Central nervous system involvement in nephropathic cystinosis.

Nephropathic cystinosis, an autosomal recessive lysosomal storage disorder due to impaired cystine transport, causes damage to multiple organs that results in end-stage renal disease, hypothyroidism, and retinopathy, usually in childhood. Dialysis and renal transplantation now frequently enable patients with cystinosis to live into adulthood. Examinations at autopsy of a 28-year-old man who died of complications of this disease showed deposits of cystine crystals in multiple organs. There was severe cerebral involvement with multifocal cystic necrosis, dystrophic calcification, spongy change, and vacuolization that had produced profound neurologic deficits. Electron microscopy of the brain documented cytoplasmic deposition of cystine crystals in membrane bound vacuoles within the cytoplasm of pericytes and within parenchymal cells of the white matter. While affected patients who have received renal transplants may no longer die from renal failure, serious, potentially life-threatening, neurologic complications of this disorder may supervene.

Analysis of methyl mercury binding sites on tubulin subunits and microtubules.

We have studied the localization and affinity of methyl mercury hydroxide (MeHg) binding sites on microtubules. There is one class of binding sites for MeHg on tubulin, a high affinity class with fifteen sites. MeHg binds to tubulin stoichiometrically within microtubules, and does not induce microtubule disassembly at this low binding ratio. MeHg binds in microtubules either in the presence or absence of free tubulin subunits but free subunits act as uncompetitive inhibitors for MeHg binding to the polymer. These stoichiometric polymer surface binding sites for MeHg apparently do not interfere with subsequent polymerization, in contrast to the multiple sites in the free dimer whose occupation blocks subsequent assembly. In assembly cycles that follow MeHg binding to polymers, we continue to find MeHg bound to microtubules at substoichiometric ratios. Dimers with higher levels of MeHg binding are rendered assembly incompetent. These results show MeHg to have one class of binding site on tubulin, and the MeHg binding site, both to the polymer surface and to the free dimer, to be the same.

Methylmercury effects on cell cycle kinetics.

Methylmercury (MeHg) effects on cell cycle kinetics were investigated to help identify its mechanisms of action. Flow cytometric analysis of normal human fibroblasts grown in vitro in the presence of BrdU allowed quantitation of the proportion of cells in G1, S, G2 and the next G1 phase. This technique provides a rapid and easily performed method of characterizing phase lengths and transition rates for the complete cell cycle. After first exposure to MeHg the cell cycle time was lengthened due to a prolonged G1. At 3 microM MeHg the G1 phase length was 25% longer than the control. The G1/S transition rate was also decreased in a dose-related manner. Confluent cells exposed to MeHg and replated with MeHg respond in the same way as cells which have not been exposed to MeHg before replating. Cells exposed for long times to MeHg lost a detectable G1 effect, and instead showed an increase in the G2 percentage, which was directly related to MeHg concentration and length of exposure. After 8 days at 5 microM MeHg, 45% of the population was in G2. The G2 accumulation was reversible up to 3 days, but at 6 days the cells remained in G2 when the MeHg was removed. Cell counts and viability indicated that there was not a selective loss of cells from the MeHg. MeHg has multiple effects on the cell cycle which include a lengthened G1 and decreased transition probability after short term exposure of cycling cells, and a G2 accumulation after a longer term exposure. There were no detectable S phase effects. It appears that mitosis (the G2 accumulation) and probably synthesis of some macromolecules in G1 (the lengthened G1 and lowered transition probability) are particularly susceptible to MeHg.

The effects of methyl mercury binding to microtubules.

The effects of methyl mercury hydroxide (MeHg) on the in vitro polymerization and depolymerization of microtubules were studied. Polymerization was totally inhibited at 3.0 X 10(-5) M MeHg and depolymerization occurred at concentrations above 1.0 X 10(-5) M MeHg, reaching a maximal rate of -0.33%/min at 5.0 X 10(-5) M MeHg. At or above 1.0 X 10(-4) M MeHg, a mercury-protein aggregate formed in both the polymerization and depolymerization systems. Fifteen free sulfhydryl groups per tubulin dimer were determined, and MeHg bound to all 15. When MeHg bound to only 2 free sulfhydryl groups per dimer, it inhibited polymerization. MeHg bound to free sulfhydryl groups exposed uniquely on the surface of microtubules, as well as those free sulfhydryl groups exposed on the ends. These results show MeHg in vitro to be a potent microtubule assembly inhibitor at ratios stoichiometric with the tubulin dimer. The effects of MeHg on microtubules are presumably mediated through MeHg binding to free sulfhydryl groups both on the ends and on the surface of microtubules. The presence of binding sites (free sulfhydryl groups) on the microtubule surface suggests multiple classes of binding sites for MeHg.