Mapping the adhesive domains of the myelin Po protein.

The Po protein holds PNS myelin compact at the intraperiod line by homophilic interactions of its single immunoglobulin (Ig)-like domain. Using transfected Chinese hamster ovary (CHO) cells expressing Po we can monitor this adhesion in vitro and have shown that the cells expressing Po when incubated as a single-cell suspension form large aggregates, whereas control-transfected cells do not. To precisely map the domains of Po responsible for Po:Po-mediated membrane adhesion, the ability of a number of antibodies raised to peptides corresponding to segments of the Ig-domain of Po, and the ability of the Po-peptides themselves, to inhibit aggregation was assessed. Both antibodies to Po-peptide, SDNGT, corresponding to amino acids Po 91-95, and the peptide itself, were able to block adhesion completely. Furthermore, within this Po sequence, amino acids Asp 92 and Gly 94 are conserved in a large number of V-like Ig-domains. To determine if these two amino acids are important for Po-mediated adhesion, the nucleotides coding for Asp 92 and Gly 94 were mutated to encode glutamate and alanine, respectively. Although the mutated Po reached the surface in transfected CHO cells and was glycosylated, the cells did not aggregate. These results suggest that the sequence SDNGT in the extracellular domain of Po is important for adhesion. In addition, antibodies to a second sequence, Po 74-82, and the peptide itself, also partially inhibited Po: Po-mediated adhesion indicating that there is more than one adhesive domain on Po-protein.

The effects of cisplatin on the concentration of protein thiols and glutathione in the rat kidney.

The effect of a nephrotoxic dose of cisplatin (5 mg/kg) on the concentrations in the rat kidney of both glutathione and protein-bound thiols was investigated. Total glutathione and oxidised glutathione were measured in the cortex and outer medulla using specific enzyme-based assays. The high-molecular-weight thiols were quantified in cells of the proximal tubule using a cyto-chemical technique. The concentration of total glutathione (oxidised and reduced) in the kidney cortex and outer medulla was significantly higher than that of controls at 1 h following cisplatin administration. The amount by which the concentration in treated animals exceeded that in controls increased to 50% at 72 h and remained significantly elevated for 120 h following treatment. This increase was mainly attributable to an increase in the concentration of reduced glutathione. In contrast, the concentration of protein thiols in the proximal tubules decreased significantly at 8 h after dosing, reaching a nadir 29% below that of controls at 120 h, thus coinciding with the maximal functional disturbance in the kidney as reflected by the concentration of blood urea. The decrease in protein thiols could not be correlated stoichiometrically with the platinum concentration in the cortex and outer medulla, which reached a peak of 16.3 +/- 0.3 micrograms/g wet tissue at 72 h after treatment. Evidently cisplatin perturbs the equilibrium that is said to exist between the concentration of reduced glutathione and that of protein thiols. This perturbation occurs well before the onset of overt functional disturbance of the kidney and is evident before the point at which the damage to the kidney caused by cisplatin becomes irreversible.