Lipoma and liposarcoma: evaluation using CT and MR imaging

The multi-subunit NADH-ubiquinone oxidoreductase (complex I) is the first enzyme complex in the electron transport chain of mitochondria. A small number of NADH-ubiquinone oxidoreductase subunits are the products of mitochondrial genes (subunits 1-7), while the remainder are nuclear encoded and imported from the cytoplasm. We have isolated and sequenced five subunits of the human complex I from a human heart lambda ZAP Express cDNA library. Comparison of the deduced amino acid sequences of the human subunits with the corresponding bovine sequences revealed greater than 80% amino acid identity. The high degree of similarity between human and bovine sequences suggests functional conservation of these subunits in the complex I. In silico Northern analysis revealed that two of the subunits were expressed ubiquitously while the remainder may have more restricted patterns of expression.     

hTom34: a novel translocase for the import of proteins into human mitochondria

Most mitochondrial proteins are nuclear encoded, synthesized on cytosolic ribosomes, and imported into the mitochondria. We have identified and characterized a 309 amino acid human protein with a molecular weight of 34 kDa that functions as a subunit of the translocase for the import of such proteins. hTom34 (34-kDa Translocase of the Outer Mitochondrial Membrane) is displayed on the surface of mitochondria and is resistant to extraction under alkaline conditions. Antibodies raised against hTom34 specifically inhibit in vitro import of the mitochondrial precursor protein preornithine transcarbamylase into mitochondria isolated from rat liver. Based on trypsin digestion experiments, the receptor has a large (27 kDa) C-terminal domain exposed to the cytosol. This novel component of the protein import machinery possesses a 62 residue motif conserved with the Tom70 family of mitochondrial receptors but otherwise appears to have no counterpart so far characterized in the mitochondria of any other species.     

Fragmented mitochondrial DNA is the predominant carrier of oxidized DNA bases

Previous analyses indicated a high level of oxidative base modification in mitochondrial DNA, the extent of which raised questions about the methodological validity and biological implications. In the present study DNA was isolated from rat liver mitochondria under carefully controlled conditions, and the extent of base oxidation, DNA fragmentation, and nuclear DNA contamination were analyzed. DNA isolated from intact mitochondria treated with DNase consisted of 16.3 kilobase pairs, mostly circular, mitochondrial DNA molecules and a mixture of nuclear and mitochondrial DNA fragments, as identified by agarose gel electrophoresis and hybridization. High-performance liquid chromatography in combination with electrochemical detection confirmed that the overall level of 8-hydroxy-2'-deoxyguanosine, a marker commonly used in the analysis of base oxidation, is higher in mitochondrial than in nuclear DNA. Importantly, 8-hydroxy-2'-deoxyguanosine is relatively scarce in the 16.3 kilobase pair mitochondrial DNA molecules (0.051 pmol/microgram) but is present in high levels in mitochondrial DNA fragments (0.741 pmol/microgram). The fragments constitute about 18% of total mitochondrial DNA. The antitumor agent bleomycin, which binds to DNA, forms an iron complex capable of transferring electrons from Fe2+ to molecular oxygen. Exposure of mitochondria to bleomycin and iron resulted in nicking but not in a significant increase in base oxidation of 16.3 kilobase pair mitochondrial DNA, whereas the amount and the oxidation level of fragmented mitochondrial DNA significantly increased. These findings are relevant for a better understanding of the role of mitochondria in aging and various diseases and are consistent with the notion that despite the overall high DNA oxidation level, mitochondria can faithfully proliferate.     

Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice

This study characterizes mitochondria isolated from livers of Sod2(-/+) and Sod2(+/+) mice. A 50% decrease in manganese superoxide dismutase (MnSOD) activity was observed in mitochondria isolated from Sod2(-/+) mice compared with Sod2(+/+) mice, with no change in the activities of either glutathione peroxidase or copper/zinc superoxide dismutase. However, the level of total glutathione was 30% less in liver mitochondria of the Sod2(-/+) mice. The reduction in MnSOD activity in Sod2(-/+) mice was correlated to an increase in oxidative damage to mitochondria: decreased activities of the Fe-S proteins (aconitase and NADH oxidoreductase), increased carbonyl groups in proteins, and increased levels of 8-hydroxydeoxyguanosine in mitochondrial DNA. In contrast, there were no significant changes in oxidative damage in the cytosolic proteins or nuclear DNA. The increase in oxidative damage in mitochondria was correlated to altered mitochondrial function. A significant decrease in the respiratory control ratio was observed in mitochondria isolated from Sod2(-/+) mice compared with Sod2(+/+) mice for substrates metabolized by complexes I, II, and III. In addition, mitochondria isolated from Sod2(-/+) mice showed an increased rate of induction of the permeability transition. Therefore, this study provides direct evidence correlating reduced MnSOD activity in vivo to increased oxidative damage in mitochondria and alterations in mitochondrial function.     

Assignment of an oligomycin-resistance locus to human chromosome 10

An oligomycin-resistant variant of human fibrosarcoma HT1080 was isolated and characterized as nuclear and codominant. The mutant was stable, was not cross-resistant to respiratory inhibitors, and it contained a mitochondrial ATPase which was less sensitive to oligomycin. Hybrids formed between the human mutant and a mouse cell line expressed the resistance phenotype. By a detailed karyotypic analysis of these hybrids using trypsin-Giemsa banding it was found that resistance to oligomycin correlated with the retention of two human chromosomes 10. The hybrid lines contained only mouse mitochondrial DNA as shown by analyses of mitochondrially synthesized proteins and mitochondrial DNA. The study assigns an ATPase oligomycin-resistance locus to human chromosome 10 and suggests that mouse and human subunits can combine in a functional enzyme complex.     

Simultaneous transfer of mitochondrial DNA and single chromosomes in somatic cells: a novel approach for the study of defects in nuclear-mitochondrial communication

The assembly and function of respiratory-competent mitochondria in eukaryotic cells depends on collaboration between the nuclear and mitochondrial genomes, but the molecular mechanisms underlying such cross-talk are poorly understood. Microcell-mediated chromosome transfer has been used to transfer intact chromosomes from one mammalian cell to another, helping to map loci implicated in different diseases and in the senescence process. In the present work, we show that microcells have a significant number of mitochondria which can be transferred to another cell simultaneously with a limited number of chromosomes. By fusing microcells from a colon carcinoma cell line with a mitochondrial DNA (mtDNA)-less osteosarcoma cell line, we were able to isolate transmitochondrial hybrids containing only one of three selectable chromosomes and mtDNA from the donor cell. The proportion of transmitochondrial hybrids containing one chromosomal marker with respect to the total transmitochondrial hybrids and cybrids was approximately 1% and no hybrids were isolated containing more than one nuclear marker. The genetic data correlated well with the composition and structure of the microcell preparations, which showed the presence of cytoplast-like structures and microcells containing mitochondria surrounding the micronuclei. Microcell-mediated mtDNA and chromosome transfer can be used to identify nuclear factors implicated in mtDNA maintenance and gene expression, as well as to investigate nuclear factors which modulate clinical phenotypes in mitochondrial disorders.     

The presence of rotenone-sensitive NADH dehydrogenase in the long slender bloodstream and the procyclic forms of Trypanosoma brucei brucei

The mitochondrial electron-transport chain present in the procyclic and long slender bloodstream forms of Trypanosoma brucei brucei was investigated by means of several experimental approaches. The oxidation of proline, glycerol and glucose in procyclic cells was inhibited 80-90% by antimycin A or cyanide, 15-19% by salicylhydroxamic acid, and 30-35% by rotenone. Cytochrom-c-reductase activity, with proline or glycerol 3-phosphate as substrate, in a mitochondrial fraction isolated from these cells was inhibited by antimycin and rotenone, but not by malonate, while cytochrome-c-reductase activity with succinate as substrate was inhibited by antimycin A and malonate, but not by rotenone. In addition, the reduction of dichloroindophenol by NADH was inhibited by rotenone but not by malonate, which suggests that rotenone-sensitive NADH dehydrogenase (complex I) is present in these mitochondria. The presence of three subunits of NADH dehydrogenase was observed in immunoblots of mitochondrial proteins with specific antibodies raised against peptides corresponding to predicted antigenic regions of these proteins, which provides further evidence for the presence of NADH dehydrogenase. In long slender bloodstream forms, the oxidation of glucose or glycerol was inhibited 100% by salicyhydroxamic acid, unaffected by cyanide or antimycin A, and inhibited 40% or 75%, respectively, by rotenone, which suggests that NADH dehydrogenase is present in these cells. In a mitochondrial fraction isolated from the bloodstream forms, oxygen uptake with glycerol 3-phosphate as substrate was inhibited 65% by rotenone. Low levels of rotenone-sensitive NADH-dependent reduction of dichloroindophenol and the presence of subunits 7 and 8 of NADH dehydrogenase provided additional evidence for the presence of NADH dehydrogenase in bloodstream forms of T. brucei.     

Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales

Powerful analyses of population structure require information from multiple genetic loci. To help develop a molecular toolbox for obtaining this information, we have designed universal oligonucleotide primers that span conserved intron-exon junctions in a wide variety of animal phyla. We test the utility of exon-primed, intron-crossing amplifications by analyzing the variability of actin intron sequences from humpback, blue, and bowhead whales and comparing the results with mitochondrial DNA (mtDNA) haplotype data. Humpback actin introns fall into two major clades that exist in different frequencies in different oceanic populations. It is surprising that Hawaii and California populations, which are very distinct in mtDNAs, are similar in actin intron alleles. This discrepancy between mtDNA and nuclear DNA results may be due either to differences in genetic drift in mitochondrial and nuclear genes or to preferential movement of males, which do not transmit mtDNA to offspring, between separate breeding grounds. Opposing mtDNA and nuclear DNA results can help clarify otherwise hidden patterns of structure in natural populations.     

Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA

To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells. To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells. Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker. Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events. Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating. In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell. This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission. We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.     

Steroidogenic acute regulatory protein (StAR) acts on the outside of mitochondria to stimulate steroidogenesis

Steroidogenic acute regulatory protein (StAR) facilitates delivery of cholesterol to the inner mitochondrial membranes. StAR is imported into mitochondria and processed to a mature form by cleavage of the N-terminal mitochondrial targeting sequence. We produced His-tagged (His-tag StAR) constructs lacking the N-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells, whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. We established an assay system using mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in E. coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import.     

Mammalian mitochondrial ribosomal proteins. N-terminal amino acid sequencing, characterization, and identification of corresponding gene sequences

The integrity of healthy mitochondria is supposed to depend largely on proper mitochondrial protein biosynthesis. Mitochondrial ribosomal proteins (MRPs) are directly involved in this process. To identify mammalian mitochondrial ribosomal proteins and their corresponding genes, we purified mature rat MRPs and determined 12 different N-terminal amino acid sequences. Using this peptide information, data banks were screened for corresponding DNA sequences to identify the genes or to establish consensus cDNAs and to characterize the deduced MRP open reading frames. Eight different groups of corresponding mammalian MRPs constituted from human, mouse, and rat origin were identified. Five of them show significant sequence similarities to bacterial and/or yeast mitochondrial ribosomal proteins. However, MRPs are much less conserved in respect to the amino acid sequence among species than cytoplasmic ribosomal proteins of eukaryotes and bacteria.     

Intrusiveness of illness and quality of life in young women with breast cancer

Respiratory chain complex I is a complicated enzyme of mitochondria, that couples electron transfer from NADH to ubiquinone to the proton translocation across the inner membrane of the organelle. The fungus Neurospora crassa has been used as one of the main model organisms to study this enzyme. Complex I is composed of multiple polypeptide subunits of dual genetic origin and contains several prosthetic groups involved in its activity. Most subunits have been cloned and those binding redox centres have been identified. Yet, the functional role of certain complex I proteins remains unknown. Insight into the possible origin and the mechanisms of complex I assembly has been gained. Several mutant strains of N. crassa, in which specific subunits of complex I were disrupted, have been isolated and characterised. This review concerns many aspects of the structure, function and biogenesis of complex I that are being elucidated.     

Changes of mitochondrial cytochrome c oxidase and FoF1 ATP synthase subunits in rat cerebral cortex during aging

The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for gamma ATP synthase, while the alpha and beta subunits increased progressively up to 26 months.     

Cloning and analysis of the genes for a novel electron-transferring flavoprotein from Megasphaera elsdenii. Expression and characterization of the recombinant protein

The genes that encode the two different subunits of the novel electron-transferring flavoprotein (ETF) from Megasphaera elsdenii were identified by screening a partial genomic DNA library with a probe that was generated by amplification of genomic sequences using the polymerase chain reaction. The cloned genes are arranged in tandem with the coding sequence for the beta-subunit in the position 5' to the alpha-subunit coding sequence. Amino acid sequence analysis of the two subunits revealed that there are two possible dinucleotide-binding sites on the alpha-subunit and one on the beta-subunit. Comparison of M. elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates that while homology occurs with the mitochondrial ETF and bacterial ETFs, the greatest similarity is with the putative ETFs from clostridia and with fixAB gene products from nitrogen-fixing bacteria. The recombinant ETF was isolated from extracts of Escherichia coli. It is a heterodimer with subunits identical in size to the native protein. The isolated enzyme contains approximately 1 mol of FAD, but like the native protein it binds additional flavin to give a total of about 2 mol of FAD/dimer. It serves as an electron donor to butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.     

Isolation of mitochondria and mitochondrial RNA from Crithidia fasciculata

Two methods were used to isolate mitochondria from Crithidia fasciculata. In the first method, cells were weakened by exposure to hypotonic conditions and then disrupted by blending; mitochondria were subsequently isolated using disodium 3,5-diacetoamido-2,4,6-triiodobenzoate gradients. In the second, cells were treated with digitonin before disruption; mitochondria were purified by differential centrifugation. Both preparations were examined with the electron microscope and were also shown to possess several characteristic biochemical properties of mitochondria. Kinetoplast DNA was present in the mitochondria, uncontaminated by nuclear DNA. Analysis by polyacrylamide gel electrophoresis showed two RNA components of molecular weights of 0-47 X 10(6) and 0-22 X 10(6), in addition to cytoplasmic RNA contamination. Four mitochondrial components with sedimentation coefficients of 14-6S, 11-4S, 10-1S and 9-9S were identified on sucrose density gradients. Ethidium bromide abolished the incorporation of [5-3H]uridine into the presumed mitochondrial RNA.     

Mitochondrial distribution and inheritance

Mechanisms mediating the inheritance of mitochondria are poorly understood, but recent studies with the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have begun to identify components that facilitate this essential process. These components have been identified through the analysis of conditional yeast mutants that display aberrant mitochondrial distribution at restrictive conditions. The analysis of these mutants has uncovered several novel proteins that are localized either to cytoskeletal structures or to the mitochondria themselves. Many mitochondrial inheritance mutants also show altered mitochondrial morphology and defects in maintenance of the mitochondrial genome. Although some inheritance components and mechanisms appear to function specifically in certain types of cells, other conserved proteins are likely to mediate mitochondrial behavior in all eukaryotic cells.     

Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria

Mitochondrial precursor proteins made in the cytosol bind to a hetero-oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This 'trans' domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N-terminal targeting sequence of a translocation-arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration-driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3- to 8-fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.