The copper concentration was investigated in the cultured astrocytes from macular mice, an animal model of Menkes disease. An excessive amount of copper was accumulated in the astrocytes as copper-metallothionein. These results show that the underlying genetic defect of the macular mouse is expressed in the astrocytes. A similar situation may exist in Menkes disease and cause a failure of copper transport to neurones.

Direct batch addition of sterile Chelex ion-exchange resin to Dubecco's modified Eagle's medium supplemented with 10% fetal calf serum with gentle stirring removed a very wide variety of trace metal ions from the medium to varying extents dependent upon Chelex content (between 0.01 and 4% w/v), exposure time (between 5 min and 10 days) and temperature (4, 25 and 37 degrees C). Prolonged treatment (10 days) with 4% w/v Chelex at 4 degrees C reduced the concentration of zinc, strontium, aluminum, copper, manganese, nickel and chromium from 100 to 2.7, 12.1, 7.7, 22.6, 13.0, 14.7 and 53.3% of their original concentrations, respectively. Re-supplementation of the metal depleted medium with a defined cocktail of metals restored the growth potential of the medium which was then capable of supporting growth over at least three subcultures without a decrease in fibroblast cell yield, demonstrating its suitability in cell culture studies on trace metal ions.

The composition of LB broth (tryptone, yeast extract and NaCl) was investigated by 1H,31P-NMR spectroscopy, FPLC and gel electrophoresis. An unexpected finding was the high level of 2'3'-cyclic nucleotides, detected by characteristic 31P-NMR resonances in the region 20-21 ppm, originating from the yeast component. 31P-NMR resonances for cyclic nucleotides were observed during the autolysis of Saccharomyces cerevisiae cells, and in model reactions of RNase with RNA.

The composition of LB broth (tryptone, yeast extract and NaCl) was investigated by 1H, 31P‐NMR spectroscopy, FPLC and gel electrophoresis. An enexpected finding was the high level of 2′,3′‐cyclic nucleotides, detected by characteristic 31P‐NMR resonances in the region 20–21 ppm, originating from the yeast component. 31P‐NMR resonances for cyclic nucleotides were observed during the autolysis of Saccharomyces cerevisiae cells, and in model reactions of RNase with RNA.

The precise nature of the variation in cellular copper load against medium copper concentration is defined using a comprehensive logarithmically incremented series of medium copper concentrations ranging from low levels (4.8 p.p.b.) through 'normal' to toxic levels (40 p.p.m.) in which fibroblasts were grown followed by determination of intracellular content. Menkes' fibroblasts showed an unexpected plateau region of stable intracellular copper content against a change in medium concentration of over 100-fold, albeit only when sufficient copper was present in the medium (0.08-8.0 p.p.m.). Thus, Menkes' cells are clearly capable of balancing uptake/efflux providing copper availability allows. Simultaneous analysis of cellular copper and zinc load at various medium copper concentrations shows an indistinguishable intracellular copper:zinc ratio between the two cell lines. The nature of non-labeled copper uptake by fibroblasts over a 40 min and 7 day period is reported. During the 40 min period copper uptake (20 p.p.m.) was essentially the same in both cell lines. However, copper absorbed was superimposed upon large pre-existing copper pools in the case of Menkes' cells only. Advantages of techniques determining non-labeled copper in copper uptake/efflux experiments are discussed in the light of these results. Fibroblast growth studies showed that, compared with normal cells, Menkes' cells are significantly (P < 0.01) more growth sensitive to extended exposure to low copper concentrations. Thus, Menkes' disease appears to be not only a result of copper maldistribution but also a direct result of an inability of Menkes' cells to function normally in low copper environments.