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Biomedical subjects

A M Kroon

Publications and source records attributed to A M Kroon.

At least 37 records · Page 2Linked to original sources
PubMed1985

The effect of long-term inhibition of mitochondrial protein synthesis on the oxidation capacity of mitochondria for NADH-linked substrates.

Experiments are presented showing that specific inhibition of mitochondrial protein synthesis by tetracyclines decreases the activity of the NADH-dehydrogenase complex in liver mitochondria, if rats are treated for long periods with these antibiotics. The corresponding inhibition of this complex in tumor cells (Zajdela hepatoma) and tumor mitochondria (Leydig cell tumor) is even more pronounced. It is concluded that the mitochondrial genetic system is involved in the assembly of the NADH-dehydrogenase complex, most likely by coding for one or more subunits. It is argued that this information, contrary to the situation for cytochrome c oxidase, the cytochrome bc1 complex and ATPsynthase, has been missed in previous experiments employing differential inhibition of mitochondrial protein synthesis, because of the circumstance that the inhibition did not reach the level at which it became rate-limiting.

Animals↗
PubMed1985

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.

Animals↗
PubMed1984

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.

Animals↗
PubMed1983

Antibacterial drugs and their interference with the biogenesis of mitochondria in animal and human cells.

Mitochondrial RNA and protein synthesis in mammalian cells is sensitive to inhibition by a variety of antibiotics which are used in medical practice. In spite of the intrinsic sensitivity of the synthetic processes to these drugs it appears that inhibition in intact cells and living organisms is not observed in all cases because the cellular membranes may act as barriers which prevent the antibiotics from reaching their intramitochondrial targets. This holds for the rifamycins, the lincomycins and a number of macrolides, but not for chloramphenicol and its analogues. Some of the toxic side-effects of the latter antibiotics can be related to their antimitochondrial action. For the tetracyclines selectivity in permeability exists in the sense that some cell types are permeable and other are not. The hypothesis is developed that the deliberate inhibition of mitochondrial protein synthesis in vivo may lead to cell proliferation arrest and offer a device in combined modality treatments of malignant growths. This hypothesis is supported by the results of two studies with experimental tumour models in rats and by retrospective and prospective clinical data.

Animals↗
PubMed1983

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.

ATP Synthetase Complexes↗
PubMed1983

Characterization of a 45-kDa polypeptide as the precursor of subunit 1 of cytochrome c oxidase in Neurospora crassa.

In a previous paper (Van 't Sant, P., Mak, J.F.C. and Kroon, A.M. (1981) Eur. J. Biochem. 121, 21-26) we showed the existence of three elongated precursor proteins (45, 36 and 25 kDa) of mitochondrial translation products in Neurospora crassa. We presented some indications that the largest precursor could be related to subunit 1 of cytochrome c oxidase. Here we present conclusive evidence that the 45-kDa polypeptide is indeed this precursor by demonstrating that an immunodetectable 45-kDa polypeptide displays the same behaviour as the labeled 45-kDa precursor; both accumulate after long incubation with cycloheximide or by decreasing the temperature and both are not tightly membrane bound. Moreover the antibody against subunit 1 of cytochrome c oxidase also recognizes, in immunoadsorption experiments, besides subunit 1, the 45-kDa polypeptide accumulated by cycloheximide incubation. Furthermore, we developed a small scale purification of antibodies against subunit 1 of cytochrome c oxidase. By means of these purified antibodies it is demonstrated that the 45-kDa polypeptide and subunit 1 have corresponding antigenic determinants. Under the various conditions tested, all three precursors are less firmly membrane-bound than the mature subunits. Finally, it is observed that in short incubations in vivo, chloramphenicol inhibits the processing of the mitochondrially synthesized precursors, under conditions where mitochondrial translation is only partially inhibited.

Electron Transport Complex IV↗
PubMed1983

Non-random patterns of nucleotide substitutions and codon strategy in the mammalian mitochondrial genes coding for identified and unidentified reading frames.

The base sequence of large part of the mitochondrial DNA of Wistar rats is presented. The sequence is compared with those of other mammalian mitochondrial DNAs. The nucleotide and amino acid homologies, codon strategy, nature and patterns of substitutions are reported. It results a very high amount of silent substitutions and, in short divergence time, a predominance of transitions on transversions. In both types of substitutions a strong bias in avoiding the use of the G in the third codon position is observed.

ATP Synthetase Complexes↗
PubMed1983

Arrest of in vivo growth of a solid Leydig cell tumor by prolonged inhibition of mitochondrial protein synthesis.

Oxytetracycline was given by means of chronic i.v. infusion, in amounts which impair specifically mitochondrial protein synthesis, to Wistar rats carrying a solid Leydig cell tumor. The prolonged inhibition of mitochondrial protein synthesis finally results in proliferation arrest of the s.c. growing tumor. As long as the tumor is growing, the energy-generating capacity of the mitochondrial becomes diluted, until it is reduced to a critical level, which results in growth arrest. This cytostatic effect is found after treatment for 8 to 12 days. During this period, the tumor volume increases to an extent comparable to 2 to 3 tumor cell divisions. The proliferation arrest found is, at least after treatment for 3 weeks, reversible. Withdrawal of oxytetracycline results in continuation of tumor growth after a latent period of about 5 days. The data confirm previous observations made in experimental systems about the usefulness of mitochondrial protein synthesis as target for cancer chemotherapy. They support, moreover, the explanation given for the marked prolongation of survival time found in tetracycline-treated patients with a squamous cell carcinoma of the larynx-pharynx. If antineoplastic therapy, based on inhibition of mitochondrial protein synthesis, is considered, the tetracyclines should be the drugs of choice, because their toxic side effects are minimal at dosages which cause tumor proliferation arrest.

Animals↗
PubMed1982

The nucleotide sequences of several tRNA genes from rat mitochondria: common features and relatedness to homologous species.

We have determined the nucleotide sequences of thirteen rat mt tRNA genes. The features of the primary and secondary structures of these tRNAs show that those for Gln, Ser, and f-Met resemble, while those for Lys, Cys, and Trp depart strikingly from the universal type. The remainder are slightly abnormal. Among many mammalian mt DNA sequences, those of mt tRNA genes are highly conserved, thus suggesting for those genes an additional, perhaps regulatory, function. A simple evolutionary relationship between the tRNAs of animal mitochondria and those of eukaryotic cytoplasm, of lower eukaryotic mitochondria or of prokaryotes, is not evident owing to the extreme divergence of the tRNA sequences in the two groups. However, a slightly higher homology does exist between a few animal mt tRNAs and those from prokaryotes or from lower eukaryotic mitochondria.

Animals↗
PubMed1982

Effects of oxytetracycline on in vivo proliferation and differentiation of erythroid and lymphoid cells in the rat.

Previous studies revealed that inhibition of mitochondrial protein synthesis by tetracyclines during a number of cell divisions results in proliferation arrest, in vitro as well as in in vivo. The tetracyclines may, therefore, be considered as cytostatics. In this context it is of interest to know the effect of the tetracyclines on rapidly dividing normal cell types, such as erythroid and lymphoid cells. To investigate the influence of the tetracyclines on the proliferation of these cells, we studied immunological responses to sheep red blood cells and recovery from severe anaemia in rats in the presence or absence of oxytetracycline (OTC). Under the experimental conditions used, effects of OTC on the immune responses were only found for events directly or indirectly related to T cell proliferation. The results presented thus strongly suggest that OTC inhibits the division of T lymphoid cells only. The proliferation of other haemopoietic cells is not influenced, most likely because these cells are not permeable to OTC.

Anemia↗
PubMed1981

The nucleotide sequence of the large ribosomal RNA gene and the adjacent tRNA genes from rat mitochondria.

We have sequenced the Eco R(1) fragment D from rat mitochondrial DNA. It contains one third of the tRNA (Val) gene (the remaining part has been sequenced from the 3' end of the Eco R(1) fragment A) the complete gene for the large mt 16S rRNA, the tRNA (Leu) gene and the 5' end of an unidentified reading frame. The mt gene for the large rRNA from rat has been aligned with the homologous genes from mouse and human using graphic computer programs. Hypervariable regions at the center of the molecule and highly conserved regions toward the 3' end have been detected. The mt gene for tRNA Leu is of the conventional type and its primary structure is highly conserved among mammals. The mt gene for tRNA(Val) shows characteristics similar to those of other mt tRNA genes but the degree of homology is lower. Comparative studies confirm that AGA and AGG are read as stop codons in mammalian mitochondria.

Animals↗
PubMed1981

The characterization of the area of rat mitochondrial DNA containing the large ribosomal RNA gene.

The base sequence of the Eco-RI D fragment from rat liver mtDNA, cloned in recombinant plasmid, has been analyzed, This fragment contains the genes for the 16SRNA of the large ribosomal subunit and the tRNA(Leu). Comparisons between these genes and corresponding regions either in other mitochondrial genomes or in E. coli DNA are presented, that allow some interesting evolutionary and phylogenetic considerations.

Animals↗
PubMed1981

Fluorometric determination of tetracyclines in small blood and tissue samples.

A reported fluorometric method to determine tetracycline in biological material was modified to determine low levels of the antibiotics when using only small amounts of starting material. The method is applicable to tissue distribution studies in small experimental animals and also can be used for serial determinations in blood samples of these animals and for biopsy material. The tetracyclines are extracted in the presence of ethylenediaminetetraacetic acid, and the phosphate precipitation steps are omitted. This omission makes the procedures considerably less time consuming. The recoveries of the tetracyclines, oxytetracycline and doxycycline, are approximately 80-100%. The values obtained by this method were consistent with those from microbiological assays, and its reliability was shown further in a comparison with the fate of radioactive tetracycline.

Bacillus cereus↗
PubMed1981

Larger precursors of mitochondrial translation products in Neurospora crassa. Indications for a precursor of subunit 1 of cytochrome c oxidase.

Specific labeling in vivo of the formylated N termini of mitochondrial translation products revealed that some mitochondrially synthesized proteins were not labeled this way. As a consequence, it was worthwhile considering that larger precursor proteins of mitochondrial translation products exist. Although we used a rapid isolation procedure, only after 2-h of labeling in the presence of cycloheximide, could three additional mitochondrial translation products (molecular mass 45, 36, and 25 kilodaltons) be detected. Preincubation with cycloheximide indicated that these proteins might be larger precursors which were no longer processed due to the prolonged presence of cycloheximide. To prevent processing of the precursors during isolation, cells of the slime mutant were directly lysed in boiling sodium dodecyl sulphate solution. In this way, the same three additional mitochondrial translation products were detected after a pulse-labeling of 1 min. These proteins behave in a precursor-like fashion. Labeling at 9 degrees C resulted in a partial accumulation of the three additional proteins. Finally protein blots treated with antibodies and 125I-labeled protein A, support the idea that the 45-kDa protein is a precursor of subunit 1 of cytochrome c oxidase; 50-80% of this precursor could be detected in the post-mitochondrial supernatant, indicating that this polypeptide is not tightly bound to the membrane.

Cycloheximide↗
PubMed1981

Arrest of in vivo proliferation of Zajdela tumor cells by inhibition of mitochondrial protein synthesis.

The proliferation of Zajdela tumor cells, grown in vivo in Wistar rats, is arrested by low amounts of oxytetracycline. Oxytetracycline is administered by means of continuous infusion in such a way that the oxytetracycline concentration in serum and ascitic fluid is maintained at a level at which only mitochondrial protein synthesis is blocked. Under these conditions, Zajdela mitochondrial tumor cells cease dividing after a few cell generations, an event which is preceded by reduction of cytochrome c oxidase activity of the tumor cells. Toxicity to host tissues is limited to the immune system which is apparently suppressed by the drug. Even so, oxytetracycline might have therapeutic potential in human cancer therapy, especially because it does not influence the hemopoietic system.

Animals↗