Inhibition Of Mitochondrial Protein Synthesis Research Articles

Specific inhibition of mitochondrial protein synthesis influences the amount of complex I in mitochondria of rat liver and Neurospora crassa directly

Specific inhibition of mitochondrial protein synthesis reduces the oxidation rate of NADH-linked substrates in rat liver as well as in Neurospora crassa mitochondria. The present study shows that this is due to the fact that inhibition of mitochondrial protein synthesis leads to a decrease of the concentration of active complex I. Therefore, these results demonstrate that at least one of the genes for the subunits of complex I is localized on mitochondrial DNA.

Effect of mitochondrial protein synthesis inhibitors on the trophic action of nerve stump in mice

We studied the effect of mitochondrial protein synthesis inhibitors on the neurotrophic action of the nerve stump in mice. The sciatic nerve was cut as close to, or as far from the extensor digitorum longus muscles as possible. At 2 and 3 days after denervation, depolarization in the resting membrane potential of muscles with long nerve stumps (14–16 mm) was significantly smaller than in muscles with very short (<2 mm) nerve stumps. Chloramphenicol (100 mg/kg, p.o.), erythromycin (4 μg/kg, i.p.), ethidium bromide, (10 μg/kg, i.p.) or acridine (10 μg/kg, i.p.), administered for 2 or 3 days after denervation, increased depolarization in the resting membrane potential of muscles with long nerve stumps; muscles with very short nerve stumps were not affected by these drugs. The administration of chloramphenicol and erythromycin resulted in an earlier increase in the depolarization than ethidium bromide and acridine. Activities of cathepsin B and L of lysosomal proteases in the nerve stumps were enhanced by nerve degeneration; they were not affected by the administration of chloramphenicol or ethidium bromide. We suggest that proteins synthesized in mitochondria of the nerve may participate in trophic actions.

RNA splicing in Neurospora mitochondria : Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1

cyt18-1 (299-9) is a nuclear mutant of Neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rRNA intron. In the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mRNA introns. Two genes were studied in detail; the cytochrome b ( cob) gene, which contains two introns, and a “long form” of the cytochrome oxidase subunit I ( coI) gene, which contains four introns. We found that splicing of both cob introns and splicing of at least two of the coI introns are strongly inhibited in the mutant, whereas splicing of col intron 1, which is excised as a 2.6 × 10 3 base circle, is relatively unaffected. The rRNA intron and both cob introns are group I introns, whereas the circular coI intron may belong to another structural class. Control experiments showed that the degree of inhibition of splicing is greater in the mutant than can be accounted for by severe inhibition of mitochondrial protein synthesis. Finally, experiments in which mutant cells were shifted from 25 °C to 37 °C showed that splicing of the large rRNA precursor and splicing of the coI mRNA precursor are inhibited with similar kinetics. Considered together, our results suggest that the cyt18-1 gene encodes a trans-acting component that is required for the splicing of group I mitochondrial DNA introns or some subclass thereof. Since Neurospora cob intron 1 has been shown to be self-splicing in vitro, defective splicing of this intron in cyt18-1 indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo.

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 bc 1 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.

Defective assembly of the mitochondrial ribosomes in yeast cells grown in the presence of mitochondrial protein synthesis inhibitors

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in the presence of mitochondrial protein synthesis inhibitors erythromycin and chloramphenicol. Yeast cells grown in the presence of erythromycin (2 mg/ml) do not appear to contain any detectable amounts of the mitochondrial small (37 S) ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S was observed; this particle could be shown to be related to the mitochondrial small ribosomal subunit by two-dimensional gel electrophoretic analysis of its protein components. Since the var1 protein is the only mitochondrial translation product known to be associated with the mitochondrial ribosome, our results suggest that this protein is essential for the assembly of the mature small subunit, and that the var1 protein enters the pathway for the assembly of the small subunit at a late step. In at least one strain of yeast the accumulation of the 30-S particle appears to be very sensitive to catabolite repression. When yeast cells are grown in the presence of chloramphenicol instead of erythromycin, assembly of the small subunit appears to be only partially inhibited, and the presence of the 30-S particle could not be clearly demonstrated. This observation is consistent with the fact that in yeast, chloramphenicol inhibits mitochondrial protein synthesis by about 95% only and that the synthesis of the var1 protein appears to be the least sensitive to this inhibition.

Protein synthesis by hepatic mitochondria isolated from carbon tetrachloride-exposed rats

Hepatic mitochondria isolated from rats 40 h after dosage with 1.1 ml/kg CCl 4 are uncoupled and display structural damage. Mitochondrial function returns during hepatic recovery. Because the products of mitochondrial protein synthesis are essential to mitochondrial structure and function, the effects of CCl 4 on the rate of mitochondrial protein synthesis, and on the products, was studied using mitochondria from CCl 4-exposed rats during the early, maximum development and resolution stages of CCl 4-induced mitochondrial damage. Rates of mitochondrial protein synthesis (incorporation of [ 35S]methionine) were elevated 300% over that of mitochondria from non-exposed rats 17 h after exposure; depressed by 50% at 40 h and above control at 113 h. When the radiolabeled products of incorporation were separated and examined by autoradiography, a novel, low-molecular-weight band, of approx. 9700, was apparent 40 h after CCl 4 exposure. A band of similar molecular weight appeared when control mitochondria were incubated without an exogenous supply of ATP. Mitochondria from exposed rats which displayed rates of protein synthesis greater than control consistently had a relative increase in a band that corresponded in size to that of cytochrome oxidase subunit I. It was concluded that the loss of mitochondrial function induced by CCl 4 could not be attributed to inhibition of mitochondrial protein synthesis, and that the mitochondria may not always synthesize protein in constant proportions.

Effect of mitochondrial inhibitors on metaphase-telophase progression and nuclear membrane formation in Chinese hamster cells.

Chinese hamster Don cells in log-phase were exposed to Colcemid during the G2 period with and without a combination of divalent cation chelators and mitochondrial inhibitors. Isolated metaphase cells were incubated as follows: (i) without Colcemid but with other agents and the progression was monitored from metaphase (M) to telophase (Tel) and to cell division; (ii) with Colcemid and other agents and the rate of micronuclei formation in the absence of anaphase was studied. Both EDTA and EGTA accelerated the progression from M to Tel, but did not affect the overall rate of cell division. Chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, blocked the effect of the chelators and also retarded the progression. An inhibitor of mitochondrial respiration, Antimycin A (AA), also retarded the progression in the absence of the chelators and prevented the promoting effect of the chelators. A stimulator of ATPase for ATP breakdown. 2,4-dinitrophenol (DNP), accelerated the M to Tel progression. Chloramphenicol (CAP) and AA, as well as DNP, appeared to have little effect on the formation of micronuclei in the presence of Colcemid. EGTA, which affects cell surface Ca2+, stimulated the formation of micronuclei. This study indicates that Ca2+ ions and mitochondrial function are involved in the regulation of a certain segment of mitosis beyond metaphase, with Ca2+ sequestration in the mitochondria and chelation of Ca2+ by EGTA as dominant factors.

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 doxycyline 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.

Identification of mitochondrial proteins on two-dimensional electrophoresis gels of extracts of adult Drosophila melanogaster

Abstract Several hundred proteins have been resolved on two-dimensional gels of extracts of [35S]methionine-labeled adult Drosophila melanogaster. 27 of these polypeptides disappear from the gel pattern after feeding the K+ ionophore nonactin. These proteins have been identified as mitochondrial, since the two-dimensional gel pattern of extracts of isolated mitochondria correlates well with the pattern of the proteins missing from that of nonactin-treated flies. Nine new proteins also appear on the two-dimensional gels of the extracts from the nonactin-treated flies. Apparently, these nine proteins are precursors of the mature mitochondrial forms. These particular data support the concept that processing of many of the cytoplasmically synthesized mitochondrial proteins requires a specific membrane potential, and that some of these proteins are modified intramitochondrially. However, using [35S]methionine incorporation techniques, not all labeled polypeptides disappear from mitochondria during such treatment. Feeding similarly radiolabeled flies with chloramphenicol, an inhibitor of mitochondrial protein synthesis, results in the disappearance of only one protein from the gel pattern with the concurrent appearance of a ‘new’ high-molecular-weight polypeptide. Collectively, these data show that a specific group of [35S]methionine-labeled mitochondrial proteins can be identified by selective inhibition of mitochondrial function in whole cell protein maps of adult D. melanogaster.

Effects of mitochondrial protein synthesis inhibitors on the incorporation of isoleucine into Plasmodium falciparum in vitro.

Plasmodium falciparum was grown in human erythrocytes in vitro and the effect of chloramphenicol, erythromycin, and tetracycline on growth and maturation of the parasites and on their ability to incorporate [3H]isoleucine into protein was observed. Exposure of rings to high concentrations of chloramphenicol had little effect on subsequent maturation of the rings whereas brief (4 h) exposure of trophozoites caused a dose-dependent inhibition of subsequent ring formation. Incorporation of [3H]isoleucine into protein was not affected during at least 6 h of exposure to high concentration of the three drugs examined, but appreciable inhibition was observed after 21 h, with chloramphenicol being the least effective inhibitor. These results suggest that there is a stage-specific effect of inhibition of mitochondrial protein synthesis on subsequent development and that the mitochondria are essential for growth and development even though they lack a functional Krebs cycle.

ACIDOSIS IS A PRESENTING FEATURE OF CHLORAMPHENICOL TOXICITY

Chloramphenicol (CAP) is an inhibitor of mitochondrial protein synthesis and impairs energy production through interference with oxidative phosphorylation. Metabolic acidosis associated with lactic acidemia has not been described as a presenting feature of CAP toxicity. Three critically ill children 6 mos to 11 y/o (congenital hypoaldosteronism, brain stem dysfunction, Reye's syndrome) received CAP (75-100 mg/kg/d) and had normal pH and stable cardiovascular, renal and pulmonary function for 48 hrs. Each developed profound acidosis (arterial pH 7.13-7.22) 60-108 hrs after beginning CAP. Initial CAP conc. were 61, 80, and 30 mcg/ml and subsequent levels decreased; CAP was 30 mcg/ml in the child with Reye's syndrome, a condition with recognized mitochondrial dysfunction. SGPT conc. were 386, 2,620, and 145 IU/1 when acidosis was recognized. Serum lactate was 6.6 and 8.8 mM in 2 patients prior to CAP and increased to 11.1 and 17.1 mM. Mean anion gap increased from 16.8 to 32.3 mM in the 3 patients. Hypotension followed acidosis by 6-19 hrs and was severe in one and mild in 2. Other signs of gray baby syndrome were absent. CAP was stopped in each patient and resolution of acidosis and hypotension paralleled the fall of serum CAP. 1) Unexplained metabolic acidosis is an early sign of CAP toxicity; 2) pre-existing lactic acidemia, hepatic dysfunction, and/or mitochondrial dysfunction may predispose to CAP toxicity; 3) toxicity may occur with CAP levels in a therapeutic range in Reye's syndrome.

Selective synthesis of mitochondrial proteins by Chinese hamster ovary cells severely starved for various amino acids.

Chinese hamster ovary (CHO) cells were subjected to severe amino acid starvation for histidine, leucine, methionine, asparagine, tyrosine, glutamine, valine, and lysine, using amino acid analogs or mutations in specific aminoacyl-tRNA synthetases. At protein synthetic rates of less than 5%, in all cases, the newly synthesized proteins were found on two-dimensional electrophoretic gels to consist of a few intensely labeled spots, with the exception of lysine. This pattern could also be produced by strong inhibition of cytoplasmic protein synthesis with cycloheximide, and was abolished by preincubation with the mitochondrial protein synthesis inhibitor chloramphenicol. It appears therefore that the spots represent mitochondrial protein synthesis and that animal cells must have separate aminoacyl-tRNA synthetases for mitochondrial tRNAs corresponding to all these amino acids except, possibly, for lysine.

Cell cycle kinetics and metabolism of Ehrlich ascites tumor cells in the presence of chloramphenicol as inhibitor of mitochondrial protein synthesis.

In the presence of 150 micrograms/ml of chloramphenicol, viability (dye exclusion test) of Ehrlich ascites tumor cells is not severely affected within 48 h; however the number of dead cells increases above this concentration; more than 10 mg/ml render all of the cells dye-positive within a culture period of 24 h. With the highest concentration tested (25 mg/ml) cells begin to lose viability 8 h after beginning of treatment. In the first passage in the presence of the antibiotic, proliferation of the cells is reduced by about 50%; in the second passage cell growth was about 65% that of controls. As is shown by flow cytometric analysis and BrdU-H33258 technique of flow cytometry, the reduction of proliferation in the second passage is caused by retardation of cell cycle progression of about 8 h; in the third passage the cell cycle is delayed by further 8 h as compared to controls. On recultivation in the third passage in the absence of the inhibitor an increase of cell number of about 75% of controls was observed. Lactate production and glucose uptake of the cells were stimulated by the inhibitor by about 20%; oxygen consumption was 60% that of controls after 24 h; however the ATP/ADP ratio of chloramphenicol treated cells was in the range of controls. Over a time period of 24 h the incorporation of [2-14C]thymidine is reduced to 34 +/- 3%, incorporation of [2-14C]uridine was 88 +/- 4% that of controls, incorporation of [U-14C]lysine was not significantly affected by 150 micrograms/ml of chloramphenicol; the same was found for the uptake of [2-14C]-alpha-aminoisobutyric acid. Electron micrographs of chloramphenicol treated cells reveal a high amplitude of swelling of all mitochondria with a translucent appearance of the inner compartment. The mitochondrial membranes remain largely intact but the number of cristae is drastically reduced.

Neurotrophic influences are not affected by miniature end-plate potentials

We investigated whether or not miniature end-plate potentials (MEPPs) play a part in neurotrophic influences. The sciatic nerve of mice, which supplies the extensor digitorum longus muscles, was cut either close to, or far from, the muscle. Denervation changes were evaluated using the resting membrane potential and dose-response curves obtained by plotting acetylcholine-induced contractures of muscles denervated 3 days earlier. The cessation of MEPPs and the manifestation of denervation changes occurred significantly later in muscles with long nerve stumps than in those with short nerve stumps. A single administration of ethidium bromide, a specific inhibitor of mitochondrial protein synthesis, accelerated the cessation of MEPPs in muscles with long nerve stumps without affecting the manifestation of denervation changes. We therefore conclude that MEPPs do not have neurotrophic effects.

Rat liver protoporphyrinogen IX oxidase: Site of synthesis and factor influencing its activity

Rat liver protoporphyrinogen IX oxidase is not formed in mitochondria in contrast to the claims made for the yeast enzyme (Poulson and Polglase, FEBS Lett. (1974) 40 , 258). Inhibition of mitochondrial protein synthesis in regenerating rat livers by thiamphenicol led, instead, to a slight increase in protoporphyrinogen oxidase activity. Protoporphyrinogen IX oxidase was not induced in rat liver by triiodothyronine, an inducer of mitochondrial protein synthesis, or by AIA, an inducer of heme synthesis. Significant increases in activity were observed to be associated with rapidly growing cells, such as regenerating livers and rat ascites hepatoma cells.

Regulation of the activity of the alternative oxidase in callus forming discs from potato tubers

Callus‐forming discs from potato tubers lose 80% of their starch during one month of incubation on nutrient medium containing either 0, 3 or 6% (w/v) sucrose. The content of soluble sugar in the discs varies from 5 mg (incubated without sucrose) to 22 mg (on 3% sucrose) and 40 mg (on 6% sucrose) per g fresh weight. The activity of the cytochrome pathway (Vcyt) increases during the first week of incubation on all media. Thereafter Vcyt decreases again on 0% sucrose medium, while it remains constant on 3 and 6% sucrose media. Alternative pathway capacity (Valt), absent in freshly sliced tissue, shows a sharp increase during the first days of incubation, independent of the sucrose concentration in the medium. This capacity further increases during prolonged incubation on 3 and 6% sucrose but decreases on 0% sucrose.The in vivo activity of the alternative pathway (the participation in uninhibited respiration, ϱValt) varies with the sucrose concentration and with the culture time. In tissue incubated for 2‐3 weeks on 6% sucrose as much as 45% of the electrons are transfered to oxygen via the alternative pathway. In this tissue the factor Q (the part of the alternative pathway capacity that is operative) is about 0.8, while in tissue incubated on 0 and 3% sucrose media p generally does not exceed 0.5.When chloramphenicol, an inhibitor of mitochondrial protein synthesis, is added to the medium together with 3% sucrose, the increase in Vyet does not occur, while the induction of Valt during the first week of incubation is the same as without chloramphenicol. A greater part of the alternative pathway capacity becomes operative in this tissue, leading to values of Q of almost 1 after prolonged incubation. Apparently, incubation on high sugar medium leads to extra participation in respiration of the energetically inefficient alternative oxidase pathway Excess sugar leads to wasteful respiration suggesting that the alternative oxidase functions as an ‘energy overflow’.

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.

The relationship of cardiolipin biosynthesis to mitochondrial protein synthesis in Tetrahymena pyriformis.

The synthesis of cardiolipin has been investigated following the inhibition of mitochondrial protein synthesis with chloramphenicol. Quantitative measurements of the amount of cardiolipin in the cell during treatment with chloramphenicol as well as pulse-labelling studies using labelled acetate were carried out. The results show that while whole cell phospholipid biosynthesis is depressed by the treatment (probably a reflection of a general cessation of growth), there is no sign of any preferential effect on cardiolipin synthesis. The data also show that as cells are reduced in size as they approach stationary phase there is a six- to seven-fold loss of total cellular phospholipids; however, the amount of cardiolipin is only reduced by four- to five-fold. There is a preferential conservation of cardiolipin as stationary phase is approached with the mole percent cardiolipin phosphorus in the cell rising from 5-7% to 10-12%.

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

Under standard conditions, liver regeneration is not 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 μg/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 μg/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.

Dependence of nucleus-directed rRNA synthesis upon mitochondrial protein synthesis in Tetrahymena.

The antibiotic chloramphenicol selectively inhibited mitochondrial protein synthesis in the ciliate protozoan Tetrahymena pyriformis GL. Secondary to the inhibition of mitochondrial protein synthesis was an inhibition of nuclear RNA synthesis at a time before inhibition of cellular protein and DNA synthesis. Of the stable non-polyadenylated RNA species in Tetrahymena, the addition of chloramphenicol resulted specifically in the inhibition of synthesis of 28S + 17S and 5S rRNA transcripts. By contrast, syntheses of 4S tRNA and 21S mitochondrial rRNA were not as extensively inhibited. The addition of 60 microM hemin before the addition of chloramphenicol partially protected against the inhibition of RNA synthesis. These data indicate that continued synthesis of nucleus-directed rRNA is linked to the synthesis of mitochondrial proteins in Tetrahymena.