Association with ZO-1 Correlates with Plasma Membrane Partitioning in Truncated Connexin45 Mutants

Zonula occludens-1 (ZO-1), the most abundant known connexin-interacting protein in osteoblastic cells, associates with the carboxyl termini of both Cx43 and Cx45. To learn more about the role of the cormexin-ZO-1 interaction, we analyzed connexin trafficking and function in ROS 17/2.8 cells that were stably transfected either with full length Cx45 or with Cx45 lacking 34 or 37 amino acids on the carboxyl terminus (Cx45t34 or Cx45t37). All three proteins were transported to appositional membranes in the transfected cells: Cx45 and Cx45t34 displayed a punctate appositional membrane-staining pattern, while Cx45t37 staining at appositional membranes was more linear. Expression of Cx45 decreased gap junction communication as assayed by dye transfer, while expression of Cx45t34 or Cx45t37 increased the amount of dye transfer seen in these cells. We found that Cx43, Cx45 and Cx45t34 co-precipitated with ZO-1 in these cells, while Cx45t37 did not. We also found that Cx45t37 was much more soluble in 1% Triton X-100 than the other connexins examined. In addition, Cx45t37 migrated to a fraction of lighter buoyant density on sucrose flotation gradients than Cx43, Cx45, ZO-1 and Cx45t34. As ZO-1 is an actin-binding protein, this suggested that the differences in Cx45t37 solubility might be due to a difference between the interaction of gap junctions and the actin cytoskeleton in the ROS/Cx45t37 and in the other transfected ROS cells. To examine this possibility, the transfected ROS cells were stained with fluorescently labeled phalloidin and demonstrated that there was a notable loss of actin stress fibers in the ROS/Cx45t37 cells. These findings suggest that association with ZO-1 alters the plasma membrane localization of Cx45 by removing it from a lipid raft compartment and rendering it Triton-insoluble, presumably by promoting an interaction with the actin cytoskeleton; they also suggest that Cx45 has a complex binding interaction with ZO-1 that involves either an extended carboxyl terminal domain or two distinct binding sites.     

Analysis of liquid extracts from tree and grass pollens by capillary electromigration methods

Capillary electromigration methods, zone electrophoresis (CZE), micellar electrokinetic chromatography (CMEKC) and isotachophoresis (CITP), have been used for analysis of water and water-buffer extracts from tree-common birch (Betula verrucosa) and grass-orchardgrass (Dactylis glomerata) pollen samples. Water extracts were analyzed by CZE using acetic acid as background electrolyte (BGE), by CMEKC in tris-phosphate BGE with anionic detergent sodium dodecyl sulfate (SDS) micellar pseudophase (TP-SDS) and by CITP in cationic mode with leading/terminating cations K+/BALA+ (beta-alanine (BALA)) and in anionic mode with leading/terminating anions Cl-/MES- (2-(N-morpholino)ethanesulphonic acid (MES)). Moreover, acetic acid extracts were analyzed by CZE using acetic acid as BGE, and alkaline water-SDS-buffer extracts were analyzed by CMEKC using TP-SDS as BGE. Extracted amounts of pollen allergens and other UV-absorbing compounds and the number of resolved components were evaluated from CZE, CMEKC and CITP analyses of the liquid extracts. Larger amounts of UV-absorbing material were found in the water-buffer pollen extracts than in the water extracts. More UV-absorbing material was found in all extracts from D. glomerata pollen than in relevant extracts from B. verrucosa pollen. It was found by CITP that the extracted amounts of anionic components and their number were much higher than those of cationic components. Concentrations of some inorganic ions (e.g. Cl-, K+, Na+, Ca2+) in pollen samples were also determined by CITP.     

Photosensitized and catalytic oxidation of DNA by metallophthalocyanine-oligonucleotide conjugates

The synthesis of new metallophthalocyanine-oligonucleotide conjugates is reported. These conjugates can cause sequence-specific photosensitized or catalytic oxidation of DNA by molecular oxygen.     

The axosomatic contacts on the bursting neuron of the snailHelix pomatia. I. ultrastructural features of the axosomatic contacts

The analysis of serial ultrathin sections of the RPAI bursting neuron of the snail Helix pomatia reveals the presence of axosomatic contacts on its surface membrane. These contacts have a number of specific features: the presynaptic axon contains synaptic vesicles and electron-dense granules, typical of peptidergic terminals; the terminal part of the axon forms many finger-like processes which invaginate the neuronal soma; the width of the cleft (80 nm) in the area of the contact is larger than that in usual synaptic contacts; and there is a system of lacoons in the region of the axosomatic contact; this system is formed by protrusions of the soma and it accompanies the contact along its extent. It is suggested that the system of lacoons which communicates with the space between the terminal and the soma may serve as a ramified synaptic cleft into which the secretion from the terminal is released. This system may contribute to a considerable prolongation of the time of action of the secretory product on the membrane of the RPAI neuron.     

Differential effects of claudin-3 and claudin-4 on alveolar epithelial barrier function

Alveolar barrier function depends critically on the claudin family tight junction proteins. Of the major claudins expressed by alveolar epithelial cells, claudin (Cldn)-3 and Cldn-4 are the most closely related by amino acid homology, yet they differ dramatically in the pattern of expression. Previously published reports have shown that Cldn-3 is predominantly expressed by type II alveolar epithelial cells; Cldn-4 is expressed throughout the alveolar epithelium and is specifically upregulated in response to acute lung injury. Using primary rat alveolar epithelial cells transduced with yellow fluorescent protein-tagged claudin constructs, we have identified roles for Cldn-3 and Cldn-4 in alveolar epithelial barrier function. Surprisingly, increasing expression of Cldn-3 decreased alveolar epithelial barrier function, as assessed by transepithelial resistance and dye flux measurements. Conversely, increasing Cldn-4 expression improved alveolar epithelial transepithelial resistance compared with control cells. Other alveolar epithelial tight junction proteins were largely unaffected by increased expression of Cldn-3 and Cldn-4. Taken together, these results demonstrate that, in the context of the alveolar epithelium, Cldn-3 and Cldn-4 have different effects on paracellular permeability, despite significant homology in their extracellular loop domains.     

Phenylarsine Oxide Binding Reveals Redox-Active and Potential Regulatory Vicinal Thiols on the Catalytic Subunit of Protein Phosphatase 2A

Our earlier finding that the activity of protein phosphatase 2A from rat brain is inhibited by micromolar concentrations of the dithiol cross-linking reagent phenylarsine oxide (PAO) has encouraged the hypothesis that the catalytic subunit (PP2Ac) of PP2A contains one or more pairs of closely-spaced (vicinal) thiol pairs that may contribute to regulation of the enzyme. The results of the present study demonstrate using immobilized PAO-affinity chromatography that PP2Ac from rat brain formed stable DTT-sensitive adducts with PAO with or without associated regulatory subunits. In addition, a subset of the PAO-binding vicinal thiols of PP2Ac was readily oxidized to disulfide bonds in vitro. Importantly, a small fraction of PP2Ac was still found to contain disulfide bonds after applying stringent conditions designed to prevent protein disulfide bond formation during homogenization and fractionation of the brains. These findings establish the presence of potentially regulatory and redox-active PAO-binding vicinal thiols on the catalytic subunit of PP2A and suggest that a population of PP2Ac may contain disulfide bonds in vivo.     

Ion channels of various types induced in lipid membranes by gramicidin a derivatives carrying a cationic sequence at their C-termini

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously (FEBS Lett., 2005, vol. 579, pp. 5247–5252) that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both β^6.3 single-stranded and β^5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.     

Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and β-Catenin Signaling

Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of β-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-β inhibitor SB431542 and the Wnt/β-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and β-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future.