K. R. Rees

The Biochemistry of Copper Deficiency. I. Enzymological Disturbances, Blood Chemistry and Excretion of Amino-Acids

The biochemistry of an uncomplicated deficiency of copper is studied. A moderate to advanced depletion of copper does not affect the activity or level of the following: liver-slice respiration, tricarboxylic acid cycle, fatty-acid oxidation, amino-acid oxidation, oxidative phosphorylation, anaerobic glycolysis, catalase, DPN-cytochrome c reductase, transmethylase, choline oxidase, isocitric dehydrogenase, succinic dehydrogenase, malic dehydrogenase, glutamic dehydrogenase, oxidation of glucose and pyruvate by brain mitochondria, urinary amino-acids, plasma protein, magnesium, calcium, sodium, potassium, or inorganic phosphate. Activities of cytochrome oxidase and succinoxidase are reduced at this stage of deficiency. The loss of succinoxidase activity is due to the depletion of cytochrome oxidase which is severe and progressive from a very early stage of deficiency. Haem α is almost completely absent from copper-deficient tissues, and it is suggested that this is the limiting component of the cytochrome oxidase system. Mitochondria from deficient rats are very susceptible to ‘ageing’. The ‘ageing’ effect is shown to be due to loss of and reversed by addition of the pyridine nucleotides, glutathione and manganese. The susceptibility to ‘ageing’ is thought to be secondary to an impaired synthesis of phospholipid. Extreme copper deficiency leads to a grave loss of the capacity of mitochondria to oxidize any substrate; this is almost certainly due both to the negligible activity of cytochrome oxidase and an acceleration oftheeffectof ‘ageing’. Isocitric dehydrogenase activity is increased and DPN-cytochrome c reductase decreased at this stage; the reasons are discussed.

The effect of the aflatoxins B1, G1, and G2 on protein and nucleic acid synthesis in rat liver

A comparison has been made of the difference spectra obtained by causing various aflatoxins (B(1), G(1) and G(2)) to interact with calf-thymus DNA. The effect of these toxins on RNA and protein synthesis by rat-liver slices has been measured. The extent of their inhibitory action on the synthetic reactions was proportional to the degree of spectral shift obtained with their interaction with DNA. It is proposed that their toxicity depends on this interaction. It was demonstrated that the RNA polymerase of nucleoli isolated from the livers of aflatoxin B(1)-poisoned rats was inhibited. This finding is in agreement with the proposed mechanism for the hepatotoxic action of aflatoxin.

The biochemistry of copper deficiency - II. Synthetic processes

The biochemistry of copper deficiency is studied in order to gain some understanding of the metabolic disturbances which lead to demyelination of the central nervous system in disease. In the preceding paper we reported our investigation of the enzyme systems, blood chemistry and amino-acid excretion in copper-deficient rats, and in this paper extend the study to investigate the syntheses of phospholipid, long-chain fatty acids, ribose nucleic acid, protein and protohaem. Phospholipid synthesis is found to be depressed considerably in copper deficiency. This is due to a failure in the process of condensation of acyl CoA with α-glycerophosphate to form phosphatidic acids. The reasons are discussed. The syntheses of long-chain fatty acids and ribose nucleic acid are normal, whilst the synthesis of protein is inconstantly affected by copper deficiency. Protohaem synthesis is depressed by a degree which exactly parallels the anaemia. The conclusion is drawn that the anaemia is due to a decrease in haematopoiesis rather than an increased destruction of red cells. The possible interrelationships of the disturbances of phospholipid synthesis and cytochrome oxidase activity and the relevance of each to demyelination of the central nervous system are discussed.

The identification of different structural classes of nucleic acids by electrophoresis in polyacrylamide gels of different concentration

Abstract By determining the way in which the electrophoretic mobility of a nucleic acid varies when analysed across a range of gel acrylamide concentrations (gel range analysis), it is possible to group the species into one of three structural classes. These classes comprise: I, single-stranded; II, double-stranded linear; and III, double-stranded circular nucleic acids. The technique of gel range analysis provides a rapid and simple way of determining to which of these structural classes a nucleic acid belongs and would be very suitable as a preliminary analytical procedure for investigating a nucleic acid of unknown structure.

The mechanism of action of aflatoxin B1 on protein synthesis; Observations on malignant, viral transformed and untransformed cells in culture

Abstract (1) Aflatoxin B1 was tested against protein and nucleic acid synthesis in a number of cell lines in culture. (2) A detailed investigation was made in CV-1 cells to determine the mechanism whereby aflatoxin B1 inhibits protein synthesis. (3) Inhibition of protein synthesis by aflatoxin B1 was not secondary to other changes in the cell but was due to a direct action of the toxin on the polysomes. The possible site of its interaction is discussed.

Effect of daunomycin on HeLa cell nucleic acid synthesis

Abstract In the presence of daunomycin (0.5 μg/ml) HeLa cell DNA synthesis was irreversibly inhibited. RNA synthesis although initially inhibited gradually reversed and returned to normal levels by 24 hr. Analysis showed that the drug selectively inhibited ribosomal RNA and only partially inhibited heterogeneous RNA which was altered in physical characteristics. Inhibition of protein synthesis was a late event indicating the continued synthesis of messenger RNA in the presence of daunomycin. A possible mechanism to explain these differential effects is discussed.

Mitochondrial RNA in mycoplasma infected hela cells

Abstract Experiments comparing RNA species extracted from mitochondria and from mycoplasma infected cells have shown that they possess many similar properties. These similarities include low G+C content, low order of methylation, location in purified mitochondrial preparations, resistance to low concentrations of actinomycin D, and some characteristics of the electrophoretic profiles on polyacrylamide gels. If a mitochondrial preparation is suspected to contain contaminating mycoplasma this can be confirmed by comparative electrophoretic methods and by use of the differential inhibitor ethidium bromide.

Action of some phenothiazine derivatives on the respiratory chain

Abstract Several phenothiazine tranquillizers and their derivatives have been tested against the respiration of liver mitochondria. Two actions have been found: Competitive inhibition of cytochrome oxidase and inhibition of DPNH-cytochrome- c reductase. The latter is manifested only in phosphorylating systems. There is some correlation between the effect on DPNH-cytochrome- c reductase and tranquillizing ability. Thus, chlorpromazine inhibits 90 per cent at 2 × 10 −4 M, at which concentration its sulphoxide has no effect. However, anomalies occur: acetylpromazine has little effect, but is a potent tranquillizer, and conversely, the primary amine of chlorpromazine is a powerful inhibitor, but is not a good tranquillizer.

The effect of aflatoxin B1 on the maturation of ribosomal and transfer RNA in monkey kidney (CV-1) cells in culture

Abstract Aflatoxin B 1 has been found to inhibit the maturation of rRNA by causing a build-up of 32S RNA in the nucleolus associated with reduced output of 28S rRNA to the cytoplasm. Processing of 18S rRNA appears normal. Other experiments on the maturation of tRNA have shown that again aflatoxin B 1 delays the processing of the precursor. The mechanism of action of the toxin and the relationship between these results and the observed inhibition of transcription are discussed.

The effect of aflatoxin B1 on translation in cells in culture

The effect of aflatoxin B1 (AFB1) upon translation has been studied in two cell lines in culture, which differ markedly in their sensitivity to the toxin. It was found that AFB1 predominantly inhibits initiation of translation in both cell lines.