Am Kroon

On the effect of chloramphenicol and oxytetracycline on the biogenesis of mammalian mitochondria.

Abstract 1. The antibiotics chloramphenicol and oxytetracycline completely blocked the formation of cytochrome c oxidase (EC 1.9.3.1) in regenerating rat liver if the serum concentration was continuously kept above 7 μg/ml serum for chloramphenicol or 6 μg/ml for oxytetracycline. 2. This inhibition was also visible in the mitochondria from regenerating livers of chloramphenicol- or oxytetracycline-treated animals. Both the specific cytochrome c oxidase activity and the concentration of cytochrome aa 3 (the hemoprotein identical to the enzyme cytochrome c oxidase) were lower than in mitochondria from control animals. 3. For both antibiotics, withdrawal caused an immediate resumption of cytochrome c oxidase and cytochrome aa 3 formation. The inhibition was therefore reversible. 4. These results are discussed in view of the known toxic effects of chloramphenicol medication and in connection with our previous findings concerning mitochondrial protein synthesis in vitro and cultured rat-heart cells.

Mitochondrial DNA from various organisms does not contain internally methylated cytosine in -CCGG- sequences

Mitochondrial DNAs from yeast, Neurospora, rat and calf do not contain internally methylated cytosine in -CCGG- sequences.

Mitochondrial biogenesis and mitochondrial activity during the progression of the cell cycle of human leukemic cells

Mitochondrial (mt) biogenesis and mt function were investigated during the cell cycle of leukemic cells. The study shows that the activity of enzymes involved in oxidative phosphorylation increases in the early G1 phase. This increase in activity precedes that of other mt enzymes such as citrate synthase and adenylate kinase. Therefore, the synthesis of mt enzymes, needed for the reduplication of the mt mass in the course of the cell cycle, occurs in a sequential order. The enzymes of the system for oxidative phosphorylation are composed of several subunits. Some of these subunits are encoded on mtDNA and synthesized by mt-specific RNA and protein synthesis. This explains why inhibition of mt protein synthesis during the progression of the cell cycle of G1-enriched cells results in an increasing shortage of ATP. This lack of ATP results first in progression delay and, subsequently, in a cell cycle block in early G1. Furthermore, shortage of ATP impairs the increase in activity of at least one mt matrix enzyme. This study offers new information about a number of aspects of mt biogenesis and mt function during cell cycle progression and elucidates the cytostatic mechanism resulting from prolonged inhibition of mt protein synthesis.

The mitochondrial genetic system as a target for chemotherapy: Tetracyclines as cytostatics

The mitochondrial genetic system is indispensable for the biosynthesis of the enzyme complexes involved in aerobic energy generation. Tetracyclines inhibit the expression of only the mitochondrial genes because they specifically block mitochondrial protein synthesis. A salient feature is that this inhibition occurs at the low concentration required for anti-bacterial treatment, provided that this concentration is maintained continuously. Evidence is presented that the growth of carcinogen-induced tumors can be inhibited by tetracyclines. It is further shown that the development in the cheek pouch of the Syrian hamster of a transplantable hypernephroma from human origin can be strongly retarded by tetracyclines as well. Therefore, the mitochondrial genetic system has to be reckoned as a target for chemotherapy and tetracyclines as cytostatic agents.

Circular DNA from mitochondria of Neurospora crassa.

Abstract Isolated mitochondria from two wild-type strains of Neurospora crassa were disrupted by osmotic shock in order to release mitochondrial DNA. In the electron microscope this DNA appeared to consist for a large part of circular molecules, most of which showed the typical supertwisted aspect, characteristic for closed circular duplex DNA. Strain 5256 contained circular molecules of 19 μm, one circle of 5.7 μm was found. Strain 5297 contained circular molecules of 19 μm as well as many small circles ranging in size from 0.5 to 7 μm. One circular molecule of 39 μm was found. The number of crossovers for molecules of both strains is about 7 per μm.

The restriction fragment map of rat-liver mitochondrial DNA: a reconsideration.

Abstract 1. Rat-liver mitochondrial DNA (mtDNA) contains at least 8 cleavage sites for the restriction endonuclease Eco RI, 6 for the restriction endonuclease Hind III, 2 for the restriction endonuclease Bam HI and 11 for the restriction endonuclease Hap II. 2. The physical map of the restriction fragments of Eco RI, Hind III, Bam HI and Hap II is constructed on the basis of: (a) the analysis of partially restricted fragments; (b) analysis of the double digests of total mtDNA; (c) the digestion of isolated restriction fragments with other restriction endonucleases; (d) the identification of fragments of complete single and double digestions and of partially digested fragments containing the base sequences complementary to the 12-S and 16-S RNAs of rat-liver mitochondrial ribosomes. 3. The genes for the ribosomal RNAs are shown to be closely linked. This result differs from data previously reported (Saccone, C., Pepe, G., Cantatore, P., Terpstra, P. and Kroon, A.M. (1976) in The Genetic Function of Mitochondrial DNA, pp. 27–36, Elsevier/North-Holland Biomedical Press, Amsterdam). 4. The origin of replication (D-loop) is localized in the vicinity of the small ribosomal RNA gene and the direction of replication is distant from this gene. 5. The mitochondrial tRNA genes are scattered over the genome as in other animal mtDNAs. The approximate minimal number of tRNA genes is 16–20. 6. We concluded previously that the Eco RI restriction fragments A and D are not adjacent and failed to show the overlap of the 16 S rRNA gene for the Eco RI fragment D and Hind III fragment A. This misinterpretation was due to the fact that the two smallest Eco RI fragments could not be detected with the methods applied and to the lower specific radioactivity of the ribosomal RNAs used in the first series of hybridization experiments.

The antitumour effect of doxycycline on a T-cell leukaemia in the rat

Previous studies showed that T-lymphoid cells are permeable to the tetracyclines, whereas B-lymphoid and erythroid cells are not. The tetracyclines impair mitochondrial protein synthesis if they have access to cells. Inhibition of mitochondrial protein synthesis during a number of cell cycles results, as a consequence, in proliferation arrest. The tetracyclines can therefore be considered as cytostatics. In the present study the effect of prolonged treatment with doxycycline on the growth of a T-cell type leukaemia of the rat was investigated. It is shown that doxycycline treatment inhibits not only tumour cell proliferation, but leads moreover to complete tumour eradication. The way by which the latter is achieved depends on the doxycycline concentration and, surprisingly, on the stage of tumour progression at which doxycycline administration is started. As, because of the permeability barrier, the proliferation of erythroid and B-lymphoid cells is not affected by the tetracyclines, the tetracyclines may provide a tool without serious side-effects in the therapy of T-type tumours.

The interference of the macrolide antibiotics with mitochondrial protein synthesis.

Abstract 1. Protein synthesis by isolated BHK-21 mitochondria was shown to be inhibited by the macrolides erythromycin, tylosin tartrate, spiramycin and carbomycin. Intact mitochondria from rat liver were insensitive to erythromycin, but inhibited by tylosin tartrate, spiramycin and carbomycin. 2. Cytochrome c oxidase formation in BHK-21 cells was unaffected by tylosin tartrate and spiramycin, but strongly inhibited by carbomycin. Mitochondrial protein synthesis in whole BHK-21 cells showed the same insensitivity to tylosin tartrate and spiramycin. 3. Peptidyl transferase activity of isolated rat-liver mitochondrial ribosomes was inhibited by tylosin tartrate, spiramycin and carbomycin, whereas chloramphenicol inhibition was reversed by erythromycin. Rather high antibiotic concentrations (200 μg/ml or higher) were necessary to obtain these effects. For Neurospora crassa mitochondrial ribosomes a similar response to these macrolides was found. Therefore mitochondrial ribosomes from N. crassa are markedly less sensitive to macrolides than yeast mitochondrial ribosomes. 4. It is shown that mammalian mitochondrial protein synthesis is sensitive to erythromycin, tylosin tartrate, spiramycin and carbomycin, but that for the first three antibiotics membrane barriers exist: for erythromycin the mitochondrial membrane is the barrier and for tylosin tartrate and spiramycin the cell membrane. 5. Amino acid incorporation by isolated mitochondria appears much more sensitive to inhibition by tylosin tartrate, spiramycin and carbomycin than the isolated 55-S ribosomes. For carbomycin this can be explained in part by the finding that carbomycin was concentrated by intact rat-liver mitochondria in vitro .

Impairment of liver regeneration during inhibition of mitochondrial protein synthesis by oxytetracycline

Under standard conditions, liver regeneration is impaired if mitochondrial protein synthesis is completely blocked. By treating rats with oxytetracycline for various periods of time directly prior to partial hepatectomy, livers were led to a condition of relative deficiency in cytochrome c oxidase and ATP synthetase. To this end, oxytetracycline was administered by means of continuous intravenous infusion up to concentrations of 20 micrograms/ml serum, giving a gradual decrease in cytochrome c oxidase activity. This activity was used as a marker for functionally capable mitochondria and as a tool to monitor the efficiency of inhibition of mitochondrial protein synthesis. It is shown that liver regeneration is strongly impaired after a period of pretreatment of 22 days or more and continuation of oxytetracycline treatment during regeneration. The mitochondrial respiratory capacity is reduced to 14% of the control value under these conditions. To obtain inhibitory levels within the regenerating liver, it was necessary to raise the serum levels slightly above 20 micrograms/ml. This measure is most likely required because of the poor vascularization of the regenerating liver. The serum levels were kept, however, far below those known to inhibit cytoplasmic protein synthesis. The results show that in normal liver the respiratory capacity must be reduced drastically before energy-requiring processes become affected. In Zajdela hepatoma cells, similar effects are found after reduction of the cytochrome c oxidase activity to 38%. This difference in sensitivity is probably based on the different mitochondrial content of liver cells and the liver-derived Zajdela cells.

A complete cleavage map of Neurospora crassa mtDNA obtained with endonucleases Eco RI and Bam HI.

A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases. The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III. The sequence of the Eco RI and Bam HI fragments has been established by analysis of partial fragments. By digestion of the Eco RI fragments with Bam HI, a complete overlapping map has been constructed. The position of the largest Hind III fragment on this map has also been determined. The map is circular and the added molecular weight of the fragments is 40 - 10(6), which is in good agreement with earlier measurements on intact DNA, using the electron microscope.