David Wilkie

Nuclear magnetic resonance studies of blood plasma and urine from subjects with chronic renal failure: identification of trimethylamine-N-oxide

We have used 1H-, 13C- and 14N-NMR spectroscopy to investigate the constituents of plasma and urine in 16 patients with chronic renal failure (CRF). Resonances not previously observed in spectra of plasma from healthy volunteers were seen in CRF plasma, including those for trimethylamine-N-oxide (TMAO) and dimethylamine (DMA). A possible analogy with the plasma of elasmobranch fishes, in which TMAO stabilizes proteins in the presence of very high urea concentrations, is noted. The intensity of the TMAO resonance for CRF subjects was correlated with the plasma concentration of urea (R = 0.55) and creatinine (R = 0.74), suggesting that the presence of TMAO is closely related to the degree of renal failure. When normal subjects ate a meal of TMAO-containing fish, TMAO appeared rapidly in the plasma and in the urine. Thus TMAO is efficiently cleared by the healthy kidney. Differences in the interaction of lactate with plasma proteins were detected by NMR, suggesting that uraemia impairs their transport roles.

Toxic and mutagenic effects of carcinogens on the mitochondria of Saccharomyces cerevisiae.

SummaryNineteen haploid yeast (Saccharomyces cerevisiae) strains were used to assess the relative growth inhibitory potencies on fermentable vs. non-fermentable media of a collection of carcinogenic and noncarcinogenic chemicals. The majority of carcinogens were distinctly more potent on the non-fermentable (glycerol) medium, where mitochondrial function is required for growth, than on the fermentable medium, where it is not. The anti-mitochondrial selectivity indicated by these growth tests was much slighter for the non-carcinogens. Similarly most carcinogens induced the cytoplasmic petite mutation whereas the non-carcinogens did not.Five carcinogens which were tested impaired the development of cytochromes aa3 and b in glucose cultures.Six carcinogens, when tested, inhibited growth on three fermentable sugars, the utilisation of which requires mitochondrial function.Out of five carcinogens which were examined, four suppressed the surface-dependent phenomenon of flocculence in a flocculating strain of yeast, at concentrations primarily affecting the mitochondrial system; the fifth had a similar but less pronounced effect.

Mitochondria and the yeast cell surface: implications for carcinogenesis

Abstract In yeast cells many chemical carcinogens primarily attack the mitochondria. Resulting lesions in the organelles affect the activity of certain nuclear genes which are involved in plasma membrane/cell surface biogenesis. The surface changes are analogous to those observed in neoplastic transformation in mammalian cells.

Mitochondrial control of cell surface characteristics in Saccharomyces cerevisiae

Defects in the inner mitochondrial membrane of petite mutants of yeast resulted not only in respiratory deficiency, but also in changes in cell surface characteristics. These were (1) concanavalin A agglutinability, (2) cell movement in a biphasic polymer system, (3) cell adhesion. Genetic analysis indicated that the control exerted by the mitochondria was on nuclear genes or on the products of these genes which were presumably specifying cell surface components. These findings ascribe a new role to mitochondria but also have implications for neoplastic transformation.

Antimitochondrial drugs in cancer chemotherapy: preliminary communication.

In initial studies with yeast cells it was found that the primary target of the antidepressant chlorimipramine (Anafranil, CIBA-Geigy) and the anti-leprosy drug clofazimine (Lamprene, CIBA-Geigy) was the mitochondrion (Hughes & Wilkie 1970, Rhodes & Wilkie 1973). Both drugs inhibited the respiratory chain, although by different mechanisms. The oxygen uptake of isolated rat liver mitochondria was affected in a similar way to that of the intact yeast mitochondria. When these studies were extended to human skin fibroblast cultures (Wilkie & Delhanty 1970, Delhanty et al. 1974, Mittwoch & Wilkie 1971), both drugs again had a direct effect on oxygen uptake in the human cells similar to that in yeast cells. The significant finding in this series of experiments was that SV-40 transformed cells (cancer type) were much more susceptible to the inhibitory effects of the drugs than the non-transformed fibroblasts. Results with chlorimipramine on transformed cells are included in the above references, while clofazimine results showed that about 2 jsg/ml in the culture medium was a lethal dose for transformed fibroblasts but about 8 jig/ml was required to kill non-transformed cultures (unpublished results). It was noted that the respiratory rate of transformed cells was significantly less than their normal counterparts in oxygen electrode studies (Wilkie & Delhanty 1970). These results indicated that depression of mitochondrial activity (already low), in the cancer type cells, by treatment with antimitochondrial agents kills these cells, while normal cells can recover from this treatment.

Mitochondrial control of sugar utilization in Saccharomyces cerevisiae.

Abstract When a number of wild-type strains of Saccharomyces cerevisiae—all capable of utilizing the three sugars galactose, maltose, and α-methyl- d -glucoside for growth—were converted by ethidium bromide (EtdBr) mutagenesis to stable cytoplasmic petite (rho−) mutants, the latter lost the ability to grow on one or more of these sugars. The actual pattern of retention (or loss) or sugar utilization by these mutants depended on the wild-type strain, but was independent of the length of exposure to EtdBr during mutagenesis. This treatment varied from 0.5 to 24 h, by which time the majority of the mutants must have been of the mitochondrial (mt) DNA-deficient rho0 type. Furthermore, with one exception—involving the ability of one set of mutants to utilize α-methyl-glucoside—all rho− mutants derived from the same wild type exhibited the same, discrete pattern of sugar utilization. Respiration-deficient mutants with defined lesions in their mtDNA (mit− mutants) exhibited the same pattern of sugar utilization as did the petite mutants of the same strain. Diploid petite strains also exhibited discrete, but less stringent, patterns of sugar utilization. For any one genotype this pattern was identical whether the mutant was generated by crossing two haploid rho− strains, themselves derived by EtdBr mutagenesis, or by EtdBr mutagenesis of the diploid obtained from a haploid wild-type × wild-type cross. In such mutant diploids the sugar-positive phenotype was usually dominant, but there were indications in some instances of modulation of this effect by virtue of nuclear gene interactions. Various respiration-deficient mutants incapable of utilizing α-methylglucoside also were unable to form α-glucosidase, but were able to do so after being rendered permeable by exposure to dimethyl sulfoxide. Arguments are advanced that respiring mitochondria generate an entity—probably not directly related to ATP production—required for the expression of nuclear genes or their products, some of which may be necessary for plasma membrane function.

Ultraviolet irradiation studies on the cytoplasmic determinant of the yeast mitochondrion

Abstract Ultraviolet induction of the cytoplasmic petite mutation in yeast was studied under a variety of growth conditions both before and after irradiation. The induction curves were found to be single-event for anaerobically-grown cells, and multiple-event for aerobic cells. By using a split dose technique, both photo-reactivation and dark-repair were investigated. No significant difference in repair ability was found between aerobic and anaerobic cells. A target theory interpretation of the induction curves suggests that mitochondrial DNA, assumed to be the genetic determinant, exists as a single heritable unit in anaerobic cells, and as multiple units in aerobic cells; moreover, the number of genetically effective copies in the aerobic cells appears to be far smaller than the number of mitochondria. These results are shown to be in accord with an independent estimate of the number of cytoplasmic determinants derived from recent genetic studies on mitochondrial recombination in aerobic and anaerobic yeast cells.

Effect of antibiotics on the transmission of mitochondrial factors in Saccharomyces cerevisiae

SummaryTransmission of mitochondrial factors was studied in zygote clones, random diploids, and zygote cell lineages. An asymmetrical distribution of both parental and recombinant classes was consistently seen. Genetic analysis indicated that a nuclear factor was controlling this pattern. Treatment of young zygotes with either cycloheximide or thiolutin for 90 minutes destroyed the pattern and randomized the distribution of parental and recombinant classes. Inhibition of mitochondrial protein synthesis with antibacterial antibiotics had no detectable effect on the pattern of distribution of mitochondrial markers.

Induction of respiratory deficiency by repression of the respiratory system in a mutant of Saccharomyces cerevisiae

Abstract Cells of a mutant strain of Saccharomyces cerevisiae produce daughter cells with respiratory deficiency of the cytoplasmic petite type when growing under conditions of respiratory repression, that is, in the presence of glucose or under anoxia, but otherwise produce respiratory normal cultures. Apart from inheriting the repressionpetite induction character, the latter are normal in other respects such as response to specific mutagens and oxygen uptake under different physiological conditions. Chloramphenicol, although specifically inhibiting the synthesis of mitochondriabound cytochromes in this strain, does not induce the petite mutation in cultures growing on a non-repressing sugar. The character of anoxia or glucose induction ( gi ) of petite appears to be under the control of a recessive nuclear gene and spontaneous reversion to normal takes place with a low frequency. Since petite cells show effective loss of mitochondrial DNA, these results are believed to provide evidence that the replication and/or transmission of the mitochondrial genetic information under respiratory repression is controlled by a system distinct from that operating under respiratory adaptation. In the latter, genetic continuity is probably achieved by auto-reproduction and passive inheritance of intact mitochondria—a mechanism not affected by chloramphenicol, while the former system is dependent on the induced activity of a nuclear gene or genes.

Studies on the genetic regulation of cytochrome P-450 production in Saccharomyces cerevisiae

SummaryAn initial survey of 18 haploid strains of Saccharomyces cerevisiae revealed that only 3 of these strains could produce a detectable level of cytochrome P-450. A cross between a cytochrome P-450 producing strain of S. cerevisiae (B/B) and a non-producing strain (D22) gave a diploid which was a non-producer and a 2:2 segregation of producers to non-producers in meiotic tetrads. Of the two producers in each tetrad, one produced a higher level of cytochrome P-450 than the other. We deduce that cytochrome P-450 production in S. cerevisiae is regulated by a single nuclear gene and that a modifier gene is also involved which can enhance the amount of cytochrome P-450 synthesized. Benzo(a)pyrene (an inducer of P-450 in yeast) had no effect on the action of the regulatory gene.