apical membrane Cl- conductance (GCl-), nasal epithelia from N1 and CF subjects were cultured on collagen membranes, and responses to isoproterenol or Ca2- ionophores [A23187 10(-6) M; ionomycin (10(-5)M)] were measured with transepithelial and intracellular techniques. Isoproterenol induced activation of an apical membrane GCl- in N1 cultures but was ineffective in CF. In contrast, in both N1 and CF amiloride-pretreated cultures, A23187 induced an increase in the equivalent short-circuit current that was associated with an activation of an apical membrane Gc1- and was bumetanide inhibitable. A23187 addition during superfusion of the lumen with a low Cl- (3 mM) solution reduced intracellular Cl- activity of CF cells. A Ca2+ ionophore of different selectivity properties, ionomycin, was also an effective Cl- secretagogue in both N1 and CF cultures. We conclude that 1) the A23187 induced Cl- secretion via activation of an apical GCl- in N1 human nasal epithelium, and 2) in contrast to an isoproterenol-dependent path, a Ca2+ -dependent path for GCl- activation is preserved in CF epithelia. PMID:2465689

  16. Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells.

    PubMed

    Beronja, Slobodan; Laprise, Patrick; Papoulas, Ophelia; Pellikka, Milena; Sisson, John; Tepass, Ulrich

    2005-05-23

    Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs. PMID:15897260

  17. Apical root resorption in orthodontically treated adults.

    PubMed

    Baumrind, S; Korn, E L; Boyd, R L

    1996-09-01

    This study analyzed the relationship in orthodontically treated adults between upper central incisor displacement measured on lateral cephalograms and apical root resorption measured on anterior periapical x-ray films. A multiple linear regression examined incisor displacements in four directions (retraction, advancement, intrusion, and extrusion) as independent variables, attempting to account for observed differences in the dependent variable, resorption. Mean apical resorption was 1.36 mm (sd +/- 1.46, n = 73). Mean horizontal displacement of the apex was -0.83 mm (sd +/- 1.74, n = 67); mean vertical displacement was 0.19 mm (sd +/- 1.48, n = 67). The regression coefficients for the intercept and for retraction were highly significant; those for extrusion, intrusion, and advancement were not. At the 95% confidence level, an average of 0.99 mm (se = +/- 0.34) of resorption was implied in the absence of root displacement and an average of 0.49 mm (se = +/- 0.14) of resorption was implied per millimeter of retraction. R2 for all four directional displacement variables (DDVs) taken together was only 0.20, which implied that only a relatively small portion of the observed apical resorption could be accounted for by tooth displacement alone. In a secondary set of univariate analyses, the associations between apical resorption and each of 14 additional treatment-related variables were examined. Only Gender, Elapsed Time, and Total Apical Displacement displayed statistically significant associations with apical resorption. Additional multiple regressions were then performed in which the data for each of these three statistically significant variables were considered separately, with the data for the four directional displacement variables. The addition of information on Elapsed Time or Total Apical Displacement did not explain a significant additional portion of the variability in apical resorption. On the other hand, the addition of information on Gender to the

  18. Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2013-11-01

    Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

  19. Microbiology and treatment of acute apical abscesses.

    PubMed

    Siqueira, José F; Rôças, Isabela N

    2013-04-01

    Acute apical abscess is the most common form of dental abscess and is caused by infection of the root canal of the tooth. It is usually localized intraorally, but in some cases the apical abscess may spread and result in severe complications or even mortality. The reasons why dental root canal infections can become symptomatic and evolve to severe spreading and sometimes life-threatening abscesses remain elusive. Studies using culture and advanced molecular microbiology methods for microbial identification in apical abscesses have demonstrated a multispecies community conspicuously dominated by anaerobic bacteria. Species/phylotypes commonly found in these infections belong to the genera Fusobacterium, Parvimonas, Prevotella, Porphyromonas, Dialister, Streptococcus, and Treponema. Advances in DNA sequencing technologies and computational biology have substantially enhanced the knowledge of the microbiota associated with acute apical abscesses and shed some light on the etiopathogeny of this disease. Species richness and abundance and the resulting network of interactions among community members may affect the collective pathogenicity and contribute to the development of acute infections. Disease modifiers, including transient or permanent host-related factors, may also influence the development and severity of acute abscesses. This review focuses on the current evidence about the etiology and treatment of acute apical abscesses and how the process is influenced by host-related factors and proposes future directions in research, diagnosis, and therapeutic approaches to deal with this disease. PMID:23554416

  20. Microbiology and Treatment of Acute Apical Abscesses

    PubMed Central

    Rôças, Isabela N.

    2013-01-01

    SUMMARY Acute apical abscess is the most common form of dental abscess and is caused by infection of the root canal of the tooth. It is usually localized intraorally, but in some cases the apical abscess may spread and result in severe complications or even mortality. The reasons why dental root canal infections can become symptomatic and evolve to severe spreading and sometimes life-threatening abscesses remain elusive. Studies using culture and advanced molecular microbiology methods for microbial identification in apical abscesses have demonstrated a multispecies community conspicuously dominated by anaerobic bacteria. Species/phylotypes commonly found in these infections belong to the genera Fusobacterium, Parvimonas, Prevotella, Porphyromonas, Dialister, Streptococcus, and Treponema. Advances in DNA sequencing technologies and computational biology have substantially enhanced the knowledge of the microbiota associated with acute apical abscesses and shed some light on the etiopathogeny of this disease. Species richness and abundance and the resulting network of interactions among community members may affect the collective pathogenicity and contribute to the development of acute infections. Disease modifiers, including transient or permanent host-related factors, may also influence the development and severity of acute abscesses. This review focuses on the current evidence about the etiology and treatment of acute apical abscesses and how the process is influenced by host-related factors and proposes future directions in research, diagnosis, and therapeutic approaches to deal with this disease. PMID:23554416

  1. Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes.

    PubMed

    Hansen, Gert H; Pedersen, Jens; Niels-Christiansen, Lise-Lotte; Immerdal, Lissi; Danielsen, E Michael

    2003-07-01

    The brush border of small intestinal enterocytes is highly enriched in cholesterol- and glycosphingolipid-containing membrane microdomains, commonly termed as lipid 'rafts'. Functionally, transcytosis of IgA and exocytosis of newly made brush-border proteins in enterocytes occur through apical lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized by the non-permeable surface marker Ruthenium Red in the brush-border region of the cells. The surface-connected tubules were labelled by antibodies to caveolin-1 and the glycolipid asialo G(M1), and they were sensitive to cholesterol depletion by methyl-beta-cyclodextrin, indicating the presence of raft microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion suggests that deep-apical tubules function as a cell-surface membrane reservoir for cholesterol and for rapid adaptive changes in the size of microvilli at the brush border. PMID:12689332

  2. CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells.

    PubMed

    Toya, Mika; Kobayashi, Saeko; Kawasaki, Miwa; Shioi, Go; Kaneko, Mari; Ishiuchi, Takashi; Misaki, Kazuyo; Meng, Wenxiang; Takeichi, Masatoshi

    2016-01-12

    Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated-spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells. PMID:26715742

  3. Molecular and Parasitological Survey of Bovine Piroplasms in the Black Sea Region, Including the First Report of Babesiosis Associated with Babesia divergens in Turkey.

    PubMed

    Aktas, M; Ozubek, S

    2015-11-01

    Clinical cases of babesiosis were evaluated, and the frequency of bovine Babesia and Theileria parasites was determined in cattle. Blood samples and thin blood smears were collected from 23 cattle exhibiting clinical signs of babesiosis. In addition, tick and blood samples were collected from 100 apparently healthy cattle cograzing from the same area. Egg masses obtained from fully engorged female ticks were included. DNA isolated from blood and tick samples was screened for Babesia and Theileria by reverse line blot assay. Piroplasms compatible with Babesia spp. were observed microscopically for symptomatic cattle as circular, oval, elongated, or pear-shaped bodies. Parasitemia ranged from 0.08 to 0.9% for Babesia bovis, 2.5 to 15.4% for Babesia bigemina, and 7.4% for Babesia divergens. Reverse line blot showed positivity in 13 (13%) of the sampled clinically normal cattle and revealed the presence of three Babesia species. Babesia bovis was the most prevalent (9/100, 9%), followed by Babesia occultans (3/100, 3%) and B. bigemina (1/100, 1%). One animal infected with B. bigemina was also infected with B. bovis. The single animal infected with B. divergens showed symptoms of babesiosis. Ticks were identified as Rhipicephalus annulatus, Rhipicephalus turanicus, and Ixodes ricinus. One female R. annulatus and its egg mass were infected with B. bigemina. Neither Theileria annulata nor Theileria buffeli/orientalis infections were observed in cattle or ticks. This is the first report of clinical babesiosis caused by B. divergens in cattle from Turkey. PMID:26336265

  4. Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation

    PubMed Central

    He, Bing; Doubrovinski, Konstantin; Polyakov, Oleg; Wieschaus, Eric

    2014-01-01

    Epithelial folding mediated by apical constriction converts flat epithelial sheets into multilayered, complex tissue structures and is employed throughout the development in most animals1. Little is known, however, how forces produced near the apical surface of the tissue are transmitted within individual cells to generate the global changes in cell shape that characterize tissue deformation. Here we apply particle tracking velocimetry in gastrulating Drosophila embryos to measure the movement of cytoplasm and plasma membrane during ventral furrow (VF) formation2, 3. We find that cytoplasmic redistribution during the lengthening phase of VF formation can be precisely described by viscous flows that quantitatively match the predictions of hydrodynamics. Cell membranes move with the ambient cytoplasm, with little resistance to or driving force on the flow. Strikingly, apical constriction produces similar flow patterns in mutant embryos that fail to form cells prior to gastrulation (“acellular” embryos), such that the global redistribution of cytoplasm mirrors the summed redistribution occurring in individual cells of wild type embryos. Our results suggest that during the lengthening phase of VF formation, hydrodynamic behavior of the cytoplasm provides the predominant mechanism transmitting apically generated forces deep into the tissue and that cell individualization is dispensable. PMID:24590071

  5. Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation.

    PubMed

    He, Bing; Doubrovinski, Konstantin; Polyakov, Oleg; Wieschaus, Eric

    2014-04-17

    Epithelial folding mediated by apical constriction converts flat epithelial sheets into multilayered, complex tissue structures and is used throughout development in most animals. Little is known, however, about how forces produced near the apical surface of the tissue are transmitted within individual cells to generate the global changes in cell shape that characterize tissue deformation. Here we apply particle tracking velocimetry in gastrulating Drosophila embryos to measure the movement of cytoplasm and plasma membrane during ventral furrow formation. We find that cytoplasmic redistribution during the lengthening phase of ventral furrow formation can be precisely described by viscous flows that quantitatively match the predictions of hydrodynamics. Cell membranes move with the ambient cytoplasm, with little resistance to, or driving force on, the flow. Strikingly, apical constriction produces similar flow patterns in mutant embryos that fail to form cells before gastrulation ('acellular' embryos), such that the global redistribution of cytoplasm mirrors the summed redistribution occurring in individual cells of wild-type embryos. Our results indicate that during the lengthening phase of ventral furrow formation, hydrodynamic behaviour of the cytoplasm provides the predominant mechanism transmitting apically generated forces deep into the tissue and that cell individualization is dispensable. PMID:24590071

  6. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia.

    PubMed

    Walters, R W; Grunst, T; Bergelson, J M; Finberg, R W; Welsh, M J; Zabner, J

    1999-04-01

    Recent identification of two receptors for the adenovirus fiber protein, coxsackie B and adenovirus type 2 and 5 receptor (CAR), and the major histocompatibility complex (MHC) Class I alpha-2 domain allows the molecular basis of adenoviral infection to be investigated. Earlier work has shown that human airway epithelia are resistant to infection by adenovirus. Therefore, we examined the expression and localization of CAR and MHC Class I in an in vitro model of well differentiated, ciliated human airway epithelia. We found that airway epithelia express CAR and MHC Class I. However, neither receptor was present in the apical membrane; instead, both were polarized to the basolateral membrane. These findings explain the relative resistance to adenovirus infection from the apical surface. In contrast, when the virus was applied to the basolateral surface, gene transfer was much more efficient because of an interaction of adenovirus fiber with its receptors. In addition, when the integrity of the tight junctions was transiently disrupted, apically applied adenovirus gained access to the basolateral surface and enhanced gene transfer. These data suggest that the receptors required for efficient infection are not available on the apical surface, and interventions that allow access to the basolateral space where fiber receptors are located increase gene transfer efficiency. PMID:10187807

  7. Mutation Conferring Apical-Targeting Motif on AE1 Exchanger Causes Autosomal Dominant Distal RTA

    PubMed Central

    Fry, Andrew C.; Su, Ya; Yiu, Vivian; Cuthbert, Alan W.; Trachtman, Howard

    2012-01-01

    Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the α-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1. PMID:22518001

  8. Mutation conferring apical-targeting motif on AE1 exchanger causes autosomal dominant distal RTA.

    PubMed

    Fry, Andrew C; Su, Ya; Yiu, Vivian; Cuthbert, Alan W; Trachtman, Howard; Karet Frankl, Fiona E

    2012-07-01

    Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the α-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1. PMID:22518001

  9. Apical Budding of a Recombinant Influenza A Virus Expressing a Hemagglutinin Protein with a Basolateral Localization Signal

    PubMed Central

    Mora, Rosalia; Rodriguez-Boulan, Enrique; Palese, Peter; García-Sastre, Adolfo

    2002-01-01

    Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA. PMID:11884578

  10. Surface area of apical and basolateral plasmalemma of epithelial cells of the ductuli efferentes testis of the rat.

    PubMed

    Wang, S; Jones, R C; Clulow, J

    1994-06-01

    Serial sectioning was used to determine the occurrence of ciliated cells, and a morphological technique was used to estimate the relative and absolute surface areas of apical and basolateral membrane of the epithelial cells lining the ductuli efferentes of the rat. It was found that the ciliated cells constitute 15% of the epithelial cells and occur as groups of mainly 1-3 cells which are distributed at random in the duct epithelium. For the non-ciliated cells it was estimated that the formation of microvilli by the apical membrane increased the surface area of that border by a factor of 37-fold. The average surface density of the basolateral membrane was 76% the surface density of the apical membrane. However, there was a 3-fold increase in surface density along the apical-basal axis of the basolateral plasmalemma. In the Discussion, the ductuli efferentes are compared to their homologue, the proximal tubules of the kidney, in the rates of fluid transport and membrane adaptations of their epithelium. PMID:8062346

  11. Pathogenesis of Apical Periodontitis: a Literature Review

    PubMed Central

    Lodiene, Greta; Maciulskiene, Vita

    2011-01-01

    ABSTRACT Objectives This review article discusses the host response in apical periodontitis with the main focus on cytokines, produced under this pathological condition and contributing to the degradation of periradicular tissues. The pace of research in this field has greatly accelerated in the last decade. Here we provide an analysis of studies published in this area during this period. Material and methods Literature was selected through a search of PubMed electronic database. The keywords used for search were pathogenesis of apical periodontitis cytokines, periapical granuloma cytokines, inflammatory infiltrate apical periodontitis. The search was restricted to English language articles, published from 1999 to December 2010. Additionally, a manual search in the cytokine production, cytokine functions and periapical tissue destruction in the journals and books was performed. Results In total, 97 literature sources were obtained and reviewed. The topics covered in this article include cellular composition of an inflammatory infiltrate in the periapical lesions, mechanisms of the formation of the innate and specific immune response. Studies which investigated cytokine secretion and functions were identified and cellular and molecular interactions in the course of apical periodontitis described. Conclusions The abundance and interactions of various inflammatory and anti-inflammatory molecules can influence and alter the state and progression of the disease. Therefore, periapical inflammatory response offers a model, suited for the study of many facets of pathogenesis, biocompatibility of different materials to periapical tissues and development of novel treatment methods, based on the regulation of cytokines expression PMID:24421998

  12. The Impact of Apical Patency in the Success of Endodontic Treatment of Necrotic Teeth with Apical Periodontitis: A Brief Review

    PubMed Central

    Machado, Ricardo; Ferrari, Carlos Henrique; Back, Eduardo; Comparin, Daniel; Tomazinho, Luiz Fernando; Vansan, Luiz Pascoal

    2016-01-01

    Accumulation of soft tissue or dentinal remnants in the apical region is a common event that can cause blockage of root canals. This event can be avoided if apical patency is performed during the root canal shaping procedures. However, there is no consensus on the role of apical patency in relation to the success of endodontic treatment of necrotic teeth with apical periodontitis. Therefore, the purpose of this paper was to conduct a brief review on the role of apical patency in guaranteeing the success of endodontic treatments of necrotic teeth with apical periodontitis considering two other key points; the root canal anatomy and microbiology. PMID:26843880

  13. Microbispora sp. LGMB259 endophytic actinomycete isolated from Vochysia divergens (Pantanal, Brazil) producing β-carbolines and indoles with biological activity.

    PubMed

    Savi, Daiani C; Shaaban, Khaled A; Vargas, Nathalia; Ponomareva, Larissa V; Possiede, Yvelise M; Thorson, Jon S; Glienke, Chirlei; Rohr, Jürgen

    2015-03-01

    Endophytic actinomycetes encompass bacterial groups that are well known for the production of a diverse range of secondary metabolites. Vochysia divergens is a medicinal plant, common in the "Pantanal" region (Brazil) and was focus of many investigations, but never regarding its community of endophytic symbionts. During a screening program, an endophytic strain isolated from the V. divergens, was investigated for its potential to show biological activity. The strain was characterized as Microbispora sp. LGMB259 by spore morphology and molecular analyze using nucleotide sequence of the 16S rRNA gene. Strain LGMB259 was cultivated in R5A medium producing metabolites with significant antibacterial activity. The strain produced 4 chemically related β-carbolines, and 3 Indoles. Compound 1-vinyl-β-carboline-3-carboxylic acid displayed potent activity against the Gram-positive bacterial strains Micrococcus luteus NRRL B-2618 and Kocuria rosea B-1106, and was highly active against two human cancer cell lines, namely the prostate cancer cell line PC3 and the non-small-cell lung carcinoma cell line A549, with IC50 values of 9.45 and 24.67 µM, respectively. 1-Vinyl-β-carboline-3-carboxylic acid also showed moderate activity against the yeast Saccharomyces cerevisiae ATCC204508, as well as the phytopathogenic fungi Phyllosticta citricarpa LGMB06 and Colletotrichum gloeosporioides FDC83. PMID:25385358

  14. Microbispora sp. LGMB259 Endophytic Actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing β-Carbolines and Indoles with Biological Activity

    PubMed Central

    Savi, Daiani C.; Shaaban, Khaled A.; Vargas, Nathalia; Ponomareva, Larissa V.; Possiede, Yvelise M.; Thorson, Jon S.; Glienke, Chirlei; Rohr, Jürgen

    2014-01-01

    Endophytic actinomycetes encompass bacterial groups that are well known for the production of a diverse range of secondary metabolites. Vochysia divergens is a medicinal plant, common in the “Pantanal” region (Brazil) and was focus of many investigations, but never regarding its community of endophytic symbionts. During a screening program, an endophytic strain isolated from the V. divergens, was investigated for its potential to show biological activity. The strain was characterized as Microbispora sp. LGMB259 by spore morphology and molecular analyze using nucleotide sequence of the 16S rRNA gene. Strain LGMB259 was cultivated in R5A medium producing metabolites with significant antibacterial activity. The strain produced 4 chemically related β-carbolines, and 3 Indoles. Compound 1-Vinyl-β-carboline-3-carboxylic acid displayed potent activity against the Gram-positive bacterial strains Micrococcus luteus NRRL B-2618 and Kocuria rosea B-1106, and was highly active against two human cancer cell lines, namely the prostate cancer cell line PC3 and the non-small-cell lung carcinoma cell line A549, with IC50 values of 9.45 and 24.67 µM, respectively. 1-Vinyl-β-carboline-3-carboxylic acid also showed moderate activity against the yeast Saccharomyces cerevisiae ATCC204508, as well as the phytopathogenic fungiPhyllosticta citricarpa LGMB06 and Colletotrichum gloeosporioides FDC83. PMID:25385358

  15. Characterization of apical potassium channels induced in rat distal colon during potassium adaptation.

    PubMed Central

    Butterfield, I; Warhurst, G; Jones, M N; Sandle, G I

    1997-01-01

    1. Chronic dietary K+ loading stimulates an active K+ secretory process in rat distal colon, which involves an increase in the macroscopic apical K+ conductance of surface epithelial cells. In the present study, the abundance and characteristics of K+ channels constituting this enhanced apical K+ conductance were evaluated using patch clamp recording techniques. 2. In isolated non-polarized surface cells, K+ channels were seen in 9 of 90 (10%) cell-attached patches in cells from control animals, and in 247 of 437 (57%) cell-attached patches in cells from K(+)-loaded animals, with a significant (P < 0.001) shift in distribution density. Similarly, recordings from cell-attached patches of the apical membrane of surface cells surrounding the openings of distal colonic crypts revealed identical K+ channels in 1 of 11 (9%) patches in control animals, and in 9 of 13 (69%) patches in K(+)-loaded animals. 3. In isolated surface cells and surface cells in situ, K+ channels had mean slope conductances of 209 +/- 6 and 233 +/- 14 pS, respectively, when inside-out patches were bathed symmetrically in K2SO4 solution. The channels were sensitive to 'cytosolic' Ca2+ concentration, were voltage sensitive at 'cytosolic' Ca2+ concentrations encountered in colonic epithelial cells, and were inhibited by 1 mM quinidine, 20 mM TEA or 5 mM Ba2+ ions. 4. The data show that dietary K+ loading increases the abundance of Ca(2+)- and voltage-sensitive large-conductance K+ channels in the apical membrane of surface cells in rat distal colon. These channels constitute the enhanced macroscopic apical K+ conductance previously identified in these cells, and are likely to play a critical role in the active K+ secretory process that typifies this model of colonic K+ adaptation. PMID:9218214

  16. Light sheet microscopy for tracking single molecules on the apical surface of living cells.

    PubMed

    Li, Yu; Hu, Ying; Cang, Hu

    2013-12-12

    Single particle tracking is a powerful tool to study single molecule dynamics in living biological samples. However, current tracking techniques, which are based mainly on epifluorescence, confocal, or TIRF microscopy, have difficulties in tracking single molecules on the apical surface of a cell. We present here a three-dimensional (3D) single particle tracking technique that is based on prism coupled light-sheet microscopy (PCLSM). This novel design provides a signal-to-noise ratio comparable to confocal microscopy while it has the capability of illuminating at arbitrary depth. We demonstrate tracking of single EGF molcules on the apical surface of live cell membranes from their binding to EGF receptors until they are internalized or photobleached. We found that EGF exhibits multiple diffusion behaviors on live A549 cell membranes. At room temperature, the average diffusion coefficient of EGF on A549 cells was measured to be 0.13 μm(2)/s. Depletion of cellular cholesterol with methyl-β-cyclodextrin leads to a broader distribution of diffusion coefficients and an increase of the average diffusion coefficient at room temperature. This light-sheet based 3D single particle tracking technique solves the technique difficulty of tracking single particles on apical membranes and is able to document the whole "lifetime" of a particle from binding till photobleaching or internalization. PMID:23895420

  17. Junctionally restricted RhoA activity is necessary for apical constriction during phase 2 inner ear placode invagination.

    PubMed

    Sai, Xiaorei; Yonemura, Shigenobu; Ladher, Raj K

    2014-10-15

    After induction, the inner ear is transformed from a superficially located otic placode into an epithelial vesicle embedded in the mesenchyme of the head. Invagination of this epithelium is biphasic: phase 1 involves the expansion of the basal aspect of the otic cells, and phase 2, the constriction of their apices. Apical constriction is important not only for otic invagination, but also the invagination of many other epithelia; however, its molecular basis is still poorly understood. Here we show that phase 2 otic morphogenesis, like phase 1 morphogenesis, results from the activation of myosin-II. However unlike the actin depolymerising activity observed basally, active myosin-II results in actomyosin contractility. Myosin-II activation is triggered by the accumulation of the planar cell polarity (PCP) core protein, Celsr1 in apical junctions (AJ). Apically polarized Celsr1 orients and recruits the Rho Guanine exchange factor (GEF) ArhGEF11 to apical junctions, thus restricting RhoA activity to the junctional membrane where it activates the Rho kinase ROCK. We suggest that myosin-II and RhoA activation results in actomyosin dependent constriction in an apically polarised manner driving otic epithelium invagination. PMID:25173873

  18. [The technology of apical infection control].

    PubMed

    Qing, Yu; Yang, Yang; Bei, Chang

    2014-10-01

    Root canal therapy is the most efficient way to treat pulptitis and periapical inflammation, which can clear infections of root canal systems, fill the root canal firmly, and avoid reinfection. However, the variations in root canal morphology and complexity of infection confer difficulty in thoroughly eliminating microorganisms and their by-products in the root canal system, especially in the root apex area (including the top one-third of the root canal and periapical tissue), which is described as the hardest area to clean during endodontic treatment. Infection is difficult to remove entirely because the apex area is hard to approach using dental instruments and because of the existence of special morphological structures, such as apical ramification, intercanal anastomoses, and lateral branch of root canal. This review gives a brief introduction of the characteristics and difficulties of apical infection and knowledge on how to control such infections, including root apex preparation, irrigation and disinfection, and root canal filling. PMID:25490815

  19. Frequency-dependent signal processing in apical dendrites of hippocampal CA1 pyramidal cells.

    PubMed

    Watanabe, H; Tsubokawa, H; Tsukada, M; Aihara, T

    2014-10-10

    Depending on an animal's behavioral state, hippocampal CA1 pyramidal cells receive distinct patterns of excitatory and inhibitory synaptic inputs. The time-dependent changes in the frequencies of these inputs and the nonuniform distribution of voltage-gated channels lead to dynamic fluctuations in membrane conductance. In this study, using a whole-cell patch-clamp method, we attempted to record and analyze the frequency dependencies of membrane responsiveness in Wistar rat hippocampal CA1 pyramidal cells following noise current injection directly into dendrites and somata under pharmacological blockade of all synaptic inputs. To estimate the frequency-dependent properties of membrane potential, membrane impedance was determined from the voltage response divided by the input current in the frequency domain. The cell membrane of most neurons showed low-pass filtering properties in all regions. In particular, the properties were strongly expressed in the somata or proximal dendrites. Moreover, the data revealed nonuniform distribution of dendritic impedance, which was high in the intermediate segment of the apical dendritic shaft (∼220-260μm from the soma). The low-pass filtering properties in the apical dendrites were more enhanced by membrane depolarization than those in the somata. Coherence spectral analysis revealed high coherence between the input signal and the output voltage response in the theta-gamma frequency range, and large lags emerged in the distal dendrites in the gamma frequency range. Our results suggest that apical dendrites of hippocampal CA1 pyramidal cells integrate synaptic inputs according to the frequency components of the input signal along the dendritic segments receiving the inputs. PMID:25135353

  20. The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia

    PubMed Central

    Perez Bay, Andres E; Schreiner, Ryan; Mazzoni, Francesca; Carvajal-Gonzalez, Jose M; Gravotta, Diego; Perret, Emilie; Lehmann Mantaras, Gullermo; Zhu, Yuan-Shan; Rodriguez-Boulan, Enrique J

    2013-01-01

    Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis. PMID:23749212

  1. Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells

    PubMed Central

    Jerdeva, Galina V.; Wu, Kaijin; Yarber, Francie A.; Rhodes, Christopher J.; Kalman, Daniel; Schechter, Joel E.; Hamm-Alvarez, Sarah F.

    2006-01-01

    Summary The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 μM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (p≤0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t½) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 min) and ML-7 (40 μM, 15 min), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate

  2. Inflammatory Myofibroblastic Tumor Mimicking Apical Periodontitis.

    PubMed

    Adachi, Makoto; Kiho, Kazuki; Sekine, Genta; Ohta, Takahisa; Matsubara, Makoto; Yoshida, Takakazu; Katsumata, Akitoshi; Tanuma, Jun-ichi; Sumitomo, Shinichiro

    2015-12-01

    Inflammatory myofibroblastic tumors (IMTs) are rare. IMTs of the head and neck occur in all age groups, from neonates to old age, with the highest incidence occurring in childhood and early adulthood. An IMT has been defined as a histologically distinctive lesion of uncertain behavior. This article describes an unusual case of IMT mimicking apical periodontitis in the mandible of a 42-year-old man. At first presentation, the patient showed spontaneous pain and percussion pain at teeth #28 to 30, which continued after initial endodontic treatment. Panoramic radiography revealed a radiolucent lesion at the site. Cone-beam computed tomographic imaging showed osteolytic lesions, suggesting an aggressive neoplasm requiring incisional biopsy. Histopathological examination indicated an IMT. The lesion was removed en bloc under general anesthesia, and the patient manifested no clinical evidence of recurrence for 24 months. Lesions of nonendodontic origin should be included in the differential diagnosis of apical periodontitis. Every available diagnostic tool should be used to confirm the diagnosis. Cone-beam computed tomographic imaging is very helpful for differential diagnosis in IMTs mimicking apical periodontitis. PMID:26602450

  3. Cingulin and actin mediate midbody-dependent apical lumen formation during polarization of epithelial cells

    PubMed Central

    Mangan, Anthony J.; Sietsema, Daniel V.; Li, Dongying; Moore, Jeffrey K.; Citi, Sandra; Prekeris, Rytis

    2016-01-01

    Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis. PMID:27484926

  4. Cingulin and actin mediate midbody-dependent apical lumen formation during polarization of epithelial cells.

    PubMed

    Mangan, Anthony J; Sietsema, Daniel V; Li, Dongying; Moore, Jeffrey K; Citi, Sandra; Prekeris, Rytis

    2016-01-01

    Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis. PMID:27484926

  5. miR-219 regulates neural progenitors by dampening apical Par protein-dependent Hedgehog signaling.

    PubMed

    Hudish, Laura I; Galati, Domenico F; Ravanelli, Andrew M; Pearson, Chad G; Huang, Peng; Appel, Bruce

    2016-07-01

    The transition of dividing neuroepithelial progenitors to differentiated neurons and glia is essential for the formation of a functional nervous system. Sonic hedgehog (Shh) is a mitogen for spinal cord progenitors, but how cells become insensitive to the proliferative effects of Shh is not well understood. Because Shh reception occurs at primary cilia, which are positioned within the apical membrane of neuroepithelial progenitors, we hypothesized that loss of apical characteristics reduces the Shh signaling response, causing cell cycle exit and differentiation. We tested this hypothesis using genetic and pharmacological manipulation, gene expression analysis and time-lapse imaging of zebrafish embryos. Blocking the function of miR-219, a microRNA that downregulates apical Par polarity proteins and promotes progenitor differentiation, elevated Shh signaling. Inhibition of Shh signaling reversed the effects of miR-219 depletion and forced expression of Shh phenocopied miR-219 deficiency. Time-lapse imaging revealed that knockdown of miR-219 function accelerates the growth of primary cilia, revealing a possible mechanistic link between miR-219-mediated regulation of apical Par proteins and Shh signaling. Thus, miR-219 appears to decrease progenitor cell sensitivity to Shh signaling, thereby driving these cells towards differentiation. PMID:27226318

  6. MicroRNAs as key regulators of GTPase-mediated apical actin reorganization in multiciliated epithelia

    PubMed Central

    Mercey, Olivier; Kodjabachian, Laurent; Barbry, Pascal; Marcet, Brice

    2016-01-01

    ABSTRACT Multiciliated cells (MCCs), which are present in specialized vertebrate tissues such as mucociliary epithelia, project hundreds of motile cilia from their apical membrane. Coordinated ciliary beating in MCCs contributes to fluid propulsion in several biological processes. In a previous work, we demonstrated that microRNAs of the miR-34/449 family act as new conserved regulators of MCC differentiation by specifically repressing cell cycle genes and the Notch pathway. Recently, we have shown that miR-34/449 also modulate small GTPase pathways to promote, in a later stage of differentiation, the assembly of the apical actin network, a prerequisite for proper anchoring of centrioles-derived neo-synthesized basal bodies. We characterized several miR-34/449 targets related to small GTPase pathways including R-Ras, which represents a key and conserved regulator during MCC differentiation. Direct RRAS repression by miR-34/449 is necessary for apical actin meshwork assembly, notably by allowing the apical relocalization of the actin binding protein Filamin-A near basal bodies. Our studies establish miR-34/449 as central players that orchestrate several steps of MCC differentiation program by regulating distinct signaling pathways. PMID:27144998

  7. Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA-regulated apical chloride channels in cortical collecting duct.

    PubMed

    Lu, Ming; Dong, Ke; Egan, Marie E; Giebisch, Gerhard H; Boulpaep, Emile L; Hebert, Steven C

    2010-03-30

    The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK. PMID:20231442

  8. Minimal Apical Enlargement for Penetration of Irrigants to the Apical Third of Root Canal System: A Scanning Electron Microscope Study

    PubMed Central

    Srikanth, P; Krishna, Amaravadi Gopi; Srinivas, Siva; Reddy, E Sujayeendranatha; Battu, Someshwar; Aravelli, Swathi

    2015-01-01

    Background: The aim of this study was to determine minimal apical enlargement for irrigant penetration into apical third of root canal system using scanning electron microscope (SEM). Materials and Methods: Distobuccal canals of 40 freshly extracted human maxillary first molar teeth were instrumented using crown-down technique. The teeth were divided into four test groups according to size of their master apical file (MAF) (#20, #25, #30, #35 0.06% taper), and two control groups. After final irrigation, removal of debris and smear layer from the apical third of root canals was determined under a SEM. Data was analyzed using Kruskal–Wallis and Mann–Whitney tests. Results: Smear layer removal in apical third for MAF size #30 was comparable with that of the control group (size #40). Conclusion: Minimal apical enlargement for penetration of irrigants to the apical third of root canal system is #30 size. PMID:26124608

  9. Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish

    USGS Publications Warehouse

    Hiroi, J.; Yasumasu, S.; McCormick, S.D.; Hwang, P.-P.; Kaneko, T.

    2008-01-01

    Cation-chloride cotransporters, such as the Na+/K +/2Cl- cotransporter (NKCC) and Na+/Cl - cotransporter (NCC), are localized to the apical or basolateral plasma membranes of epithelial cells and are involved in active ion absorption or secretion. The objectives of this study were to clone and identify 'freshwater-type' and 'seawater-type' cation-chloride cotransporters of euryhaline Mozambique tilapia (Oreochromis mossambicus) and to determine their intracellular localization patterns within mitochondria-rich cells (MRCs). From tilapia gills, we cloned four full-length cDNAs homologous to human cation-chloride cotransporters and designated them as tilapia NKCC1a, NKCC1b, NKCC2 and NCC. Out of the four candidates, the mRNA encoding NKCC1a was highly expressed in the yolk-sac membrane and gills (sites of the MRC localization) of seawater-acclimatized fish, whereas the mRNA encoding NCC was exclusively expressed in the yolk-sac membrane and gills of freshwater-acclimatized fish. We then generated antibodies specific for tilapia NKCC1a and NCC and conducted whole-mount immunofluorescence staining for NKCC1a and NCC, together with Na+/K+-ATPase, cystic fibrosis transmembrane conductance regulator (CFTR) and Na+/H+ exchanger 3 (NHE3), on the yolk-sac membrane of tilapia embryos acclimatized to freshwater or seawater. The simultaneous quintuple-color immunofluorescence staining allowed us to classify MRCs clearly into four types: types I, II, III and IV. The NKCC1a immunoreactivity was localized to the basolateral membrane of seawater-specific type-IV MRCs, whereas the NCC immunoreactivity was restricted to the apical membrane of freshwater-specific type-II MRCs. Taking account of these data at the level of both mRNA and protein, we deduce that NKCC1a is the seawater-type cotransporter involved in ion secretion by type-IV MRCs and that NCC is the freshwater-type cotransporter involved in ion absorption by type-II MRCs. We propose a novel ion-uptake model by MRCs in

  10. Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish.

    PubMed

    Hiroi, Junya; Yasumasu, Shigeki; McCormick, Stephen D; Hwang, Pung-Pung; Kaneko, Toyoji

    2008-08-01

    Cation-chloride cotransporters, such as the Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and Na(+)/Cl(-) cotransporter (NCC), are localized to the apical or basolateral plasma membranes of epithelial cells and are involved in active ion absorption or secretion. The objectives of this study were to clone and identify ;freshwater-type' and ;seawater-type' cation-chloride cotransporters of euryhaline Mozambique tilapia (Oreochromis mossambicus) and to determine their intracellular localization patterns within mitochondria-rich cells (MRCs). From tilapia gills, we cloned four full-length cDNAs homologous to human cation-chloride cotransporters and designated them as tilapia NKCC1a, NKCC1b, NKCC2 and NCC. Out of the four candidates, the mRNA encoding NKCC1a was highly expressed in the yolk-sac membrane and gills (sites of the MRC localization) of seawater-acclimatized fish, whereas the mRNA encoding NCC was exclusively expressed in the yolk-sac membrane and gills of freshwater-acclimatized fish. We then generated antibodies specific for tilapia NKCC1a and NCC and conducted whole-mount immunofluorescence staining for NKCC1a and NCC, together with Na(+)/K(+)-ATPase, cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+)/H(+) exchanger 3 (NHE3), on the yolk-sac membrane of tilapia embryos acclimatized to freshwater or seawater. The simultaneous quintuple-color immunofluorescence staining allowed us to classify MRCs clearly into four types: types I, II, III and IV. The NKCC1a immunoreactivity was localized to the basolateral membrane of seawater-specific type-IV MRCs, whereas the NCC immunoreactivity was restricted to the apical membrane of freshwater-specific type-II MRCs. Taking account of these data at the level of both mRNA and protein, we deduce that NKCC1a is the seawater-type cotransporter involved in ion secretion by type-IV MRCs and that NCC is the freshwater-type cotransporter involved in ion absorption by type-II MRCs. We propose a novel ion-uptake model

  11. cAMP stimulates apical exocytosis of the renal Na(+)-K(+)-2Cl(-) cotransporter NKCC2 in the thick ascending limb: role of protein kinase A.

    PubMed

    Caceres, Paulo S; Ares, Gustavo R; Ortiz, Pablo A

    2009-09-11

    The apical renal Na(+)-K(+)-2Cl(-) cotransporter NKCC2 mediates NaCl absorption by the thick ascending limb (TAL) of Henle's loop. cAMP stimulates NKCC2 by enhancing steady-state apical membrane levels of this protein; however, the trafficking and signaling mechanisms by which this occurs have not been studied. Here, we report that stimulation of endogenous cAMP levels with either forskolin/3-isobutyl-1-methylxanthine (IBMX) or the V2 receptor agonist [deamino-Cys(1),d-Arg(8)]vasopressin increases steady-state surface NKCC2 and that the protein kinase A (PKA) inhibitor H-89 blocks this effect. Confocal imaging of apical surface NKCC2 in isolated perfused TALs confirmed a stimulatory effect of cAMP on apical trafficking that was blocked by PKA inhibition. Selective stimulation of PKA with the agonist N(6)-benzoyl-cAMP (500 microm) stimulated steady-state surface NKCC2, whereas the Epac-selective agonist 8-p-chlorophenylthio-2'-O-methyl-cAMP (100 and 250 microm) had no effect. To explore the trafficking mechanism by which cAMP increases apical NKCC2, we measured cumulative apical membrane exocytosis and NKCC2 exocytic insertion in TALs. By monitoring apical FM1-43 fluorescence, we observed rapid stimulation of apical exocytosis (2 min) by forskolin/IBMX. We also found constitutive exocytic insertion of NKCC2 in TALs over time, which was increased by 3-fold in the presence of forskolin/IBMX. PKA inhibition blunted cAMP-stimulated exocytic insertion but did not affect the rate of constitutive exocytosis. We conclude that cAMP stimulates steady-state apical surface NKCC2 by stimulating exocytic insertion and that this process is highly dependent on PKA but not Epac. PMID:19592485

  12. Vectorial transport of fexofenadine across Caco-2 cells: involvement of apical uptake and basolateral efflux transporters.

    PubMed

    Ming, Xin; Knight, Beverly M; Thakker, Dhiren R

    2011-10-01

    Fexofenadine is a nonsedative antihistamine that exhibits good oral bioavailability despite its zwitterionic chemical structure and efflux by P-gp. Evidence exists that multiple uptake and efflux transporters play a role in hepatic disposition of fexofenadine. However, the roles of specific transporters and their interrelationship in intestinal absorption of this drug are unclear. This study was designed to elucidate vectorial absorptive transport of fexofenadine across Caco-2 cells involving specific apical uptake and efflux transporters as well as basolateral efflux transporters. Studies with cellular models expressing single transporters showed that OATP2B1 expression stimulated uptake of fexofenadine at pH 6.0. Apical uptake of fexofenadine into Caco-2 cells was decreased by 45% by pretreatment with estrone 3-sulfate, an OATP inhibitor, at pH 6.0 but not at pH 7.4, indicating that OATP2B1 mediates apical uptake of fexofenadine into these cells. Examination of fexofenadine efflux from preloaded Caco-2 cells in the presence or absence of (i) the MRP inhibitor MK-571 and (ii) the P-gp inhibitor GW918 showed that apical efflux is predominantly mediated by P-gp, with a small contribution by MRP2, whereas basolateral efflux is predominantly mediated by MRP3. These results also showed that while OSTαβ is functionally active in the basolateral membrane of Caco-2 cells, it does not play a role in the export of fexofenadine. MK-571 decreased the absorptive transport of fexofenadine by 17%. However, the decrease in absorptive transport by MK-571 was 42% when P-gp was inhibited by GW918. The results provide a novel insight into a vectorial transport system mainly consisting of apical OATP2B1 and basolateral MRP3 that may play an important role in delivering hydrophilic anionic and zwitterionic drugs such as pravastatin and fexofenadine into systemic circulation upon oral administration. PMID:21780830

  13. Apical accumulation of MARCKS in neural plate cells during neurulation in the chick embryo

    PubMed Central

    Zolessi, Flavio R; Arruti, Cristina

    2001-01-01

    Background The neural tube is formed by morphogenetic movements largely dependent on cytoskeletal dynamics. Actin and many of its associated proteins have been proposed as important mediators of neurulation. For instance, mice deficient in MARCKS, an actin cross-linking membrane-associated protein that is regulated by PKC and other kinases, present severe developmental defects, including failure of cranial neural tube closure. Results To determine the distribution of MARCKS, and its possible relationships with actin during neurulation, chick embryos were transversely sectioned and double labeled with an anti-MARCKS polyclonal antibody and phalloidin. In the neural plate, MARCKS was found ubiquitously distributed at the periphery of the cells, being conspicuously accumulated in the apical cell region, in close proximity to the apical actin meshwork. This asymmetric distribution was particularly noticeable during the bending process. After the closure of the neural tube, the apically accumulated MARCKS disappeared, and this cell region became analogous to the other peripheral cell zones in its MARCKS content. Actin did not display analogous variations, remaining highly concentrated at the cell subapical territory. The transient apical accumulation of MARCKS was found throughout the neural tube axis. The analysis of another epithelial bending movement, during the formation of the lens vesicle, revealed an identical phenomenon. Conclusions MARCKS is transiently accumulated at the apical region of neural plate and lens placode cells during processes of bending. This asymmetric subcellular distribution of MARCKS starts before the onset of neural plate bending. These results suggest possible upstream regulatory actions of MARCKS on some functions of the actin subapical meshwork. PMID:11329360

  14. Shank2 contributes to the apical retention and intracellular redistribution of NaPiIIa in OK cells.

    PubMed

    Dobrinskikh, Evgenia; Lanzano, Luca; Rachelson, Joanna; Cranston, DeeAnn; Moldovan, Radu; Lei, Tim; Gratton, Enrico; Doctor, R Brian

    2013-03-01

    In renal proximal tubule (PT) cells, sodium-phosphate cotransporter IIa (NaPiIIa) is normally concentrated within the apical membrane where it reabsorbs ∼70% of luminal phosphate (Pi). NaPiIIa activity is acutely regulated by moderating its abundance within the apical membrane. Under low-Pi conditions, NaPiIIa is retained within the apical membrane. Under high-Pi conditions, NaPiIIa is retrieved from the apical membrane and trafficked to the lysosomes for degradation. The present study investigates the role of Shank2 in regulating the distribution of NaPiIIa. In opossum kidney cells, a PT cell model, knockdown of Shank2 in cells maintained in low-Pi media resulted in a marked decrease in NaPiIIa abundance. After being transferred into high-Pi media, live-cell imaging showed that mRFP-Shank2E and GFP-NaPiIIa underwent endocytosis and trafficked together through the subapical domain. Fluorescence cross-correlation spectroscopy demonstrated that GFP-NaPiIIa and mRFP-Shank2 have indistinguishable diffusion coefficients and migrated through the subapical domain in temporal synchrony. Raster image cross-correlation spectroscopy demonstrated these two proteins course through the subapical domain in temporal-spatial synchrony. In the microvilli of cells under low-Pi conditions and in the subapical domain of cells under high-Pi conditions, fluorescence lifetime imaging microscopy-Forster resonance energy transfer analysis of Cer-NaPiIIa and EYFP-Shank2E found these fluors reside within 10 nm of each other. Demonstrating a complexity of functions, in cells maintained under low-Pi conditions, Shank2 plays an essential role in the apical retention of NaPiIIa while under high-Pi conditions Shank2 remains associated with NaPiIIa and escorts NaPiIIa through the cell interior. PMID:23325414

  15. Histochemical evidence for generation of active oxygen species on the apical surface of cigarette-smoke-exposed tracheal explants.

    PubMed Central

    Hobson, J.; Wright, J.; Churg, A.

    1991-01-01

    Cigarette smoke is known to contain many types of free radicals, and solutions of smoke tar have been shown to liberate hydrogen peroxide as well as superoxide radical. To further investigate the relationship of smoke exposure and generation of active oxygen species, the authors exposed rat tracheal explants to varying amounts of smoke for 10 minutes in a humidified chamber. After smoke exposure was completed, tracheal segments were incubated in a modification of the ultrastructural cerium chloride technique that was devised by Briggs et al. to demonstrate hydrogen peroxide production. Smoke dose-dependent deposition of cerium-containing reaction product was found on the cilia and the apical membranes; with low-dose smoke, the reaction product appeared as individual dots along the apical surface, but with greater amounts of smoke, heavy linear deposits of reaction product were found along the apical membranes. Smoke produced focal dose-related cell damage with blebbing of the apical membranes, loss of cilia, and focal cell necrosis. Catalase prevented both the positive histochemical reaction and the cell damage; if the catalase was first boiled, its protective effect was destroyed. Similarly, after smoke exposure was completed, tracheal segments were covered with a solution of nitroblue tetrazolium to demonstrate production of superoxide anion. A positive reaction was observed by light microscopy on the surface of tracheas that was exposed to smoke but not that exposed to air; the reaction could be prevented by addition of superoxide dismutase. The authors conclude that exposure of tracheal explants to cigarette smoke in vitro is associated with histochemical evidence of continuing production of both hydrogen peroxide and superoxide anion at the apical cell membrane. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1653519

  16. H(+)/solute-induced intracellular acidification leads to selective activation of apical Na(+)/H(+) exchange in human intestinal epithelial cells.

    PubMed

    Thwaites, D T; Ford, D; Glanville, M; Simmons, N L

    1999-09-01

    The intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces - transepithelial ion gradients - are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pH(i)) regulation in response to H(+)-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na(+)/H(+) exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na(+)/H(+) exchange was determined by a combination of pH(i), (22)Na(+) influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H(+)/solute-induced acidification (using glycylsarcosine or beta-alanine) led to Na(+)-dependent, EIPA-inhibitable pH(i) recovery or EIPA-inhibitable (22)Na(+) influx at the apical membrane only. Selective activation of apical (but not basolateral) Na(+)/H(+) exchange by H(+)/solute cotransport demonstrates that coordinated activity of H(+)/solute symport with apical Na(+)/H(+) exchange optimizes the efficient absorption of nutrients and Na(+), while maintaining pH(i) and the ion gradients involved in driving transport. PMID:10487777

  17. PAR3 acts as a molecular organizer to define the apical domain of chick neuroepithelial cells.

    PubMed

    Afonso, Cristina; Henrique, Domingos

    2006-10-15

    Neural progenitors in the vertebrate nervous system are fully polarized epithelial cells, with intercellular junctions at the apical region. These progenitor cells remain within the neuroepithelium throughout neurogenesis, and will ultimately give rise to all the neurons in the mature nervous system. We have addressed the role of the PAR polarity complex in vertebrate neuroepithelial polarity and show that PAR3 functions as the initial scaffold to assemble and organize the PAR complex at the apical region of neuroepithelial cells, coordinating also the recruitment of additional polarity complexes and junction-associated proteins to the same region, while restricting other polarity proteins to the basolateral membrane. We propose that PAR3 acts as a molecular organizer to connect the acquisition of apico-basal polarity with the positioning and formation of junctional structures in neuroepithelial cells, a function of upmost importance for the morphogenesis of embryonic neural tissue and the process of neurogenesis. PMID:17003110

  18. 76 FR 77375 - Airworthiness Directives; Apical Industries, Inc., (Apical) Emergency Float Kits

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-13

    ... Register on December 7, 2010 (75 FR 75934). That action proposed to require, for certain model helicopters... Regulatory Policies and Procedures (44 FR 11034, February 26, 1979); 3. Will not affect intrastate aviation...-038-AD; Amendment 39-16877; AD 2011-25-01] RIN 2120-AA64 Airworthiness Directives; Apical...

  19. 75 FR 75934 - Airworthiness Directives; Apical Industries Inc. (Apical) Emergency Float Kits

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-07

    ... FR 19477-78). Examining the Docket You may examine the docket that contains the proposed AD, any... ``significant rule'' under the DOT Regulatory Policies and Procedures (44 FR 11034, February 26, 1979); and 3... Federal Aviation Administration 14 CFR Part 39 RIN 2120-AA64 Airworthiness Directives; Apical...

  20. Apical hypertrophic cardiomyopathy presenting as acute coronary syndrome.

    PubMed

    Abdin, Amr; Eitel, Ingo; de Waha, Suzanne; Thiele, Holger

    2016-06-01

    Apical hypertrophic cardiomyopathy is a rare variant of hypertrophic cardiomyopathy. It is characterized by a local hypertrophy of the apical segments and displays typical electrocardiographic and imaging patterns. The clinical manifestations are variable and range from an asymptomatic course to sudden cardiac death. The most frequent symptom is chest pain and thus apical hypertrophic cardiomyopathy can mimic the symptoms and repolarization disturbances indicative of acute coronary syndrome. PMID:26628684

  1. Aldosterone increases the apical Na sup + permeability of toad bladder by two different mechanisms

    SciTech Connect

    Asher, C.; Garty, H. )

    1988-10-01

    The aldosterone-induced augmentation of Na{sup +} transport in toad bladder was analyzed by comparing the hormonal actions on the transepithelial short-circuit current and on the amiloride-sensitive {sup 22}Na{sup +} uptake in isolated membrane vesicles. Incubating bladders with 0.5 {mu}M aldosterone for 3 hr evoked more than a 2-fold increase of the short-circuit current but had no effect on the amiloride-sensitive Na{sup +} transport in apical vesicles derived from the treated tissue. A longer incubation produced an additional augmentation of the short-circuit current, which was accompanied by about a 3-fold increase of the channel activity in isolated membranes. The stimulatory effect of aldosterone sustained in vesicles was inhibited by the antagonist spironolactone and the protein synthesis inhibitor cycloheximide. It is suggested that aldosterone elevates the apical Na{sup +} permeability of target epithelia by two different mechanisms: a relatively fast effect which is insensitive to triiodothyronine or butyrate and is not sustained by the isolated membrane, and a slower or later response blocked by these reagents, which is preserved by the isolated membrane. The data also indicate that these processes are mediated by different nuclear receptors.

  2. Apical ballooning syndrome following exercise treadmill testing

    PubMed Central

    Irwin, RB; Mamas, MA; El-Omar, M

    2011-01-01

    Transient left ventricular apical ballooning syndrome is an increasingly recognized cause of acute coronary syndrome, particularly in postmenopausal women, and is the subject of increasing interest to both clinicians and researchers. Emotional and physical stressors are often implicated in its development and, while excess sympathetic drive appears to act as a primary trigger, the exact mechanism remains controversial. The clinical presentation is characterized by transient, often severe, left ventricular dysfunction affecting the mid and apical myocardium. By definition, no significant coronary artery lesions are present, although this may not be recognized at initial presentation. While recovery of function with evidence of limited myocardial necrosis is common, significant complications may manifest in the acute phase. A case involving an elderly patient who developed classical features of the syndrome following an exercise treadmill test is presented. To the authors’ knowledge, the present case is the only such report that meets the recently proposed diagnostic criteria. The present case serves to highlight a rare but important complication of exercise testing in an elderly patient. Recent large systematic reviews have provided valuable insights into the clinical features of this condition. The current article examines the data from these studies and others to provide a comprehensive clinical overview. PMID:21747667

  3. Apical leakage of four endodontic sealers.

    PubMed

    Pommel, Ludovic; About, Imad; Pashley, David; Camps, Jean

    2003-03-01

    The purpose of this study was to evaluate the sealing properties of four root canal sealers. Forty-eight maxillary central incisors were instrumented with Profile rotary instruments. They were randomly divided into four groups (n = 12) and filled using lateral condensation with one of the four sealers: Sealapex, Pulp Canal Sealer, AH 26, and Ketac-Endo. The apical leakage was measured with a fluid filtration method and expressed as L s(-1) KPa(-1). The teeth filled with Sealapex displayed a higher apical leakage (8.42 +/- 4.2 10(-11) L s(-1) KPa(-1)) than those filled with AH 26 (2.10 +/- 1.39 10(-11) L s(-1) KPa(-1)), Pulp Canal Sealer (0.17 +/- 0.09 10(-11) L s(-1) KPa(-1)) or Ketac-Endo (0.32 +/- 0.24 10(-1) L s(-1) KPa(-1)) (p < 0.01). No statistically significant difference was found among AH 26, Pulp Canal Sealer, and Ketac-Endo. No correlation was found between the sealing efficiency of the four sealers and their adhesive properties recorded in a previous study. PMID:12669883

  4. CLIC4 regulates apical exocytosis and renal tube luminogenesis through retromer- and actin-mediated endocytic trafficking

    PubMed Central

    Chou, Szu-Yi; Hsu, Kuo-Shun; Otsu, Wataru; Hsu, Ya-Chu; Luo, Yun-Cin; Yeh, Celine; Shehab, Syed S.; Chen, Jie; Shieh, Vincent; He, Guo-an; Marean, Michael B.; Felsen, Diane; Ding, Aihao; Poppas, Dix P.; Chuang, Jen-Zen; Sung, Ching-Hwa

    2016-01-01

    Chloride intracellular channel 4 (CLIC4) is a mammalian homologue of EXC-4 whose mutation is associated with cystic excretory canals in nematodes. Here we show that CLIC4-null mouse embryos exhibit impaired renal tubulogenesis. In both developing and developed kidneys, CLIC4 is specifically enriched in the proximal tubule epithelial cells, in which CLIC4 is important for luminal delivery, microvillus morphogenesis, and endolysosomal biogenesis. Adult CLIC4-null proximal tubules display aberrant dilation. In MDCK 3D cultures, CLIC4 is expressed on early endosome, recycling endosome and apical transport carriers before reaching its steady-state apical membrane localization in mature lumen. CLIC4 suppression causes impaired apical vesicle coalescence and central lumen formation, a phenotype that can be rescued by Rab8 and Cdc42. Furthermore, we show that retromer- and branched actin-mediated trafficking on early endosome regulates apical delivery during early luminogenesis. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating the formation of branched actin on early endosomes. PMID:26786190

  5. Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals.

    PubMed

    Hovater, Michael B; Olteanu, Dragos; Hanson, Elizabeth L; Cheng, Nai-Lin; Siroky, Brian; Fintha, Attila; Komlosi, Peter; Liu, Wen; Satlin, Lisa M; Bell, P Darwin; Yoder, Bradley K; Schwiebert, Erik M

    2008-06-01

    mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca(2+) derived from both extracellular and intracellular stores. This flow-induced Ca(2+) signal was less robust in cilium-deficient monolayers. Flow-induced Ca(2+) signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca(2+). Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na(+)) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases. PMID:18368523

  6. Unique apicomplexan IMC sub-compartment proteins are early markers for apical polarity in the malaria parasite

    PubMed Central

    Poulin, Benoit; Patzewitz, Eva-Maria; Brady, Declan; Silvie, Olivier; Wright, Megan H.; Ferguson, David J. P.; Wall, Richard J.; Whipple, Sarah; Guttery, David S.; Tate, Edward W.; Wickstead, Bill; Holder, Anthony A.; Tewari, Rita

    2013-01-01

    Summary The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite. PMID:24244852

  7. Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

    PubMed

    Ratner, Martha A; Decker, Sarah E; Aller, Stephen G; Weber, Gerhard; Forrest, John N

    2006-03-01

    In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action. PMID:16432888

  8. PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells

    PubMed Central

    Arkhipenko, Alexander; Syan, Sylvie; Victoria, Guiliana Soraya

    2016-01-01

    The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures. PMID:27389581

  9. PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells.

    PubMed

    Arkhipenko, Alexander; Syan, Sylvie; Victoria, Guiliana Soraya; Lebreton, Stéphanie; Zurzolo, Chiara

    2016-01-01

    The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures. PMID:27389581

  10. Diagnosis of apical hypertrophic cardiomyopathy: T-wave inversion and relative but not absolute apical left ventricular hypertrophy☆

    PubMed Central

    Flett, Andrew S.; Maestrini, Viviana; Milliken, Don; Fontana, Mariana; Treibel, Thomas A.; Harb, Rami; Sado, Daniel M.; Quarta, Giovanni; Herrey, Anna; Sneddon, James; Elliott, Perry; McKenna, William; Moon, James C.

    2015-01-01

    Background Diagnosis of apical HCM utilizes conventional wall thickness criteria. The normal left ventricular wall thins towards the apex such that normal values are lower in the apical versus the basal segments. The impact of this on the diagnosis of apical hypertrophic cardiomyopathy has not been evaluated. Methods We performed a retrospective review of 2662 consecutive CMR referrals, of which 75 patients were identified in whom there was abnormal T-wave inversion on ECG and a clinical suspicion of hypertrophic cardiomyopathy. These were retrospectively analyzed for imaging features consistent with cardiomyopathy, specifically: relative apical hypertrophy, left atrial dilatation, scar, apical cavity obliteration or apical aneurysm. For comparison, the same evaluation was performed in 60 healthy volunteers and 50 hypertensive patients. Results Of the 75 patients, 48 met conventional HCM diagnostic criteria and went on to act as another comparator group. Twenty-seven did not meet criteria for HCM and of these 5 had no relative apical hypertrophy and were not analyzed further. The remaining 22 patients had relative apical thickening with an apical:basal wall thickness ratio > 1 and a higher prevalence of features consistent with a cardiomyopathy than in the control groups with 54% having 2 or more of the 4 features. No individual in the healthy volunteer group had more than one feature and no hypertension patient had more than 2. Conclusion A cohort of individuals exist with T wave inversion, relative apical hypertrophy and additional imaging features of HCM suggesting an apical HCM phenotype not captured by existing diagnostic criteria. PMID:25666123

  11. Iterative sorting of apical and basolateral cargo in Madin-Darby canine kidney cells.

    PubMed

    Treyer, Aleksandr; Pujato, Mario; Pechuan, Ximo; Müsch, Anne

    2016-07-15

    For several decades, the trans-Golgi network (TGN) was considered the most distal stop and hence the ultimate protein-sorting station for distinct apical and basolateral transport carriers that reach their respective surface domains in the direct trafficking pathway. However, recent reports of apical and basolateral cargoes traversing post-Golgi compartments accessible to endocytic ligands before their arrival at the cell surface and the post-TGN breakup of large pleomorphic membrane fragments that exit the Golgi region toward the surface raised the possibility that compartments distal to the TGN mediate or contribute to biosynthetic sorting. Here we describe the development of a novel assay that quantitatively distinguishes different cargo pairs by their degree of colocalization at the TGN and by the evolution of colocalization during their TGN-to-surface transport. Keys to the high resolution of our approach are 1) conversion of perinuclear organelle clustering into a two-dimensional microsomal spread and 2) identification of TGN and post-TGN cargo without the need for a TGN marker that universally cosegregates with all cargo. Using our assay, we provide the first evidence that apical NTRp75 and basolateral VSVG in Madin-Darby canine kidney cells still undergo progressive sorting after they exit the TGN toward the cell surface. PMID:27226480

  12. Efficacy of Biodentine as an Apical Plug in Nonvital Permanent Teeth with Open Apices: An In Vitro Study.

    PubMed

    Bani, Mehmet; Sungurtekin-Ekçi, Elif; Odabaş, Mesut Enes

    2015-01-01

    The aim of this study was to evaluate the apical microleakage of Biodentine and MTA orthograde apical plugs and to compare the effect of thickness of these biomaterials on their sealing ability. A total of eighty maxillary anterior teeth were used. The apices were removed by cutting with a diamond disc (Jota, Germany) 2 mm from the apical root end in an attempt to standardize the working length of all specimens to 15 ± 1 mm. Both materials were placed in 1-4 mm thickness as apical plugs root canal. Root canal leakage was evaluated by the fluid filtration technique. One-way ANOVA was used in order to determine normality of dispersal distribution of parameters; thereafter, results were analyzed by Kolmogorov-Smirnov test. Overall, between microleakage values of MTA and Biodentine regardless of apical plug thickness, no difference was observed. In terms of plug thickness, a statistically significant difference was observed between the subgroups of MTA and Biodentine (p < 0.05). The apical sealing ability of Biodentine was comparable to MTA at any apical plug thickness. PMID:26436090

  13. Bacterial community profiling of cryogenically ground samples from the apical and coronal root segments of teeth with apical periodontitis.

    PubMed

    Alves, Flávio R F; Siqueira, José F; Carmo, Flávia L; Santos, Adriana L; Peixoto, Raquel S; Rôças, Isabela N; Rosado, Alexandre S

    2009-04-01

    Bacteria located at the apical part of infected root canals are arguably directly involved in the pathogenesis of apical periodontitis. This study was conducted to profile and further compare the bacterial communities established at the apical and middle/coronal segments of infected root canals. Extracted teeth with attached apical periodontitis lesions were sectioned so as to obtain two root fragments representing the apical third and the coronal two thirds. Root fragments were subjected to a cryogenic grinding approach. DNA was extracted from root powder samples and used as a template for bacterial community profiling using a 16S ribosomal RNA gene-based seminested polymerase chain reaction/denaturing gradient gel electrophoresis approach. The mean number of bands in apical samples from teeth with primary infections was 28, ranging from 18 to 48, whereas in the middle/coronal samples, it was also 28, ranging from 19 to 36. Findings showed that the profile of bacterial community colonizing the apical third of infected root canals is as diverse as that occurring at the middle/coronal thirds. A high variability was observed for both interindividual (samples from the same region but from different patients) and intraindividual (samples from different regions of the same tooth) comparisons. The methodology used to prepare and analyze samples was highly effective in disclosing a previously unanticipated broad diversity of endodontic bacterial communities, especially at the apical part of infected root canals. PMID:19345792

  14. Efficacy of Biodentine as an Apical Plug in Nonvital Permanent Teeth with Open Apices: An In Vitro Study

    PubMed Central

    Bani, Mehmet; Sungurtekin-Ekçi, Elif; Odabaş, Mesut Enes

    2015-01-01

    The aim of this study was to evaluate the apical microleakage of Biodentine and MTA orthograde apical plugs and to compare the effect of thickness of these biomaterials on their sealing ability. A total of eighty maxillary anterior teeth were used. The apices were removed by cutting with a diamond disc (Jota, Germany) 2 mm from the apical root end in an attempt to standardize the working length of all specimens to 15 ± 1 mm. Both materials were placed in 1–4 mm thickness as apical plugs root canal. Root canal leakage was evaluated by the fluid filtration technique. One-way ANOVA was used in order to determine normality of dispersal distribution of parameters; thereafter, results were analyzed by Kolmogorov-Smirnov test. Overall, between microleakage values of MTA and Biodentine regardless of apical plug thickness, no difference was observed. In terms of plug thickness, a statistically significant difference was observed between the subgroups of MTA and Biodentine (p < 0.05). The apical sealing ability of Biodentine was comparable to MTA at any apical plug thickness. PMID:26436090

  15. Mechanisms of lysophosphatidic acid (LPA) mediated stimulation of intestinal apical Cl-/OH- exchange.

    PubMed

    Singla, Amika; Dwivedi, Alka; Saksena, Seema; Gill, Ravinder K; Alrefai, Waddah A; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

    2010-02-01

    Lysophosphatidic acid (LPA), a potent bioactive phospholipid, is a natural component of food products like soy and egg yolk. LPA modulates a number of epithelial functions and has been shown to inhibit cholera toxin-induced diarrhea. Antidiarrheal effects of LPA are known to be mediated by inhibiting chloride secretion. However, the effects of LPA on chloride absorption in the mammalian intestine are not known. The present studies examined the effects of LPA on apical Cl(-)/OH(-) exchangers known to be involved in chloride absorption in intestinal epithelial cells. Caco-2 cells were treated with LPA, and Cl(-)/OH(-) exchange activity was measured as DIDS-sensitive (36)Cl(-) uptake. Cell surface biotinylation studies were performed to evaluate the effect of LPA on cell surface levels of apical Cl(-)/OH(-) exchangers, downregulated in adenoma (DRA) (SLC26A3), and putative anion transporter-1 (SLC26A6). Treatment of Caco-2 cells with LPA (100 muM) significantly stimulated Cl(-)/OH(-) exchange activity. Specific agonist for LPA2 receptor mimicked the effects of LPA. LPA-mediated stimulation of Cl(-)/OH(-) exchange activity was dependent on activation of phosphatidylinositol 3-kinase/Akt signaling pathway. Consistent with the functional activity, LPA treatment resulted in increased levels of DRA on the apical membrane. Our results demonstrate that LPA stimulates apical Cl(-)/OH(-) exchange activity and surface levels of DRA in intestinal epithelial cells. This increase in Cl(-)/OH(-) exchange may contribute to the antidiarrheal effects of LPA. PMID:19910524

  16. Potassium transport across rat alveolar epithelium: evidence for an apical Na+-K+ pump.

    PubMed

    Basset, G; Bouchonnet, F; Crone, C; Saumon, G

    1988-06-01

    1. Experiments were performed on rat lungs into which various solutions were instilled whilst the lungs were perfused with either whole blood or Ringer solution. Instillation of ion-free glucose solution led to a net flux of fluid and ions into the alveolar spaces. K+ ions entered faster than Na+ ions and reached a concentration about twice that in the perfusate. Ouabain in the perfusate (basolateral side) prevented the rise in alveolar K+ concentration above that in the perfusate, indicating a transcellular pathway. Ba2+ in the instillate (apical side) hindered the entry of K+ into alveoli, suggesting the presence of apical K+ channels. 2. When Ringer solution was instilled, K+ was continuously removed from the alveoli and the K+ concentration in the instillate remained constant or decreased slightly depending on the rate of fluid absorption. The net K+ efflux from alveoli to blood was 0.23 pmol/(cm2 s). When Ba2+ was added to the instillate the net K+ efflux increased to 0.36 pmol/(cm2 s). Apical ouabain reversed the K+ flux resulting in a net K+ flux of 0.19 pmol/(cm2 s) into the alveoli. This suggests the presence of an Na+-K+-ATPase located in the apical membrane of some alveolar cells. 3. The K+ transport from instillate (Ringer solution) to perfusate was traced by means of 86Rb which was added to the instillate. Ouabain in the instillate did not affect fluid absorption but reduced the apparent 86Rb permeability by 50% although the paracellular permeability (estimated with [3H]mannitol) was unaffected. This also indicates the presence of an apical Na+-K+-ATPase. When ouabain was added to the perfusate, the apparent 86Rb permeability doubled. These findings indicate that recirculation of 86Rb (and K+) occurs due to the activity of both apical and basolateral Na+-K+-ATPases. 4. When ouabain was placed on both sides of the epithelium, preventing transcellular transport, the passive 86Rb permeability was 10.3 x 10(-8) cm/s (assuming an alveolar surface area of

  17. Apical surgery: A review of current techniques and outcome

    PubMed Central

    von Arx, Thomas

    2010-01-01

    Apical surgery is considered a standard oral surgical procedure. It is often a last resort to surgically maintain a tooth with a periapical lesion that cannot be managed with conventional endodontic (re-)treatment. The main goal of apical surgery is to prevent bacterial leakage from the root-canal system into the periradicular tissues by placing a tight root-end filling following root-end resection. Clinicians are advised to utilize a surgical microscope to perform apical surgery to benefit from magnification and illumination. In addition, the application of microsurgical techniques in apical surgery, i.e., gentle incision and flap elevation, production of a small osteotomy, and the use of sonic- or ultrasonic driven microtips, will result in less trauma to the patient and faster postsurgical healing. A major step in apical surgery is to identify possible leakage areas at the cut root face and subsequently to ensure adequate root-end filling. Only a tight and persistent apical obturation will allow periapical healing with good long-term prognosis. The present paper describes current indications, techniques and outcome of apical surgery. PMID:24151412

  18. Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

    PubMed Central

    Silvis, Mark R.; Bertrand, Carol A.; Ameen, Nadia; Golin-Bisello, Franca; Butterworth, Michael B.; Bradbury, Neil A.

    2009-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells. PMID:19244346

  19. Apical control of conidiation in Aspergillus nidulans.

    PubMed

    Oiartzabal-Arano, Elixabet; Perez-de-Nanclares-Arregi, Elixabet; Espeso, Eduardo A; Etxebeste, Oier

    2016-05-01

    The infection cycle of filamentous fungi consists of two main stages: invasion (growth) and dispersion (development). After the deposition of a spore on a host, germination, polar extension and branching of vegetative cells called hyphae allow a fast and efficient invasion. Under suboptimal conditions, genetic reprogramming of hyphae results in the generation of asexual spores, allowing dissemination to new hosts and the beginning of a new infection cycle. In the model filamentous fungus Aspergillus nidulans, asexual development or conidiation is induced by the upstream developmental activation (UDA) pathway. UDA proteins transduce signals from the tip, the polarity site of hyphae, to nuclei, where developmental programs are transcriptionally activated. The present review summarizes the current knowledge on this tip-to-nucleus communication mechanism, emphasizing its dependence on hyphal polarity. Future approaches to the topic will also be suggested, as stimulating elements contributing to the understanding of how apical signals are coupled with the transcriptional control of development and pathogenesis in filamentous fungi. PMID:26782172

  20. Electron-microscopic study of the apical region of the toad bladder epithelial cell.

    PubMed

    Sasaki, J; Tilles, S; Condeelis, J; Carboni, J; Meiteles, L; Franki, N; Bolon, R; Robertson, C; Hays, R M

    1984-09-01

    Antidiuretic hormone (ADH) promotes fusion of cytoplasmic tubules with the luminal membrane and delivery of particles from the tubules to the membrane. The particles are believed to be the water-conducting elements in the membrane. We have employed several scanning (SEM) and transmission electron-microscopic (TEM) techniques to study the relationship of the cytoplasmic tubules to the luminal membrane and to the apical cytoskeleton of the toad bladder epithelial cell. This paper reports the results of freeze-crack SEM and tannic acid-fixed TEM studies, as well as studies with a resinless method of embedding. Freeze-cracked epithelial cells reveal that the tubules are anchored in a matrix of cytoskeleton and granules just below the luminal membrane, and many, if not all, retain their anchorage to the matrix after ADH-induced fusion. Tannic acid-fixed specimens show that the tubules in unstimulated cells lie horizontally. Fusion appears to involve an angulation of the tubules, and this may be the major mode of ADH-induced tubule movement. There are suggestions in the tannic acid sections of filamentous attachments of tubules to the surrounding cytoskeleton. In addition there are prominent microfilament bundles running down the microvilli and a dense concentration of filaments just below the luminal membrane. The presence of these filaments is confirmed in the resinless sections, and their possible role in ADH action is discussed. PMID:6433717

  1. Shot and Patronin polarise microtubules to direct membrane traffic and biogenesis of microvilli in epithelia

    PubMed Central

    Khanal, Ichha; Elbediwy, Ahmed; Diaz de la Loza, Maria del Carmen; Fletcher, Georgina C.

    2016-01-01

    ABSTRACT In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. Here, we examine the hierarchy of events during cytoskeletal polarisation in Drosophila melanogaster epithelia. Core apical-basal polarity determinants polarise the spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1, CAMSAP2 and CAMPSAP3 in humans) and Shortstop [Shot; MACF1 and BPAG1 (also known as DST) in humans] to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf–Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV (also known as Didum in Drosophila) actin motor to deliver the key microvillar determinant Cadherin 99C to the apical membrane to organise the biogenesis of actin microvilli. PMID:27231092

  2. Shot and Patronin polarise microtubules to direct membrane traffic and biogenesis of microvilli in epithelia.

    PubMed

    Khanal, Ichha; Elbediwy, Ahmed; Diaz de la Loza, Maria Del Carmen; Fletcher, Georgina C; Thompson, Barry J

    2016-07-01

    In epithelial tissues, polarisation of microtubules and actin microvilli occurs along the apical-basal axis of each cell, yet how these cytoskeletal polarisation events are coordinated remains unclear. Here, we examine the hierarchy of events during cytoskeletal polarisation in Drosophila melanogaster epithelia. Core apical-basal polarity determinants polarise the spectrin cytoskeleton to recruit the microtubule-binding proteins Patronin (CAMSAP1, CAMSAP2 and CAMPSAP3 in humans) and Shortstop [Shot; MACF1 and BPAG1 (also known as DST) in humans] to the apical membrane domain. Patronin and Shot then act to polarise microtubules along the apical-basal axis to enable apical transport of Rab11 endosomes by the Nuf-Dynein microtubule motor complex. Finally, Rab11 endosomes are transferred to the MyoV (also known as Didum in Drosophila) actin motor to deliver the key microvillar determinant Cadherin 99C to the apical membrane to organise the biogenesis of actin microvilli. PMID:27231092

  3. Detection of Treponema Denticola in Symptomatic Apical Periodontitis and in Symptomatic Apical Abscesses by Real-Time PCR

    PubMed Central

    Ozbek, Selcuk M.; Ozbek, Ahmet; Erdogan, Aziz S.

    2009-01-01

    Objectives: The aim of this study was to investigate the presence of Treponema denticola in symptomatic apical periodontitis and in symptomatic apical abscesses by real-time polymerase chain reaction (PCR) method. Methods: Microbial samples were collected from 60 single-rooted teeth having carious lesions and necrotic pulps. For each tooth, clinical data including patient symptoms were recorded. Teeth were categorized by diagnosis as having symptomatic apical periodontitis or symptomatic apical abscess. Aseptic microbial samples were collected using paper points from 30 infected root canals and from aspirates of 30 abscesses. DNA was extracted from the samples by using a QIAamp® DNA mini-kit and analyzed with real-time PCR. Results: T. denticola was detected in 24 of 30 cases diagnosed as symptomatic apical abscesses (80%), and 19 of 30 cases diagnosed as symptomatic apical periodontitis (63.3%). In general T. denticola was found in 43 of 60 cases (71.6%). Conclusions: Our findings suggest that T. denticola can participate in the pathogenesis of symptomatic apical abscesses. PMID:19421390

  4. The phospholipid flippase ATP8B1 mediates apical localization of the cystic fibrosis transmembrane regulator.

    PubMed

    van der Mark, Vincent A; de Jonge, Hugo R; Chang, Jung-Chin; Ho-Mok, Kam S; Duijst, Suzanne; Vidović, Dragana; Carlon, Marianne S; Oude Elferink, Ronald P J; Paulusma, Coen C

    2016-09-01

    Progressive familial intrahepatic cholestasis type 1 (PFIC1) is caused by mutations in the gene encoding the phospholipid flippase ATP8B1. Apart from severe cholestatic liver disease, many PFIC1 patients develop extrahepatic symptoms characteristic of cystic fibrosis (CF), such as pulmonary infection, sweat gland dysfunction and failure to thrive. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel essential for epithelial fluid transport. Previously it was shown that CFTR transcript levels were strongly reduced in livers of PFIC1 patients. Here we have investigated the hypothesis that ATP8B1 is important for proper CFTR expression and function. We analyzed CFTR expression in ATP8B1-depleted intestinal and pulmonary epithelial cell lines and assessed CFTR function by measuring short-circuit currents across transwell-grown ATP8B1-depleted intestinal T84 cells and by a genetically-encoded fluorescent chloride sensor. In addition, we studied CFTR surface expression upon induction of CFTR transcription. We show that CFTR protein levels are strongly reduced in the apical membrane of human ATP8B1-depleted intestinal and pulmonary epithelial cell lines, a phenotype that coincided with reduced CFTR activity. Apical membrane insertion upon induction of ectopically-expressed CFTR was strongly impaired in ATP8B1-depleted cells. We conclude that ATP8B1 is essential for correct apical localization of CFTR in human intestinal and pulmonary epithelial cells, and that impaired CFTR localization underlies some of the extrahepatic phenotypes observed in ATP8B1 deficiency. PMID:27301931

  5. Monoclonal Antibodies to the Apical Chloride Channel in Necturus Gallbladder Inhibit the Chloride Conductance

    NASA Astrophysics Data System (ADS)

    Finn, Arthur L.; Tsai, Lih-Min; Falk, Ronald J.

    1989-10-01

    Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.

  6. AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption.

    PubMed

    Whiting, Jennifer L; Ogier, Leah; Forbush, Katherine A; Bucko, Paula; Gopalan, Janani; Seternes, Ole-Morten; Langeberg, Lorene K; Scott, John D

    2016-07-26

    Filtration through the kidney eliminates toxins, manages electrolyte balance, and controls water homeostasis. Reabsorption of water from the luminal fluid of the nephron occurs through aquaporin-2 (AQP2) water pores in principal cells that line the kidney-collecting duct. This vital process is impeded by formation of an "actin barrier" that obstructs the passive transit of AQP2 to the plasma membrane. Bidirectional control of AQP2 trafficking is managed by hormones and signaling enzymes. We have discovered that vasopressin-independent facets of this homeostatic mechanism are under the control of A-Kinase Anchoring Protein 220 (AKAP220; product of the Akap11 gene). CRISPR/Cas9 gene editing and imaging approaches show that loss of AKAP220 disrupts apical actin networks in organoid cultures. Similar defects are evident in tissue sections from AKAP220-KO mice. Biochemical analysis of AKAP220-null kidney extracts detected reduced levels of active RhoA GTPase, a well-known modulator of the actin cytoskeleton. Fluorescent imaging of kidney sections from these genetically modified mice revealed that RhoA and AQP2 accumulate at the apical surface of the collecting duct. Consequently, these animals are unable to appropriately dilute urine in response to overhydration. We propose that membrane-proximal signaling complexes constrained by AKAP220 impact the actin barrier dynamics and AQP2 trafficking to ensure water homeostasis. PMID:27402760

  7. Development of Apical Blebbing in the Boar Epididymis

    PubMed Central

    Hughes, Jennifer; Berger, Trish

    2015-01-01

    Microvesicles are of increasing interest in biology as part of normal function of numerous systems; from the immune system (T cell activation) to implantation of the embryo (invasion of the trophoblasts) and sperm maturation (protein transfer in the epididymis). Yet, the mechanisms involved in the appearance of apical blebbing from healthy cells as part of their normal function remain understudied. Microvesicles are produced via one of two pathways: exocytosis or apical blebbing also termed ectocytosis. This work quantifies the histological appearance of apical blebbing in the porcine epididymis during development and examines the role of endogenous estrogens in regulating this blebbing. Apical blebbing appears at puberty and increases in a linear manner into sexual maturity suggesting that this blebbing is a mature phenotype. Endogenous estrogen levels were reduced with an aromatase inhibitor but such a reduction did not affect apical blebbing in treated animals compared with their vehicle-treated littermates. Epididymal production of apical blebs is a secretion mechanism of functionally mature principal cells regulated by factors other than estradiol. PMID:25996942

  8. Haemostatic agents in apical surgery. A systematic review

    PubMed Central

    Clé-Ovejero, Adrià

    2016-01-01

    Background Blood presence in apical surgery can prevent the correct vision of the surgical field, change the physical properties of filling materials and reduce their sealing ability. Objetive To describe which are the most effective and safest haemostatic agents to control bleeding in patients undergoing apical surgery. Material and Methods TWe carried out a systematic review, using Medline and Cochrane Library databases, of human clinical studies published in the last 10 years. Results The agents that proved more effective in bleeding control were calcium sulphate (100%) and collagen plus epinephrine (92.9%) followed by ferric sulphate (60%), gauze packing (30%) and collagen (16.7%). When using aluminium chloride (Expasyl®), over 90% of the apical lesions improved, but this agent seemed to increase swelling. Epinephrine with collagen did not significantly raise either blood pressure or heart rate. Conclusions Despite the use of several haemostatic materials in apical surgery, there is little evidence on their effectiveness and safety. The most effective haemostatic agents were calcium sulphate and epinephrine plus collagen. Epinephrine plus collagen did not seem to significantly raise blood pressure or heart rate during surgery. Aluminium chloride did not increase postoperative pain but could slightly increase postoperative swelling. Randomized clinical trials are needed to assess the haemostatic effectiveness and adverse effects of haemostatic materials in apical surgery. Key words:Haemostasis, apical surgery. PMID:27475689

  9. Healing of apical rarefaction of three nonvital open apex anterior teeth using a white portland cement apical plug

    PubMed Central

    Chakraborty, Amitabha; Dey, Bibhas; Dhar, Reema; Sardar, Prabir

    2012-01-01

    The major challenge of performing root canal treatment in an open apex pulp-less tooth is to obtain a good apical seal. MTA has been successfully used to achieve a good apical seal, wherein the root canal obturation can be done immediately. MTA and White Portland Cement has been shown similarity in their physical, chemical and biological properties and has also shown similar outcome when used in animal studies and human trials. In our study, open apex of three non vital upper central incisors has been plugged using modified white Portland cement. 3 to 6 months follow up revealed absence of clinical symptoms and disappearance of peri-apical rarefactions. The positive clinical outcome may encourage the future use of white Portland cement as an apical plug material in case of non vital open apex tooth as much cheaper substitute of MTA. PMID:23230357

  10. Differentiation of Apical Bud Cells in a Newly Developed Apical Bud Transplantation Model Using GFP Transgenic Mice as Donor

    PubMed Central

    Sakagami, Ryuji; Yoshinaga, Yasunori; Okamura, Kazuhiko

    2016-01-01

    Rodent mandibular incisors have a unique anatomical structure that allows teeth to grow throughout the lifetime of the rodent. This report presents a novel transplantation technique for studying the apical bud differentiation of rodent mandibular incisors. Incisal apical end tissue with green fluorescent protein from transgenic mouse was transplanted to wild type mice, and the development of the transplanted cells were immunohistologically observed for 12 weeks after the transplantation. Results indicate that the green fluorescent apical end tissue replaced the original tissue, and cells from the apical bud differentiated and extended toward the incisal edge direction. The immunostaining with podoplanin also showed that the characteristics of the green fluorescent tissue were identical to those of the original. The green fluorescent cells were only found in the labial side of the incisor up to 4 weeks. After 12 weeks, however, they were also found in the lingual side. Here the green fluorescent cementocyte-like cells were only present in the cementum close to the dentin surface. This study suggests that some of the cells that form the cellular cementum come from the apical tissue including the apical bud in rodent incisors. PMID:26978064

  11. Annexin A2 Mediates Apical Trafficking of Renal Na+-K+-2Cl− Cotransporter*

    PubMed Central

    Dathe, Christin; Daigeler, Anna-Lena; Seifert, Wenke; Jankowski, Vera; Mrowka, Ralf; Kalis, Ronny; Wanker, Erich; Mutig, Kerim; Bachmann, Sebastian; Paliege, Alexander

    2014-01-01

    The furosemide-sensitive Na+-K+-2Cl− cotransporter (NKCC2) is responsible for urine concentration and helps maintain systemic salt homeostasis. Its activity depends on trafficking to, and insertion into, the apical membrane, as well as on phosphorylation of conserved N-terminal serine and threonine residues. Vasopressin (AVP) signaling via PKA and other kinases activates NKCC2. Association of NKCC2 with lipid rafts facilitates its AVP-induced apical translocation and activation at the surface. Lipid raft microdomains typically serve as platforms for membrane proteins to facilitate their interactions with other proteins, but little is known about partners that interact with NKCC2. Yeast two-hybrid screening identified an interaction between NKCC2 and the cytosolic protein, annexin A2 (AnxA2). Annexins mediate lipid raft-dependent trafficking of transmembrane proteins, including the AVP-regulated water channel, aquaporin 2. Here, we demonstrate that AnxA2, which binds to phospholipids in a Ca2+-dependent manner and may organize microdomains, is codistributed with NKCC2 to promote its apical translocation in response to AVP stimulation and low chloride hypotonic stress. NKCC2 and AnxA2 interact in a phosphorylation-dependent manner. Phosphomimetic AnxA2 carrying a mutant phosphoacceptor (AnxA2-Y24D-GFP) enhanced surface expression and raft association of NKCC2 by 5-fold upon low chloride hypotonic stimulation, whereas AnxA2-Y24A-GFP and PKC-dependent AnxA2-S26D-GFP did not. As the AnxA2 effect involved only nonphosphorylated NKCC2, it appears to affect NKCC2 trafficking. Overexpression or knockdown experiments further supported the role of AnxA2 in the apical translocation and surface expression of NKCC2. In summary, this study identifies AnxA2 as a lipid raft-associated trafficking factor for NKCC2 and provides mechanistic insight into the regulation of this essential cotransporter. PMID:24526686

  12. Use of human chorionic gonadotropin in a male Pacific walrus (Odobenus rosmarus divergens) to induce rut and achieve a pregnancy in a nulliparous female.

    PubMed

    Muraco, Holley S; Coombs, Leah D; Procter, Dianna G; Turek, Paul J; Muraco, Michael J

    2012-01-01

    Walrus in US zoos have a very low reproductive rate of 11 births in 80 years, and little is known about Pacific walrus (Odobenus rosmarus divergens) reproductive biology. To address this, we initiated a program in which detailed biological data were recorded on captive walrus. As part of a 7-year study, 1 male and 1 female 16-year-old captive Pacific walrus were carefully monitored with weekly serum hormone analysis, daily glans penis smears for spermatozoa, and abdominal ultrasound for pregnancy. The female ovulated once annually from late December through mid-January and then exhibited 9 months of sustained elevated progesterone. This nonconceptive estrous cycle profile is consistent with reports from wild walrus females. In contrast, the male's seasonal rut routinely occurred in late February through May with a serum testosterone peak in March. This profile differed from the reported adult male cycle in wild walrus of November through March. During the period of the female's ovulation, the male had nadir testosterone levels and was consistently azoospermic. Likewise, during the male's spermatogenic rut in the spring, the female was anovulatory with elevated progesterone. On this basis, the male was treated for 14 weeks with human chorionic gonadotropin (hCG) in an attempt to increase testosterone levels in synchrony with the female's annual ovulation. The treatment successfully induced rut characterized by sustained elevated serum testosterone levels and production of spermatozoa. The male and female successfully bred, and the female became pregnant. Upon discontinuation of hCG treatment, the male resumed baseline testosterone levels. We theorize that the lack of synchronization of rut and ovulatory cycles is a primary reason for reproductive failure in these captive walrus. PMID:22207706

  13. Participation of IAA in transduction of gravistimulus in apical cells of moss protonema

    NASA Astrophysics Data System (ADS)

    Oksyniuk, U. A.; Khorkavtsiv, O. Y.; Lesniak, Y. I.

    carried out experiments it can be suggested that high concentrations of IAA and 1-NAA result in surplus of IAA cells led, probably, to a destruction of the apical-basal gradient in cells. Our results testify that NPA inhibits the gravitropism stronger than the growth of protonema. The peculiarity of moss protonema is that the growth orientation change is a result of a transference of growth zone in the apical cell dome caused by amyloplasts sedimentation inducing lateral asymmetry of Ca2+ and apical-basal IAA flow what in its turn manifests itself in distribution of IAA and/or Ca2+ channels in apical cell dome plasma membrane ( Schwuchow et al., 2001). The transport of IAA in apical cells, probably, functionally polarizes it and just that polarizing function is dominant in cells with tip growth.

  14. microRNA Expression in Rat Apical Periodontitis Bone Lesion

    PubMed Central

    Gao, Bo; Zheng, Liwei

    2013-01-01

    Apical periodontitis, dominated by dense inflammatory infiltrates and increased osteoclast activities, can lead to alveolar bone destruction and tooth loss. It is believed that miRNA participates in regulating various biological processes, osteoclastogenesis included. This study aims to investigate the differential expression of miRNAs in rat apical periodontitis and explore their functional target genes. Microarray analysis was used to identify differentially expressed miRNAs in apical periodontitis. Bioinformatics technique was applied for predicting the target genes of differentially expressed miRNAs and their biological functions. The result provided us with an insight into the potential biological effects of the differentially expressed miRNAs and showed particular enrichment of target genes involved in the MAPK signaling pathways. These findings may highlight the intricate and specific roles of miRNA in inflammation and osteoclastogenesis, both of which are key aspects of apical periodontitis, thus contributing to the future investigation into the etiology, underlying mechanism and treatment of apical periodontitis. PMID:26273501

  15. Rab6 Is Required for Multiple Apical Transport Pathways but Not the Basolateral Transport Pathway in Drosophila Photoreceptors

    PubMed Central

    Liu, Ziguang; Satoh, Akiko K.

    2016-01-01

    Polarized membrane trafficking is essential for the construction and maintenance of multiple plasma membrane domains of cells. Highly polarized Drosophila photoreceptors are an excellent model for studying polarized transport. A single cross-section of Drosophila retina contains many photoreceptors with 3 clearly differentiated plasma membrane domains: a rhabdomere, stalk, and basolateral membrane. Genome-wide high-throughput ethyl methanesulfonate screening followed by precise immunohistochemical analysis identified a mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with normal basolateral transport. Rapid gene identification using whole-genome resequencing and single nucleotide polymorphism mapping identified a nonsense mutation of Rab6 responsible for the apical-specific transport deficiency. Detailed analysis of the trafficking of a major rhabdomere protein Rh1 using blue light-induced chromophore supply identified Rab6 as essential for Rh1 to exit the Golgi units. Rab6 is mostly distributed from the trans-Golgi network to a Golgi-associated Rab11-positive compartment that likely recycles endosomes or transport vesicles going to recycling endosomes. Furthermore, the Rab6 effector, Rich, is required for Rab6 recruitment in the trans-Golgi network. Moreover, a Rich null mutation phenocopies the Rab6 null mutant, indicating that Rich functions as a guanine nucleotide exchange factor for Rab6. The results collectively indicate that Rab6 and Rich are essential for the trans-Golgi network–recycling endosome transport of cargoes destined for 2 apical domains. However, basolateral cargos are sorted and exported from the trans-Golgi network in a Rab6-independent manner. PMID:26890939

  16. Apical sorting of a voltage- and Ca2+-activated K+ channel alpha -subunit in Madin-Darby canine kidney cells is independent of N-glycosylation.

    PubMed

    Bravo-Zehnder, M; Orio, P; Norambuena, A; Wallner, M; Meera, P; Toro, L; Latorre, R; González, A

    2000-11-21

    The voltage- and Ca(2+)-activated K(+) (K(V,Ca)) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel K(V,Ca) alpha-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., K(V,Ca) beta-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells. PMID:11069304

  17. Apical sorting of a voltage- and Ca2+-activated K+ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

    PubMed Central

    Bravo-Zehnder, Marcela; Orio, Patricio; Norambuena, Andrés; Wallner, Martin; Meera, Pratap; Toro, Ligia; Latorre, Ramón; González, Alfonso

    2000-01-01

    The voltage- and Ca2+-activated K+ (KV,Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel KV,Ca α-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., KV,Ca β-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells. PMID:11069304

  18. Causes and management of post-treatment apical periodontitis.

    PubMed

    Siqueira, J F; Rôças, I N; Ricucci, D; Hülsmann, M

    2014-03-01

    Endodontic treatment failure is usually characterised by the presence of post-treatment apical periodontitis, which may be persistent, emergent or recurrent. The major aetiology of post-treatment disease is persistent intraradicular infection, but in some cases a secondary intraradicular infection due to coronal leakage or an extraradicular infection may be the cause of failure. Understanding the causes of endodontic treatment failure is of paramount importance for the proper management of this condition. Teeth with post-treatment apical periodontitis can be managed by either nonsurgical endodontic retreatment or periradicular surgery, both of which have very high chances of restoring the health of the periradicular tissues and maintaining the tooth function in the oral cavity. This review article focuses on the aetiological factors of post-treatment apical periodontitis and discusses the indications and basics of the procedures for optimal clinical management of this condition. PMID:24651336

  19. Phosphatidylcholine passes through lateral tight junctions for paracellular transport to the apical side of the polarized intestinal tumor cell-line CaCo2.

    PubMed

    Stremmel, Wolfgang; Staffer, Simone; Gan-Schreier, Hongying; Wannhoff, Andreas; Bach, Margund; Gauss, Annika

    2016-09-01

    Phosphatidylcholine (PC) is the most abundant phospholipid in intestinal mucus, indicative of a specific transport system across the mucosal epithelium to the intestinal lumen. To elucidate this transport mechanism, we employed a transwell tissue culture system with polarized CaCo2 cells. It was shown that PC could not substantially be internalized by the cells. However, after basal application of increasing PC concentrations, an apical transport of 47.1±6.3nmolh(-1)mMPC(-1) was observed. Equilibrium distribution studies with PC applied in equal concentrations to the basal and apical compartments showed a 1.5-fold accumulation on the expense of basal PC. Disruption of tight junctions (TJ) by acetaldehyde or PPARγ inhibitors or by treatment with siRNA to TJ proteins suppressed paracellular transport by at least 50%. Transport was specific for the choline containing the phospholipids PC, lysoPC and sphingomyelin. We showed that translocation is driven by an electrochemical gradient generated by apical accumulation of Cl(-) and HCO3(-) through CFTR. Pretreatment with siRNA to mucin 3 which anchors in the apical plasma membrane of mucosal cells inhibited the final step of luminal PC secretion. PC accumulates in intestinal mucus using a paracellular, apically directed transport route across TJs. PMID:27365309

  20. Cargo-selective apical exocytosis in epithelial cells is conducted by Myo5B, Slp4a, Vamp7, and Syntaxin 3

    PubMed Central

    Vogel, Georg F.; Klee, Katharina M.C.; Janecke, Andreas R.; Müller, Thomas

    2015-01-01

    Mutations in the motor protein Myosin Vb (Myo5B) or the soluble NSF attachment protein receptor Syntaxin 3 (Stx3) disturb epithelial polarity and cause microvillus inclusion disease (MVID), a lethal hereditary enteropathy affecting neonates. To understand the molecular mechanism of Myo5B and Stx3 interplay, we used genome editing to introduce a defined Myo5B patient mutation in a human epithelial cell line. Our results demonstrate a selective role of Myo5B and Stx3 for apical cargo exocytosis in polarized epithelial cells. Apical exocytosis of NHE3, CFTR (cystic fibrosis transmembrane conductance regulator), and GLUT5 required an interaction cascade of Rab11, Myo5B, Slp4a, Munc18-2, and Vamp7 with Stx3, which cooperate in the final steps of this selective apical traffic pathway. The brush border enzymes DPPIV and sucrase-isomaltase still correctly localize at the apical plasma membrane independent of this pathway. Hence, our work demonstrates how Myo5B, Stx3, Slp4a, Vamp7, Munc18-2, and Rab8/11 cooperate during selective apical cargo trafficking and exocytosis in epithelial cells and thereby provides further insight into MVID pathophysiology. PMID:26553929

  1. Dental Apical Papilla as Therapy for Spinal Cord Injury.

    PubMed

    De Berdt, P; Vanacker, J; Ucakar, B; Elens, L; Diogenes, A; Leprince, J G; Deumens, R; des Rieux, A

    2015-11-01

    Stem cells of the apical papilla (SCAP) represent great promise regarding treatment of neural tissue damage, such as spinal cord injury (SCI). They derive from the neural crest, express numerous neurogenic markers, and mediate neurite outgrowth and axonal targeting. The goal of the present work was to investigate for the first time their potential to promote motor recovery after SCI in a rat hemisection model when delivered in their original stem cell niche-that is, by transplantation of the human apical papilla tissue itself into the lesion. Control groups consisted of animals subjected to laminectomy only (shams) and to lesion either untreated or injected with a fibrin hydrogel with or without human SCAP. Basso-Beattie-Bresnahan locomotor scores at 1 and 3 d postsurgery confirmed early functional decline in all SCI groups. This significant impairment was reversed, as seen in CatWalk analyses, after transplantation of apical papilla into the injured spinal cord wound, whereas the other groups demonstrated persistent functional impairment. Moreover, tactile allodynia did not develop as an unwanted side effect in any of the groups, even though the SCAP hydrogel group showed higher expression of the microglial marker Iba-1, which has been frequently associated with allodynia. Notably, the apical papilla transplant group presented with reduced Iba-1 expression level. Masson trichrome and human mitochondria staining showed the preservation of the apical papilla integrity and the presence of numerous human cells, while human cells could no longer be detected in the SCAP hydrogel group at the 6-wk postsurgery time point. Altogether, our data suggest that the transplantation of a human apical papilla at the lesion site improves gait in spinally injured rats and reduces glial reactivity. It also underlines the potential interest for the application of delivering SCAP in their original niche, as compared with use of a fibrin hydrogel. PMID:26341974

  2. Robustness of sensory-evoked excitation is increased by inhibitory inputs to distal apical tuft dendrites

    PubMed Central

    Egger, Robert; Schmitt, Arno C.; Wallace, Damian J.; Sakmann, Bert; Oberlaender, Marcel; Kerr, Jason N. D.

    2015-01-01

    Cortical inhibitory interneurons (INs) are subdivided into a variety of morphologically and functionally specialized cell types. How the respective specific properties translate into mechanisms that regulate sensory-evoked responses of pyramidal neurons (PNs) remains unknown. Here, we investigated how INs located in cortical layer 1 (L1) of rat barrel cortex affect whisker-evoked responses of L2 PNs. To do so we combined in vivo electrophysiology and morphological reconstructions with computational modeling. We show that whisker-evoked membrane depolarization in L2 PNs arises from highly specialized spatiotemporal synaptic input patterns. Temporally L1 INs and L2–5 PNs provide near synchronous synaptic input. Spatially synaptic contacts from L1 INs target distal apical tuft dendrites, whereas PNs primarily innervate basal and proximal apical dendrites. Simulations of such constrained synaptic input patterns predicted that inactivation of L1 INs increases trial-to-trial variability of whisker-evoked responses in L2 PNs. The in silico predictions were confirmed in vivo by L1-specific pharmacological manipulations. We present a mechanism—consistent with the theory of distal dendritic shunting—that can regulate the robustness of sensory-evoked responses in PNs without affecting response amplitude or latency. PMID:26512104

  3. Robustness of sensory-evoked excitation is increased by inhibitory inputs to distal apical tuft dendrites.

    PubMed

    Egger, Robert; Schmitt, Arno C; Wallace, Damian J; Sakmann, Bert; Oberlaender, Marcel; Kerr, Jason N D

    2015-11-10

    Cortical inhibitory interneurons (INs) are subdivided into a variety of morphologically and functionally specialized cell types. How the respective specific properties translate into mechanisms that regulate sensory-evoked responses of pyramidal neurons (PNs) remains unknown. Here, we investigated how INs located in cortical layer 1 (L1) of rat barrel cortex affect whisker-evoked responses of L2 PNs. To do so we combined in vivo electrophysiology and morphological reconstructions with computational modeling. We show that whisker-evoked membrane depolarization in L2 PNs arises from highly specialized spatiotemporal synaptic input patterns. Temporally L1 INs and L2-5 PNs provide near synchronous synaptic input. Spatially synaptic contacts from L1 INs target distal apical tuft dendrites, whereas PNs primarily innervate basal and proximal apical dendrites. Simulations of such constrained synaptic input patterns predicted that inactivation of L1 INs increases trial-to-trial variability of whisker-evoked responses in L2 PNs. The in silico predictions were confirmed in vivo by L1-specific pharmacological manipulations. We present a mechanism-consistent with the theory of distal dendritic shunting-that can regulate the robustness of sensory-evoked responses in PNs without affecting response amplitude or latency. PMID:26512104

  4. Effects of DIDS on the chick retinal pigment epithelium. I. Membrane potentials, apparent resistances, and mechanisms.

    PubMed

    Gallemore, R P; Steinberg, R H

    1989-06-01

    While little is known about the transport properties of the retinal pigment epithelium (RPE) basal membrane, mechanisms for anion movement across the basal membrane appear to be present (Miller and Steinberg, 1977; Hughes et al., 1984; Miller and Farber, 1984). This work examines the electrophysiological effects of the anion conductance blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) on the basal membrane of an in vitro preparation of chick retina-RPE-choroid. DIDS (10-125 microM), added to the choroidal bath, decreased the transtissue potential by decreasing the potential across the RPE. Intracellular RPE recordings showed that DIDS affected the membrane potential in 2 phases, initially hyperpolarizing the basal membrane and then, after prolonged exposure, depolarizing the apical membrane. Resistance assessment by transtissue current pulses and intracellular c-wave recordings suggested that DIDS increased basal membrane resistance (Rba) during the first phase and increased apical membrane resistance (Rap) during the second phase. Measurements of intracellular Cl- activity (aiCl) showed that Cl- was actively accumulated by the chick RPE since it was distributed above equilibrium across both the apical and basal membranes. Perfusion of the basal membrane with 50 microM DIDS significantly increased aiCl-. The DIDS-induced basal membrane hyperpolarization, apparent increase in Rba, and increase in aiCl- are all consistent with Cl- -conductance blockade. During the second phase, apical membrane responsiveness to the light-evoked decrease in subretinal [K+]o (Oakley, 1977) was reduced an average of 58%. This finding, given the second-phase apical membrane depolarization and apparent increase in Rap, is consistent with a decrease in apical membrane K+ conductance.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2723761

  5. Chapter Four - Shoot apical meristem form and function. In:

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The shoot apical meristem (SAM) generates above-ground aerial organs throughout the lifespan of higher plants. In order to fulfill this function, the meristem must maintain a balance between the self-renewal of a reservoir of central stem cells and organ initiation from peripheral cells. The activit...

  6. [Nonsurgical retreatment in a case of a radiolucent apical lesion].

    PubMed

    Vicente Gómez, A; Rodríguez Ponce, A

    1989-01-01

    We present a case of failure that was helpful solved without surgical endodontic treatment. We don't achieve clinical success besides endodontic treatment was twice remade. Finally we decided to put a temporary filling with calcium hydroxide and wait until apical radiolucency disappear and complete our treatment with gutta-percha, sealer and lateral condensation. PMID:2640036

  7. Echocardiographic assessment of takotsubo cardiomyopathy: beyond apical ballooning.

    PubMed

    Okura, Hiroyuki

    2016-03-01

    It has been >25 years since the first report of the takotsubo cardiomyopathy (TC). Although left ventriculography was originally used to depict its typical and impressive wall motion abnormality mimicking "takotsubo", or octopus pot, echocardiography plays a pivotal role in detecting not only its left ventricular (LV) wall motion abnormality, apical ballooning, but also various other findings. First of all, apical ballooning is not an essential finding for TC anymore. Mid-ventricular LV asynergy with or without apical involvement is a basic pattern of the LV wall motion abnormality. Distribution and time course of the asynergy may be best detected by echocardiography and echo provides useful information to differentiate between TC and acute coronary syndrome or acute myocarditis. In addition to the wall motion assessment, echo detects complications of TC such as systolic anterior motion of the mitral leaflet with or without LV outflow obstruction, mitral regurgitation, LV thrombus, right ventricular (RV) involvement. In particular, RV involvement is not an uncommon finding and is associated with worse short-term as well as long-term prognosis. Finally, coronary flow measurements and speckle tracking by echo may offer additional and useful information about pathophysiology and prognosis of TC. In conclusion, echocardiography is a standard imaging modality for detecting various dynamic findings beyond apical ballooning in patients with TC. PMID:26694809

  8. Apical and basolateral ATP stimulates tracheal epithelial chloride secretion via multiple purinergic receptors.

    PubMed

    Hwang, T H; Schwiebert, E M; Guggino, W B

    1996-06-01

    Stimulation of Cl- secretion across the airway epithelium by ATP or UTP as agonists has therapeutic implications for cystic fibrosis. Our results demonstrate that ATP stimulates Cl- secretion in rat tracheal epithelial cell monolayers in primary culture from the apical or basolateral side of the monolayer. Multiple types of ATP-sensitive Cl- conductances in intact monolayers were elucidated through inhibition by Cl- channel-blocking drugs. Multiple Cl- conductances stimulated by ATP and adenosine 3',5'-cyclic monophosphate (cAMP) (tested for comparison) were also deciphered more specifically by nystatin permeabilization of the basolateral membrane, subsequent imposition of symmetrical Cl-, I-, or Br- solutions to test halide permselectivity, inhibition by Cl- channel-blocking drugs, and construction of current-voltage plots to study time and voltage dependence of the currents. Apical ATP stimulates Cl- secretion through P2U (or P2Y2) purinergic receptors via both intracellular Ca2+ (Ca(2+)i)-dependent and Cai(2+)-independent signaling pathways by opening outwardly rectifying Cl- channels (ORCCs), cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, and Cai(2+)-dependent Cl- channels. Basolateral ATP stimulates Cl- secretion via a combination of receptor subtypes (P2T and P2U) or a novel type of receptor (P2Y3), independent of Cai2+ or cAMP signaling by opening only CFTR channels. cAMP also stimulated multiple types of Cl- conductances, consistent with simultaneous activation of CFTR and ORCCs. Together, these results suggest that ATP as an agonist stimulates Cl- secretion via multiple purinergic receptors and multiple signal transduction pathways activated in different membrane domains of tracheal epithelia. PMID:8764143

  9. chaoptin, prominin, eyes shut and crumbs form a genetic network controlling the apical compartment of Drosophila photoreceptor cells

    PubMed Central

    Gurudev, Nagananda; Yuan, Michaela; Knust, Elisabeth

    2014-01-01

    ABSTRACT The apical surface of epithelial cells is often highly specialised to fulfil cell type-specific functions. Many epithelial cells expand their apical surface by forming microvilli, actin-based, finger-like membrane protrusions. The apical surface of Drosophila photoreceptor cells (PRCs) forms tightly packed microvilli, which are organised into the photosensitive rhabdomeres. As previously shown, the GPI-anchored adhesion protein Chaoptin is required for the stability of the microvilli, whereas the transmembrane protein Crumbs is essential for proper rhabdomere morphogenesis. Here we show that chaoptin synergises with crumbs to ensure optimal rhabdomere width. In addition, reduction of crumbs ameliorates morphogenetic defects observed in PRCs mutant for prominin and eyes shut, known antagonists of chaoptin. These results suggest that these four genes provide a balance of adhesion and anti-adhesion to maintain microvilli development and maintenance. Similar to crumbs mutant PRCs, PRCs devoid of prominin or eyes shut undergo light-dependent retinal degeneration. Given the observation that human orthologues of crumbs, prominin and eyes shut result in progressive retinal degeneration and blindness, the Drosophila eye is ideally suited to unravel the genetic and cellular mechanisms that ensure morphogenesis of PRCs and their maintenance under light-mediated stress. PMID:24705015

  10. Cultured enterocytes internalise bacteria across their basolateral surface for, pathogen-inhibitable, trafficking to the apical compartment

    PubMed Central

    Dean, Paul; Quitard, Sabine; Bulmer, David M.; Roe, Andrew J.; Kenny, Brendan

    2015-01-01

    In vitro- and in vivo-polarised absorptive epithelia (enterocytes) are considered to be non-phagocytic towards bacteria with invasive pathogenic strains relying on virulence factors to ‘force’ entry. Here, we report a serendipitous discovery that questions these beliefs. Thus, we uncover in well-established models of human small (Caco-2; TC-7) and large (T84) intestinal enterocytes a polarization-dependent mechanism that can transfer millions of bacteria from the basal to apical compartment. Antibiotic-protection assays, confocal imaging and drug inhibitor data are consistent with a transcellular route in which internalized, basolateral-membrane enclosed bacteria are trafficked to and across the apical surface. Basal-to-apical transport of non-pathogenic bacteria (and inert beads) challenged the idea of pathogens relying on virulence factors to force entry. Indeed, studies with Salmonella demonstrated that it’s entry-forcing virulence factor (SPI-I) was not required to enter via the basolateral surface but to promote another virulence-associated event (intra-enterocyte accumulation). PMID:26612456