Association of advanced glycation end products with A549 cells, a human pulmonary epithelial cell line, is mediated by a receptor distinct from the scavenger receptor family and RAGE.

Cellular interactions with advanced glycation end products (AGE)-modified proteins are known to induce several biological responses, not only endocytic uptake and degradation, but also the induction of cytokines and growth factors, combined responses that may be linked to the development of diabetic vascular complications. In this study we demonstrate that A549 cells, a human pulmonary epithelial cell line, possess a specific binding site for AGE-modified bovine serum albumin (AGE-BSA) (K(d) = 27.8 nM), and additionally for EN-RAGE (extracellular newly identified RAGE binding protein) (K(d) = 118 nM). Western blot and RT-PCR analysis showed that RAGE (receptor for AGE) is highly expressed on A549 cells, while the expression of other known AGE-receptors such as galectin-3 and SR-A (class A scavenger receptor), are below the level of detection. The binding of (125)I-AGE-BSA to these cells is inhibited by unlabeled AGE-BSA, but not by EN-RAGE. In contrast, the binding of (125)I-EN-RAGE is significantly inhibited by unlabeled EN-RAGE and soluble RAGE, but not by AGE-BSA. Our results indicate that A549 cells possess at least two binding sites, one specific for EN-RAGE and the other specific for AGE-BSA. The latter receptor on A549 cells is distinct from the scavenger receptor family and RAGE.

Development of vitellogenin assay for endocrine disrupters using medaka (Oryzias latipes).

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of the egg yolk precursor vitellogenin (VTG) in the liver of medaka (Oryzias latipes) and was employed to establish an in vivo testing system for estrogen and estrogenic compounds using liver homogenates. Results of 3-month-old fish exposed to three reference chemicals (ethynylestradiol, methyltestosterone and flutamide) recommended by Organisation for Economic Co-operation and Development (OECD) for the validation showed the induction and decrease of vitellogenic responses, making the assay using the liver VTG of medaka a possible screening method for not only estrogens but also androgens. In addition, 21-day exposure of male fish to 4-tert-octylphenol and 4-nonylphenol produced concentration-dependent inductions of liver vitellogenin, with the lowest observed effect concentrations of 64.1 microg/L and 22.5 microg/L, respectively, while no significant VTG responses were observed in male and female fish exposed to tributyltin chloride and dibutyl phthalate. This study demonstrates that the VTG assay using liver homogenates from small fish such as medaka can be used as a screening method for environmental estrogens. This is because the sensitivity of the VTG response in medaka may be almost the same as that of other fish using blood samples.

Calnexin Delta 185-520 partially reverses the misprocessing of the Delta F508 cystic fibrosis transmembrane conductance regulator.

Abnormal retention of Delta F508 CFTR (cystic fibrosis transmembrane conductance regulator) in the endoplasmic reticulum is a major cause of cystic fibrosis (CF). We show that calnexin Delta 185-520 but not calnexin can partially reverse the mislocalization of Delta F508 CFTR. This 256-amino acid protein has neither the transmembrane domain nor the P domain of calnexin. Calnexin Delta 185-520 interacted with CFTR directly, and was secreted into the extracellular compartment over time. Forty-eight hours after transfection into CHO cells, calnexin Delta 185-520 increased the conversion of immature Delta F508 CFTR into mature Delta F508 CFTR. In immortalized human CF cell lines expressing Delta F508 CFTR, a halide efflux assay showed that calnexin Delta 185-520 partially restored CFTR function. These data indicate that calnexin Delta 185-520 may give a clue to develop the therapeutic way of cystic fibrosis with Delta F508 CFTR.