OBJECTIVETo study the effect of supernatants from silicon dioxide(SiO2) stimulated pulmonary alveolar macrophages(PAM) on the localization of connexin 43(Cx43) so as to explore the inhibition level of SiO2 on alveolar epithelial cellular gap-junctional communication(GJIC).

METHODSThe supermatants from the primary cultured PAM were prepared, and then added 5% (v/v) SiO2 into 2% (v/v) NBS RPMI 1640 to stimulate the normal mink lung epithelial cell line CCL-64 for 24 hours. The localizations of Cx43 in CCL-64 were analyzed by indirect immunofluorescence histochemistry and laser confocal scanning microscopy(LCSM).

RESULTSThe normal cultured CCL-64 cells displayed bright membrane-associated Cx43 plaques labeling and formed dashes at regions of intercellular junction. Being exposed to supernatants from SiO2-stimulated PAM, the CCL-64 cells retained a relative low degree of Cx43 labeling at the cell periphery, localized in cytoplasm, and the individual spot, rather than plaques, were smaller compared to normal cultured cells. Along with the increase of the concentrations of SiO2, the cells displayed a different staining pattern, with clear cluster labeling aggregating towards the nucleus.

CONCLUSIONThe altered localization of the gap-junctional protein Cx43 in alveolar epithelial cells, mediated by SiO2, indicated that the internalization of Cx43 may contribute to the inhibition on GJIC in silica-induced lung epithelium injury.

Animals ; Cell Communication ; drug effects ; Cell Line ; Connexin 43 ; analysis ; Epithelial Cells ; chemistry ; drug effects ; Fluorescent Antibody Technique, Indirect ; Gap Junctions ; drug effects ; Microscopy, Confocal ; Mink ; Pulmonary Alveoli ; chemistry ; drug effects ; Silicon Dioxide ; toxicity

OBJECTIVETo explore the effect of silica dioxide(SiO2) on proliferation and downregulation of gap junctional intercellular communication (GJIC) in pulmonary alveolar epithelial cells (CCL-64 cells).

METHODSThe pulmonary alveolar macrophages(PAMs) were incubated in the serum-free RPMI 1640 containing the various concentration of SiO2 for 24 hours. The supernatants were prepared and added 5% (V/V) into 2% (V/V) NBS RPMI 1640 to stimulate the proliferation of CCL-64 cells for 24 hours. A set of "blank control", run in parallel, contained RPMI 1640 + 2% (V/V) NBS alone. The proliferation of CCL-64 cells was detected using MTT assay(to show as the absorbency, A570nm). GJIC function was measured using the fluorescence redistribution after photobleaching(FRAP) assay [to express as the transfer rate of the fluorescence, K (x 10(-3)/s)], with a laser scanning confocal microscope(LSCM, Leica TCS SP).

RESULTSThe silica-exposed PAM supernatants could induce both the proliferation(F = 9.679, P < 0.01) and downregulation of GJIC(F = 20.587, P < 0.01) of CCL-64 cells. In the range of 50-500 micrograms/ml SiO2 concentrations, the proliferation (A570nm values) and GJIC(the transfer rate, K) were fitted well in a dose-dependent manner(proliferation: r = 0.891, P < 0.05; GJIC: r = -0.943, P < 0.05).

CONCLUSIONBy way of stimulating the PAM, SiO2 could inhibit GJIC function in lung alveolar epithelial cells, and induce epithelial cell proliferation. In the pathogenesis of silicosis, the downregulation of GJIC of the pulmonary epithelial cells may play an important role in silica-mediated alveolar epithelial cell injury.

Cell Communication ; drug effects ; Cell Proliferation ; drug effects ; Epithelial Cells ; drug effects ; Gap Junctions ; drug effects ; Pulmonary Alveoli ; drug effects ; Silicon Dioxide ; toxicity ; Silicosis ; etiology

OBJECTIVETo express and identify soluble liver antigen (SLA) and cytochrome P-450 (CYP 2D6).

METHODSSLA cDNA and CYP 2D6 cDNA were obtained from human liver tissue poly (A)+RNA by RT-PCR. The cDNAs were inserted into fusion expression vector PQE-30 site of BamH I and Hind III. SLA and CYP 2D6 were identified by the SDS-PAGE and Western blot.

RESULTSSDS-PAGE analysis showed that there was a very strong stained band at about 47 kd and 50 kd, respectively. The products could specifically band to anti-SLA or anti-CYP 2D6 autoantibodies.

CONCLUSIONSThe clone and expression of SLA and CYP 2D6 provide useful substances for the diagnosis and research of pathogenesis on autoimmune hepatitis.

Autoantigens ; genetics ; Cytochrome P-450 Enzyme System ; genetics ; DNA, Complementary ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Hepatitis, Autoimmune ; genetics ; Humans ; Liver ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism