Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds

The flavonoid genistein and the benzo[c]quinolizinium MPB-07 have been shown to activate the cystic fibrosis transmembrane conductance regulator (CFTR), the protein that is defective in cystic fibrosis. Lead-based combinatorial and parallel synthesis yielded 223 flavonoid, quinolizinium, and related heterocyclic compounds. The compounds were screened for their ability to activate CFTR at 50 microm concentration by measurement of the kinetics of iodide influx in Fisher rat thyroid cells expressing wild-type or G551D CFTR together with the green fluorescent protein-based halide indicator YFP-H148Q. Duplicate screenings revealed that 204 compounds did not significantly affect CFTR function. Compounds of the 7,8-benzoflavone class, which are structurally intermediate between flavones and benzo[c]quinoliziniums, were effective CFTR activators with the most potent being 2-(4-pyridinium)benzo[h]4H-chromen-4-one bisulfate (UCcf-029). Compounds of the novel structural class of fused pyrazolo heterocycles were also strong CFTR activators with the most potent being 3-(3-butynyl)-5-methoxy-1-phenylpyrazole-4-carbaldehyde (UCcf-180). A CFTR inhibitor was also identified. The active compounds did not induce iodide influx in null cells deficient in CFTR. Short-circuit current measurements showed that the CFTR activators identified by screening induced strong anion currents in the transfected cell monolayers grown on porous supports. Compared with genistein, the most active compounds had up to 10 times greater potency in activating wild-type and/or G551D-CFTR. The activators had low cellular toxicity and did not elevate cellular cAMP concentration or inhibit phosphatase activity, suggesting that CFTR activation may involve a direct interaction. These results establish an efficient screening procedure to identify CFTR activators and inhibitors and have identified 7,8-benzoflavones and pyrazolo derivatives as novel classes of CFTR activators.     

Novel lectin-like proteins on the surface of human monocytic leukemia cell line THP-1 cells that recognize oxidized cells

Presence of lectin-like receptors on the membranes of human monocytic leukemia cell line THP-1 cells for clustered sialylated poly-N-acetyllactosaminyl sugar chains on the membranes of oxidized erythrocytes and T-lympoid cells was investigated. Membranes of THP-1 cells differentiated into macrophages were solubilized, and the membrane proteins obtained by affinity chromatographies using lactoferrin-Sepharose and band 3-Sepharose were purified by successive DE column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins of 50, 60, and 80 kDa with specificity to bind to sialylated poly-N-acetyllactosaminyl sugar chains were detected in the chromatographic fractions. A 50-kDa protein was isolated in a pure form. N-Terminal amino acid sequence of the protein was Lys-Gln-Lys-Val-Ala-Gly-Lys-Gln-Pro-Val-, which has not been found in the N-terminal regions of the hitherto known proteins. The antibody, raised against the chemially synthesized peptide composed of the N-terminal amino acid sequence, bound to 50-, 60-, and 80-kDa proteins as analyzed by immunoblotting and immunoprecipitation, indicating that these proteins had the same N-terminal amino acid sequence. The results demonstrate that THP-1 cells have novel 50-, 60-, and 80-kDa lectin-like proteins with the same N-terminal amino acid sequence on the cell surface which would bind to clustered sialylated poly-N-acetyllactosaminyl sugar chains generated on oxidized erythrocytes and T-lymphoid cells.     

Conversion of gastric mucosa to intestinal metaplasia in Cdx2-expressing transgenic mice

Gastric intestinal metaplasia occurs as a pathological condition in the gastric mucosa. To clarify how an intestine-specific homeobox gene, Cdx2, affects the morphogenesis of gastric mucosa, we generated transgenic mice expressing Cdx2 in parietal cells. Until Day 18 after birth, the number of parietal cells inthegastric mucosa of transgenic mice was the same as for their normal littermates. However, at Day 19, we detected several glands in which parietal cells disappeared and the proliferating zone moved from the isthmus to the base of the glands. Thereafter, parietal cells decreased gradually and disappeared at Day 37. All of the gastric mucosal cells, except for enterochromaffin-like (ECL) cells, were completely replaced by intestinal metaplasia, consisting of goblet cells, enteroendocrine cells, and absorptive cells expressing alkaline phosphatase. Pseudopyloric gland metaplasia was also formed. The transgenic mouse is a very useful model for clarifying physiological differentiation of gastric and intestinal cell lineages and analyzing the molecular events from intestinal metaplasia to adenocarcinoma.     

Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii

Ciliates of Euplotes species constitutively secrete pleiotropic protein pheromones, which are capable to function as prototypic autocrine growth factors as well as paracrine inducers of mating processes. This paper reports the amino acid sequence and the NMR structure of the pheromone En-6 isolated from the antarctic species Euplotes nobilii. The 63-residue En-6 polypeptide chain forms three alpha-helices in positions 18-25, 36-40 and 46-56, which are arranged in an up-down-up three-helix bundle forming the edges of a distorted trigonal pyramid. The base of the pyramid is covered by the N-terminal heptadecapeptide segment, which includes a 3(10)-turn of residues 3-6. This topology is covalently anchored by four long-range disulfide bonds. Comparison with the smaller pheromones of E. raikovi, a closely related species living in temperate waters, shows that the two-pheromone families have the same three-helix bundle architecture. It then appears that cold-adaptation of the En proteins is primarily related to increased lengths of the chain-terminal peptide segments and the surface-exposed loops connecting the regular secondary structures, and to the presence of solvent-exposed clusters of negatively charged side-chains.     

Enhanced suicidal death of erythrocytes from gene-targeted mice lacking the Cl-/HCO(3)(-) exchanger AE1

Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl^– removal, and energy depletion activate Ca²⁺-permeable cation channels with Ca²⁺-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1^–/– mice) than in their wild-type littermates (AE1^+/+ mice) despite increased percentages of reticulocytes (AE1^–/–: 49%, AE1^+/+: 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1^–/–erythrocytes/reticulocytes (10%) than in AE1^+/+ erythrocytes (1%). Osmotic shock (addition of 400 mM sucrose), Cl^– removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1^–/– erythrocytes/reticulocytes than in AE1^+/+ erythrocytes. The increase of annexin binding following exposure to the Ca²⁺ ionophore ionomycin (1 µM) was, however, similar in AE1^–/– and in AE1^+/+ erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca²⁺ permeability in AE1^–/– erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1^–/– mice than in AE1^+/+ mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca²⁺ entry and subsequent scrambling of cell membrane phospholipids. annexin; cell volume; osmolarity; phosphatidylserine; energy depletion     

First isolation and molecular characterization of pseudorabies virus detected in Turkey

Background

Pigs are the main host species for the pseudorabies virus. It causes fatal encephalitis in many species, including humans. This article aims to report the first clinical case of pseudorabies as well as isolation and molecular characterization of the virus from a hunting dog in Bursa province, Turkey.

Methods and results

The dog shows clinical signs including pruritus and neurological signs such as stumbling and inability to stand up compatible with pseudorabies. The virus isolates were obtained from the supernatant of fresh tissue samples from the cerebellum, cornu ammonis, spleen, salivary gland, conjunctival swab, serum, and PBMC samples. The glycoprotein C region is targeted for viral DNA amplification. Pseudorabies virus genome detected both in fresh tissues and supernatants of third passage on Vero cells. The number of PCR positive samples was dramatically increased after cell culture inoculations. Genome sequencing of strain Bursa-10303, which was isolated from a non-endemic area, identified it to belong to clade A.

Conclusions

This study confirms the possible presence of pseudorabies infection in the wildlife reservoirs in Turkey. Future studies may clarify the importance of the infection in Turkey region, where there is no prevalent pig production.

     

Depletion with PC61 mAb before Toxoplasma gondii infection eliminates mainly Tregs in BALB/c mice, but activated cells in C57BL/6J mice

Analysis of regulatory T cells (Tregs) in vivo during infection is crucial for the understanding of immune response modulation. Depletion experiments using anti-CD25 monoclonal antibody (mAb) in order to eliminate Tregs have been widely used for this purpose despite the fact that this approach may also lead to the elimination of activated T cells. We show in this paper that treatment with anti-CD25 mAb before Toxoplasma gondii infection eliminates a different pattern of cell subsets in the resistant BALB/c and the susceptible C57BL/6J mouse strain. Injection with PC61 mAb leads to the elimination of most Tregs in BALB/c mice, while in C57BL/6J animals, treatment depletes other activated subsets [natural killer (NK), B and CD4(+) T cells]. This difference is a consequence of the dramatic cell activation observed in the latter, but not in the former strain. The different effect of the depletion reported here demonstrates that careful analysis in each model is mandatory in order to avoid misleading conclusions.     

Interleukin-1β-induced interleukin-6 production in A549 cells is mediated by both phosphatidylinositol 3-kinase and interleukin-1 receptor-associated kinase-4

The aim of this study is to investigate whether PI3K (phosphatidylinositol-3-kinase) is involved in IL-1β (interleukin-1β)-induced IL-6 production in A549 (human lung adenocarcinoma epithelial cell) and human RASF (rheumatoid arthritis synovial fibroblast). PI3K inhibitor, LY294002 significantly reduced IL-1β-induced IL-6 production in A549 cells but not in RASF, indicating that IL-1β-induced IL-6 production was partially mediated by PI3Kin A549 cells but not in RASF. siRNA (small interfering RNA) of IRAK4 (IL-1 receptor-associated kinase 4) treatment decreased IRAK4 mRNA level by up to 90% in A549 cells. In this condition, IL-1β-induced increase of IL-6 mRNA and protein level was decreased by up to 93% and 70%, respectively. Furthermore, the combination of IRAK4 siRNA and LY294002 treatment decreased protein induction level of IL-6 in A549 cells compared with that of IRAK4 siRNA or LY294002 alone. These results indicate that IL-1β-induced IL-6 production in A549 cells is mediated by both PI3K and IRAK4 and suggest that involvement of PI3K in the IL-1-induced IL-6 production is cell type specific.