Effects of mercuric chloride exposure on the glutamate uptake by cultured retinal pigment epithelial cells.

The cytotoxicity of mercuric chloride and the effects of mercuric chloride on glutamate and calcium uptake and the factors regulating glutamate uptake were studied in retinal pigment epithelium (RPE) cell cultures. RPE cells isolated from pig eyes and human RPE cell line (D407) cells were cultured to confluency and further subcultured according to the test protocol in question. The cytotoxicity caused by 15 min of exposure to mercuric chloride (0.01--1000 microM) was evaluated by WST-1 assay based on the activity of mitochondrial dehydrogenases. [(3)H]Glutamate uptake was measured after the cells were exposed to 0.1--100 microM mercuric chloride and the selected regulators of protein kinase C (PKC) pathway: PKC activator SC10, PKC inhibitor chelerythrine chloride, phospholipase A(2)/C inhibitor manoalide, tyrosine kinase inhibitor lavendustin A, competitive NMDA receptor antagonist AP7 and IP(3) receptor antagonist heparin. Intracellular calcium was monitored with Fluo-3 probe starting immediately after the exposure to 1--1000 microM mercuric chloride. Mercuric chloride showed concentration-dependent effects on cell viability, on glutamate uptake and on intracellular calcium concentration. The results give some support to the concept that glutamate uptake is affected by PKC. The PKC inhibitor chelerythrine chloride decreased glutamate uptake by 25%, but the PKC activator SC10 could partly prevent the inhibitory effect of mercuric chloride. Lavendustin A, manoalide and heparin had smaller, but statistically significant, effects. All these substances act on mediators which can regulate the activity of PKC. However, PKC is not likely to be the only regulator of glutamate uptake. The rise observed in [Ca(2+)](i) may initiate various cellular events during mercury intoxication.

Effects of mercury, methylmercury and aluminium on glial fibrillary acidic protein expression in rat cerebellar astrocyte cultures.

The effects of mercuric chloride, methylmercury chloride and aluminium chloride on glial fibrillary acidic protein (GFAP) expression in primary cerebellar astrocyte cultures were studied. GFAP has been found to be a quantitative marker of neuronal injuries on the central nervous system in vivo. The GFAP content of the astrocytes was examined by sandwich ELISA after exposure for 1 wk to various metal concentrations in the culture medium. Cellular injuries were assessed by quantifying the lactate dehydrogenase (LDH) leakage into the culture medium. The expression of GFAP was found at lower dose levels than the leakage of LDH, indicating that the expression of GFAP could be a more sensitive marker of neurotoxicity than LDH leakage. In morphological examination, staining with monoclonal antibody showed GFAP induction after mercury exposure.

Molecular cloning and nucleotide sequence of chicken avidin-related genes 1-5.

Using avidin cDNA as a hybridisation probe, we detected a gene family whose putative products are related to the chicken egg-white avidin. Two overlapping genomic clones were found to contain five genes (avidin-related genes 1-5, avr1-avr5), which have been cloned, characterized and sequenced. All of the genes have a four-exon structure with an overall identity with the avidin cDNA of 88-92%. The genes appear to have no pseudogenic features and, in fact, two of these genes have been shown to be transcribed. The putative proteins share a sequence identity of 68-78% with avidin. The amino acid residues responsible for the biotin-binding activity of avidin and the bacterial biotin-binding protein, streptavidin, are highly conserved. Since avidin is induced in both a progesterone-specific manner and in connection with inflammation, these genes offer a valuable tool to study complex gene regulation in vivo.