Candida albicans and Candida parapsilosis Rapidly Up-Regulate Galectin-3 Secretion by Human Gingival Epithelial Cells

Galectin-3 is a β-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin-3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heat-killed C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion.     

Sequence of EndoA gene encoding mouse cytokeratin and its methylation state in the CpG-rich region.

A genomic clone obtained from mouse liver DNA using a mouse cytokeratin EndoA cDNA probe revealed the complete sequence of the EndoA gene. The gene is divided into nine exons and the exon-intron pattern has been conserved compared to that of other type-II cytokeratin-encoding genes. The 5' upstream, 3' downstream and first and third introns contain potential regulatory sequences, including polyoma virus enhancer motifs (PEA1 and PEA3) and AP-1 elements. The 5' regions upstream of the EndoA, EndoB and Ck8 genes contain homologous sequences surrounding the TATA boxes. In addition, a CpG dinucleotide cluster region was located around the first exon. This CpG cluster region was found to be hypomethylated in endodermal PYS-2 cells, retinoic acid-treated F9 cells, and F9 embryonal carcinoma cells, but hypermethylated in BALB/C 3T3 fibroblast cells that do not express EndoA. These findings may provide a clue to understanding the molecular mechanisms of EndoA gene expression.     

Human atrial natriuretic polypeptide in plasma of patients with anorexia nervosa

To examine the effects of chronic dehydration and starvation on plasma levels of human atrial natriuretic polypeptide (hANP) in human subjects, the basal level and saline-induced rise of plasma hANP in 7 patients with anorexia nervosa were compared with those in age-matched healthy subjects. The unstimulated level of plasma hANP was markedly high in the patients with anorexia nervosa (patients vs. control; 55.4 +/- 9.0 pg/ml vs. 11.4 +/- 6.1 pg/ml, P less than 0.01). However, no significant increase of plasma hANP in the anorectic patients was observed in response to saline-infusion, while a 3-fold increase over the basal level of plasma hANP was noted in the saline-infused normal young subjects. These results show that hANP may be secreted to an inadequate extent, hence the release would be resistant to volume-loading. The pathophysiological meaning of such a high plasma concentrations of hANP in anorexia nervosa is the subject of ongoing studies.     

Theoretical analysis of a site-specific chemiluminescence reaction and its application to quantitation of lipid hydroperoxides

A system was designed for chemiluminescent measurement of lipid hydroperoxides by their site-specific reaction in sodium dodecylsulfate micelles. Ferrous ion-induced decomposition of lipid hydroperoxides in the sodium dodecylsulfate micelles resulted in strong chemiluminescence of the Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (CLA). After addition of ferrous sulfate to the micelles containing lipid hydroperoxide and luciferin, the chemiluminescence intensity reached a maximum rapidly and then decreased. The sequence of this reaction was elucidated by theoretical analysis, which demonstrated that the maximum chemiluminescence intensity is proportional to the initial concentration of hydroperoxide. Good linear relationships were observed between the maximum counts of chemiluminescence and the amounts of hydroperoxides of linoleic acid, phosphatidylcholine, choresterol (5 alpha), cumene and tert-butyl and hydrogen peroxide. This chemiluminescence method was simple and sensitive enough to detect picomole levels of linoleic acid and phosphatidylcholine hydroperoxides.     

Physicochemical and immunochemical properties of a thermo-labile antigen (TLA a) from yeast cell surface

The physicochemical and immunochemical properties of a thermo-labile antigen (TLA a) which is located on the cell surface of Saccharomyces cerevisiae were studied. The sedimentation constant (S20,W) and molecular weight (sedimentation equilibrium method) were 6.26S and 68,800, respectively. The circular dichroic (CD) spectrum of TLA a had negative maxima at 210 and 221 nm, indicating the presence of alpha-structure of a polypeptide chain. The molar ratio of antibody to antigen which gave maximum precipitation was 2.7. Approximately 50% of the antigenic activity of heat-denatured TLA a was recovered when denatured molecules were dissolved in 6 M guanidine hydrochloride followed by 25-fold dilution with H2O. The amount of TLA a existing on the yeast cell surface was estimated to be 37.5 micrograms per 10.5 mg of fresh cells, corresponding to 0.36% by weight of the fresh yeast.     

Purification of retinol dehydrogenase from bovine retinal rod outer segments

We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, concanavalin A-Sepharose CL-4B, and Sepharose CL-6B column chromatography in the presence of NADP. We obtained 13-fold purification of retinol dehydrogenase with specific activity of 61.8 nmol/min/mg and 3.8% recovery. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that retinol dehydrogenase had a molecular mass of 37,000 daltons. The Km values of purified retinol dehydrogenase for all-trans retinol and all-trans retinal were 6.6 mM and 0.085 mM, respectively. The purified enzyme reacted with the all-trans retinal but not with 13-, 11-, and 9-cis compounds. In addition, we prepared antibody to retinol dehydrogenase using rat. The anti-retinol dehydrogenase antibody precipitated retinol dehydrogenase activity and was confirmed to bind to 37-kDa protein by Western blotting. We also found that anti-retinol dehydrogenase antibody bound to bovine rod outer segments specifically by immunohistochemical technique. The molar ratio of retinol dehydrogenase to opsin in rod outer segments estimated by enzyme-linked immunosorbent assay was 1:140.     

Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug resistant cells.

It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells. In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins. The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL). Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine. P-gp in these cells was the only protein specifically bound to [3H]azidopine in photoaffinity experiments. The specific photoaffinity labeling of P-gp by [3H]azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively. The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein. Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.