Search

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 ofthe 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. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the occurrence and determinants of non-publication of clinical drug trials in the Netherlands. All clinical drug trials reviewed by the 28 Institutional Review Boards (IRBs) in the Netherlands in 2007 were followed-up from approval to publication. Candidate determinants were the sponsor, phase, applicant, centers, therapeutic effect expected, type of trial, approval status of the drug(s), drug type, participant category, oncology or other disease area, prospective registration, and early termination. The main outcome was publication as peer reviewed article. The percentage of trials that were published, crude and adjusted odds ratio (OR), and 95% confidence interval (CI) were used to quantify the associations between determinants and publication. In 2007, 622 clinical drug trials were reviewed by IRBs in the Netherlands. By the end of follow-up, 19 of these were rejected by the IRB, another 19 never started inclusion, and 10 were still running. Of the 574 trials remaining in the analysis, 334 (58%) were published as peer-reviewed article. The multivariable logistic regression model identified the following determinants with a robust, statistically significant association with publication: phase 2 (60% published; adjusted OR 2.6, 95% CI 1.1-5.9), phase 3 (73% published; adjusted OR 4.1, 95% CI 1.7-10.0), and trials not belonging to phase 1-4 (60% published; adjusted OR 3.2, 95% CI 1.5 to 6.5) compared to phase 1 trials (35% published); trials with a company or investigator as applicant (63% published) compared to trials with a Contract Research Organization (CRO) as applicant (50% published; adjusted OR 1.7; 95% CI 1.1-2.8); and multicenter trials also conducted in other EU countries (68% published; adjusted OR 2.2, 95% CI 1.1-4.4) or also outside the European Union (72% published; adjusted OR 2.0, 95% CI 1.0-4.0) compared to single-center trials (45% published). Trials that were not prospectively registered (48% published) had a lower likelihood of publication compared to prospectively registered trials (75% published; adjusted OR 0.5, 95% CI 0.3-0.8), as well as trials that were terminated early (33% published) compared to trials that were completed as planned (64% published; adjusted OR 0.2, 95% CI 0.1-0.3). The non-publication rate of clinical trials seems to have improved compared to previous inception cohorts, but is still far from optimal, in particular among phase 1, single-center, not prospectively registered, and early terminated trials.

The assembly of cytochrome-c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using two-dimensional PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subunits of cytochrome-c oxidase. Proteins were also visualized using metabolic labeling followed by two-dimensional electrophoresis and fluorography. These techniques allowed identification of two assembly intermediates of cytochrome-c oxidase. Assembly of the 13 subunits of cytochrome-c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and either VIIa or VIIb.

Two sons and one daughter of healthy consanguineous parents presented with fatal hepatic failure in association with severe depletion of mitochondrial (mt)DNA in liver; a third son is healthy. Other published cases of mtDNA depletion concern single members of a family, which excludes the use of haplotype analysis. In the family presented here, the inheritance of the genes for mitochondrial transcription factor A (mtTFA), nuclear respiratory factor 1 (NRF-1), mitochondrial single-stranded DNA-binding protein (mtSSBP), and endonuclease G (EndoG) was studied using microsatellite markers linked to these genes. The inheritance of the gene for mtDNA polymerase (pol gamma) was studied using a polymorphic CAG repeat present within the coding region of the gene. EndoG and mtSSBP were excluded, but mtTFA remains a candidate. Pol gamma or NRF-1 involvement would be compatible only with autosomal dominant inheritance. Coding sequence analysis of NRF-1 and mtTFA revealed no novel mutations in affected individuals.

We describe a family in which three children of consanguineous parents died of hepatic failure before the age of 3 months. The first child had clinical symptoms of liver disease with hypoglycemia that were evident at birth. The second child was healthy and has normal development. The third child had severe liver dysfunction noted a few days after birth. Liver failure also developed in the fourth child soon after birth. Recently a mitochondrial disorder was considered as a possible cause. Deficiency of respiratory chain enzymes that contain polypeptides encoded by mitochondrial DNA (mtDNA) and depletion of mtDNA were found in the liver of the fourth child, but mitochondrial abnormalities were absent in muscle of the third child. The similarities in clinical presentation suggest that liver-specific depletion of mtDNA was the cause of the hepatic failure in all three children. We conclude that liver dysfunction with onset in the perinatal period can be caused by depletion of mtDNA.

To study the assembly of mitochondrial F1F0 ATP synthase, cultured human cells were labeled with [35S]methionine in pulse-chase experiments. Next, two-dimensional electrophoresis and fluorography were used to analyze the assembly pattern. Two assembly intermediates could be demonstrated. First the F1 part appeared to be assembled, and next an intermediate product that contained F1 and subunit c. This product probably also contained subunits b, F6 and OSCP, but not the mitochondrially encoded subunits a and A6L. Both intermediate complexes accumulated when mitochondrial protein synthesis was inhibited, suggesting that mitochondrially encoded subunits are indispensable for the formation of a fully assembled ATP synthase complex, but not for the formation of the intermediate complexes. The results and methods described in this study offer an approach to study the effects of mutations in subunits of mitochondrial ATP synthase on the assembly of this complex. This might be of value for a better understanding of deficiencies of ATP synthase activity in mitochrondrial diseases.

The electron transfer activity of Complex I of the respiratory chain and Complex I-linked ATP synthesis were investigated in leukocytes of four males affected by Leber hereditary optic neuropathy and a mutation in the ND6 gene at nucleotide position 14,484 of mtDNA. The electron transfer activity in leukocytes of the patients was about 35% of that in control leukocytes, whereas the Complex I-linked ATP synthesis showed a decrease of only about 20%. This demonstrates that all three mtDNA mutations that are clearly associated with Leber hereditary optic neuropathy result in deficiency of Complex I. However, the relationship between these mtDNA mutations, the function of Complex I and the phenotypic profile remains elusive.

The electrophoretic method of Schägger and von Jagow (Anal. Biochem. 199, 233-231 (1991) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes. The cells were fractionated with digitonin and mitoplasts were isolated and used for electrophoresis. The purification of mitoplasts and the resolution by electrophoresis of the oxidative phosphorylation complexes were optimal when 0.8-1.6 mg of digitonin/mg protein was used. Intact complexes I, III, IV, and V were clearly separated by blue native-polyacrylamide gel electrophoresis (PAGE) in the first dimension and their individual subunits by tricine-sodium dodecyl sulfate-PAGE in the second dimension. Approximately 10(6) fibroblasts or amniocytes (0.4-0.6 mg protein) were sufficient for complete analysis of the oxidative phosphorylation complexes using detection by staining and by Western blotting. Comparable resolution was obtained with other cell types. Studies of fibroblasts from patients with cytochrome c oxidase deficiency demonstrated the usefulness of the method for diagnosis of mitochondrial disorders.

Nuclear respiratory factor 1 (nrf-1) is a transcriptional activator that is most probably essential in the regulation of mitochondrial biogenesis. In studies of the expression of the NRF-1 gene in cultured human fibroblasts, using RT-PCR, we identified two distinct transcripts, one of which contained an in-frame deletion of 198 bp. Analysis of genomic DNA by sequencing, showed that the shorter mRNA is the result of alternative splicing (exon skipping). The shorter transcript will result in an isoform of the protein that lacks the carboxy-terminal part of the DNA binding domain, which might influence transcriptional activation by normal nrf-1. The alternatively spliced transcript was also present in other human cell lines and in several human tissues. A quantitative PCR analysis showed that the percentages of the alternatively spliced transcript ranged from 3 to 17%. Differences in the percentage of alternatively spliced NRF-1 pre-mRNA may influence mitochondrial biogenesis under variable physiological conditions and could play a role in distinct mitochondrial diseases.

A family is described with a T-->G mutation at position 8993 of mtDNA. This mutation is located in the ATPase 6 gene of mtDNA which encodes subunit a of the ATP-synthase complex (FlFo-ATPase). Clinically, the patients showed severe infantile lactate acidosis and encephalomyopathy in a form that was different from the classical Leigh syndrome. In 3 affected boys, ranging in age from 3 months to 8 years, the mutation was found in 95-99% of the mtDNA population. The clinical symptoms correlated with the mtDNA heteroplasmy and in the healthy mother 50% of the mtDNA was mutated. The rate of mitochondrial ATP production by cultured skin fibroblasts containing 99% of mutated mtDNA was about 2-fold lower than that in normal fibroblasts. Native electrophoresis of the mitochondrial enzyme complexes revealed instability of the FlFo-ATPase in all the tissues of the patient that were investigated (heart, muscle, kidney, liver). Only a small portion of the ATP-synthase complex was present in the complete, intact form (620 kDa). Incomplete forms of the enzyme were present as subcomplexes with approx. molecular weights of 460, 390 and 150 kDa, respectively, which differed in the content of F1 and Fo subunits. Immunochemical analysis of the subunits of the FlFo-ATPase further revealed a markedly decreased content of the Fo subunit b in mitochondria from muscle and heart, and an increased content of the Fo subunit c in muscle mitochondria, respectively. These results indicate that in this family the T-->G point mutation at position 8993 in the mitochondrial ATPase 6 gene is accompanied by structural instability and altered assembly of the enzyme complex, that are both most likely due to changes in the properties of subunit a of the membrane sector part of the ATP-synthase.

Deficiency of cytochrome c oxidase activity was established in a girl born to consanguineous parents. She showed symptoms of dysmaturity, generalized hypotonia, myoclonic seizures and progressive respiratory failure, leading to death on the seventh day of life. Structural abnormalities of the central nervous system consisted of severe cerebellar hypoplasia and optic nerve atrophy. Biochemical analysis of a muscle biopsy specimen demonstrated deficiency of cytochrome c oxidase activity. Cultured fibroblasts from this patient also showed a selective decrease in the activity of cytochrome c oxidase, excluding a muscle-specific type of deficiency. Further investigations in cultured fibroblasts revealed that synthesis, assembly and stability of both the mitochondrial and the nuclear subunits of the enzyme were entirely normal. The steady-state concentration of cytochrome c oxidase in the fibroblasts of the patient was also normal, suggesting that the kinetic properties of the enzyme were altered. Analysis of the kinetic parameters of cytochrome c oxidase demonstrated an aberrant interaction between cytochrome c oxidase and its substrate, cytochrome c, most likely because of a mutation in one of the nuclear subunits of the enzyme.

Synthesis, import, assembly and turnover of the nuclearly encoded subunits of cytochrome-c oxidase were investigated in cultured human cells depleted of mitochondrial gene products by continuous inhibition of mitochondrial protein synthesis (OP- cells). Immunoprecipitation after pulse labeling demonstrated that the synthesis of the nuclear subunits was not preferentially inhibited, implying that there is no tight regulation in the synthesis of mitochondrial and nuclear subunits of mitochondrial enzyme complexes. Quantitative analysis of the mitochondrial membrane potential in OP- cells indicated that its magnitude was about 30% of that in control cells. This explains the normal import of the nuclearly encoded subunits of cytochrome-c oxidase and other nuclearly encoded mitochondrial proteins into the mitochondria that was found in OP- cells. The turnover rate of nuclear subunits of cytochrome-c oxidase, determined in pulse-chase experiments, showed a specific increase in OP- cells. Moreover, immunoblotting demonstrated that the steady-state levels of nuclear subunits of cytochrome-c oxidase were severely reduced in these cells, in contrast to those of the F1 part of complex V. Native electrophoresis of mitochondrial enzyme complexes showed that assembly of the nuclear subunits of cytochrome-c oxidase did not occur in OP- cells, whereas the (nuclear) subunits of F1 were assembled. The increased turnover of the nuclear subunits of cytochrome-c oxidase in OP- cells is, therefore, most likely due to an increased susceptibility of unassembled subunits to intra-mitochondrial degradation.

In this paper we report the effects of meta-iodobenzylguanidine (MIBG), a structural analogue of norepinephrine, on cell proliferation and several parameters related to mitochondrial respiration in Molt-4 cells. In micromolar concentrations, MIBG completely inhibited the proliferation of Molt-4 cells. In intact Molt-4 cells, a progressive increase in the lactate to pyruvate ratio was observed after incubation of these cells with glucose and increasing concentrations of MIBG. In Molt-4 cells permeabilized with digitonin, MIBG inhibited mitochondrial ATP synthesis when malate was used as a substrate. Succinate-driven synthesis of ATP was also inhibited by MIBG, although higher concentrations were required. These results indicate that apart from inhibition of complex I, MIBG inhibits at least one other complex of the respiratory chain. Measurement of the activities of the individual enzyme complexes in the presence of MIBG revealed that complex III is the other enzyme complex susceptible to inhibition by MIBG. Although maximal inhibition of ATP synthesis was observed at a concentration of 10 microM, maximal inhibition of cell proliferation was observed at a concentration of 50 microM of MIBG. This suggests that MIBG also influences other cellular processes apart from mitochondrial oxidative phosphorylation, resulting in additional inhibition of cell proliferation.

The relationship between mitochondrial genotype and mitochondrial phenotype was investigated in lymphoblasts derived from a patient with the Pearson syndrome. In 70% of the mtDNA of this Pearson cell line a deletion from within the COX II gene to within the ND5 gene was present. The deletion led to a reduced expression of the deleted genes, but the severely lowered synthesis of e.g. subunit II of cytochrome c oxidase was not reflected in a significant decrease in the cytochrome c oxidase activity. Moreover, there were no obvious differences between control cells and Pearson cells regarding the capacity for oxidative phosphorylation. Analysis of the synthesis and assembly of both nuclearly and mitochondrially encoded subunits of cytochrome c oxidase showed that normally mtDNA-encoded polypeptides are produced in excess. This overproduction fully explained the discrepancy between the severe defect in the expression of the mitochondrial genome and the normal mitochondrial function in the Pearson cells. These data demonstrate that the expression of one or more mitochondrial genes can be reduced specifically at intermediate percentages of deleted mtDNA. However, the data also suggest that whether or not a lower expression of mitochondrial genes encoding subunits of enzymes involved in oxidative phosphorylation influences the normal function of these enzymes depends on the relative abundance of the mitochondrial subunits in tissues or cells with deleted mtDNA.

Prolonged treatment of cultured cells with ethidium bromide results in loss of the capacity for oxidative phosphorylation. Because of the tight coupling between mitochondrial beta-oxidation of fatty acids and the activity of the respiratory chain, such cells may be used to study the contribution of mitochondria and peroxisomes to fatty acid beta-oxidation. To investigate this, human skin fibroblasts were cultured in the presence of ethidium bromide for at least 10 cell generations, resulting in a virtually complete absence of oxidative phosphorylation as demonstrated directly in digitonin-permeabilized fibroblasts. The cells showed a lowered ATP/ADP ratio, most likely as the consequence of the inability to generate ATP via oxidative phosphorylation. The loss of the capacity for oxidative phosphorylation was also reflected in an increased cytosolic NADH/NAD+ ratio: the cells showed a highly elevated lactate/pyruvate ratio in the suspending medium when incubated with glucose. The beta-oxidation of octanoic and palmitic acid was dramatically decreased, suggesting that the beta-oxidation of these fatty acids takes place predominantly (> 90%) in mitochondria, at least in the cells studied. In contrast, the rates of pristanic and cerotic acid beta-oxidation were only slightly decreased, suggesting that this is mainly a peroxisomal process. The reduction of beta-oxidation of cerotic and pristanic acid, 27% and 15%, respectively, is most likely due to a lowered ATP level and an increased NADH/NAD(+)-redoxstate in these cells. We conclude that fibroblasts subjected to prolonged treatment with ethidium bromide can be used as a model system to study the substrate specificity and functional characteristics of the peroxisomal beta-oxidation system.