Mitochondrial Replication Research Articles

Genetic and Functional Mitochondrial Assessment of HIV-Infected Patients Developing HAART-Related Hyperlactatemia

Mitochondrial damage of HIV and antiretrovirals, especially nucleoside-analogue interference on mitochondrial DNA (mtDNA) replication, is reported to underlay highly active antiretroviral therapy (HAART)-related hyperlactatemia, but scarce approaches have been performed to correlate clinical manifestations and mitochondrial abnormalities. We obtained lymphocytes and monocytes of 26 HIV-infected and treated patients who developed hyperlactatemia and after recovery, 28 nonhyperlactatemic HIV subjects on HAART, 31 naive individuals, and 20 uninfected controls. Mitochondrial replication and transcription analysis were performed by quantitative real-time PCR, mitochondrial translation quantification by western blot and mitochondrial enzymatic activities by spectrophotometry. Mitochondrial parameters decreased during hyperlactatemia and improved at recovery. Mitochondrial replication and transcription species were reduced (P = 0.16 and P = 0.71), but the most significant decay was observed on mitochondrial protein content (P < 0.05) and mitochondrial complexes III and IV activities (P < 0.01 and P < 0.001). During hyperlactatemia lactate level correlated complexes III and IV function (P < 0.05). After recovery mitochondrial parameters achieved values of nonhyperlactatemic HIV individuals, which were lower than ranges of naive subjects and uninfected controls. HIV and HAART-related hyperlactatemia is associated with a general mitochondrial impairment which reverts after recovery. Mitochondrial biochemistry show a better correlation with lactate levels than mitochondrial genetics suggesting that mitochondrial function could be a better marker of hyperlactatemia development than mtDNA content.

Profound Length Heteroplasmic Alterations in Mitochondrial DNA Control Region From Primary AML Cells: Implication of Impaired Mitochondrial Replication and Demonstration of ‘vicious cycle' Hypothesis.

Abstract 3116Poster Board III-53Mitochondrial DNA (mtDNA) control region (displacement (D)-loop including HV1 and HV2) is a non-coding region of 1124 bp (nucleotide positions, np 16 024–576), which acts as a promoter for both the heavy and light strands of mtDNA, and contains essential transcription and replication elements (Blood 2004;103:4466-77). Importantly, mutations in the D-loop regulatory region might change mtDNA replication rate by modifying the binding affinity of significant trans-activating factors (Eur J Cancer 2004;40:2519-24). Thus, length heteroplasmic alterations of mtDNA control region may be related with mitochondrial dysfunction resulting in ‘vicious cycle' (Mol Med Today 2000;6:425-32). In an attempt to investigate profiling of mtDNA length heteroplasmic alterations in primary AML cells, we carried out a quantitative size-based PCR product separation by capillary electrophoresis (ABI 3130XL Genetic Analyzer and ABI Prism Genotyper version 3.1) using six targets (np 303-315 poly C, np 16184-16193 poly C, np 514-511 CA repeats, np 3566-3572 poly C, np 12385-12391 poly C and np 12418-12426 poly A). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research). Forty-eight AML bone marrow samples were collected after receiving Institutional Review Board approval and informed consent. There were profound alterations of mtGI in 303 poly C, 16184 poly C and 514 CA repeats. The length heteroplasmy pattern of 303 poly C tract in the HV2 region disclosed mixture of 7C, 8C, 9C and 10C mtDNA types. In the HV2 region, length heteroplasmy in poly-C tract at np 303 - 309 exhibited 5 variant peak patterns: 7CT6C+8CT6C (50.0%), 8CT6C+9CT6C (14.0%), 8CT6C+ 9CT6C+ 10CT6C (10.4%), 9CT6C+10CT6C+11CT6C (8.3%) 9CT6C + 10CT6C + 11CT6C+12CT6C (2.1%). The length heteroplasmy pattern of 514-523 CA repeats in the HV2 region exhibited 2 variant peak patterns: CACACACACA (56.3%) and CACACACA (43.7%). In the HV1 region, length heteroplasmy in the poly-C tract at np 16184 - 16193 exhibited 9 variant peak patterns: 5CT4C+5CT3C (31.0%), 6CT4C+6CT3C (2.1%), 9C+10C+11C+12C (16.7%), 9C+10C+11C (2.1%), T4CT4C+5CT3C (4.2%), 9C+10C+11C+12C+13C (2.1%), 3CTC4C+5CT3C (2.1%), 10C+11C+12C+13C (4.2%), 8C+9C+10+11C (2.1%). Primary AML cells revealed decreased enzyme activity in respiratory chain complex I, II and III. AML cells had about a two-fold decrease in mtDNA copy number compared with normal blood mononuclear cells. Current study demonstrates that profound length heteroplasmic alterations in mtDNA control region of primary AML cells may lead to impairment of mitochondrial biogenesis (reduction of mtDNA copy number) and derangement of mitochondrial ATP synthesis. During this perturbation, mitochondria in primary AML cells might produce a large amount of reactive oxygen species, which causes the vicious cycle observed in chronic inflammatory diseases and cancers as well. DisclosuresNo relevant conflicts of interest to declare.

The Australian fresh water isopod (Phreatoicidea: Isopoda) allows insights into the early mitogenomic evolution of isopods

The complete mitochondrial (mt) genome sequence of the Australian fresh water isopod Eophreatoicus sp.-14 has been determined. The new species is a member of the taxon Phreatoicidea, a clade of particular interest, as it is often regarded as the sister group to all other Isopoda. Although the overall genome organization of Eophreatoicus sp.-14 conforms to the typical state of Metazoa—it is a circular ring of DNA hosting the usual 37 genes and one major non-coding region—it bears a number of derived characters that fall within the scope of “genome morphology”. Earlier studies have indicated that the isopod mitochondrial gene order is not as conserved as that of other crustaceans. Indeed, the mt genome of Eophreatoicus sp.-14 shows an inversion of seven genes (including cox1), which is as far as we know unique. Even more interesting is the derived arrangement of nad1, trnL(CUN), rrnS, control region, cob, trnT, nad5 and trnF that is shared by nearly all available isopod mt genomes. A striking feature is the close proximity of the rearranged genes to the mt control region. Inferable gene translocation events are, however, more suitable to trace the evolution of mt genomes. Genes like nad1/ trnL(CUN) and nad5/trnF, which retained their adjacent position after being rearranged, were most likely translocated together. A very good example for the need to understand the mechanisms of translocations is the remolding of trnL(UUR) to trnL(CUN). Both tRNA genes are adjacent and have a high sequence similarity, probably the result of a gene duplication and subsequent anticodon mutation. Modified secondary structures were found in three tRNAs of Eophreatoicus sp.-14, which are all characterized by the loss of the DHU-arm. This is common to crustaceans for tRNA Serine(AGY), while the arm-loss in tRNA Cysteine within Malacostraca is only shared by other isopods. Modification of the third tRNA, Isoleucine, is not known from any other related species. Nucleotide frequencies of genes have been found to be indirectly correlated to the orientation of the mitochondrial replication process. In Eophreatoicus sp.-14 and in other Isopoda the associated nucleotide bias is inversed to the state of other Malacostraca. This is a strong indication for an inversion of the control region that most likely evolved in the isopod ancestor.

Mitochondrial tRNAs as light strand replication origins: Similarity between anticodon loops and the loop of the light strand replication origin predicts initiation of DNA replication

Stem-loop hairpins formed by mitochondrial light strand replication origins (OL) and by heavy strand DNA coding for tRNAs that form OL-like structures initiate mitochondrial replication. The loops are recognized by one of the two active sites of the vertebrate mitochondrial gamma polymerase, which are homologous to the active sites of class II amino-acyl tRNA synthetases. Therefore, the polymerase site recognizing the OL loop could recognize tRNA anticodon loops and sequence similarity between anticodon and OL loops should predict initiation of DNA replication at tRNAs. Strengths of genome-wide deamination gradients starting at tRNA genes estimate extents by which replication starts at that tRNA. Deaminations (A-->G and C-->T) occur proportionally to time spent single stranded by heavy strand DNA during mitochondrial light strand replication. Results show that deamination gradients starting at tRNAs are proportional to sequence similarity between OL and tRNA loops: most for anticodon-, least D-, intermediate for TpsiC-loops, paralleling tRNA synthetase recognition interactions with these tRNA loops. Structural and sequence similarities with regular OLs predict OL function, loop similarity is dominant in most tRNAs. Analyses of sequence similarity and structure independently substantiate that DNA sequences coding for mitochondrial tRNAs sometimes function as alternative OLs. Pathogenic mutations in anticodon loops increase similarity with the human OL loop, non-pathogenic polymorphisms do not. Similarity/homology alignment hypotheses are experimentally testable in this system.

The use of mitochondrial nutrients to improve the outcome of infertility treatment in older patients

We present a hypothesis emphasizing the role of mitochondrial dysfunction in reproductive senescence and suggesting the use of mitochondrial nutrients as an adjuvant treatment in older patients with infertility.

Perfluorooctanoic acid stimulated mitochondrial biogenesis and gene transcription in rats

Perfluorooctanoic acid (PFOA), used in the production of non-stick surface compounds, exhibits a worldwide distribution in the serum of humans and wildlife. In rodents PFOA transactivates PPARα and PPARγ nuclear receptors and increases mitochondrial DNA (mtDNA) copy number, which may be critical to the altered metabolic state of affected animals. A key regulator of mitochondrial biogenesis and transcription of mitochondrial genes is the PPARγ coactivator-1α (Pgc-1α) protein. The purpose of this study was to determine if Pgc-1α is implicated in the stimulation of mitochondrial biogenesis that occurs following the treatment of rats with PFOA. Livers from adult male Sprague–Dawley rats that received a 30 mg/kg daily oral dose of PFOA for 28 days were used for all experiments. Analysis of mitochondrial replication and transcription was performed by real time PCR, and proteins were detected using western blotting. PFOA treatment caused a transcriptional activation of the mitochondrial biogenesis pathway leading to a doubling of mtDNA copy number. Further, transcription of OXPHOS genes encoded by mtDNA was 3–4 times greater than that of nuclear encoded genes, suggestive of a preferential induction of mtDNA transcription. Western blot analysis revealed an increase in Pgc-1α, unchanged Tfam and decreased Cox II and Cox IV subunit protein expression. We conclude that PFOA treatment in rats induces mitochondrial biogenesis at the transcriptional level with a preferential stimulation of mtDNA transcription and that this occurs by way of activation of the Pgc-1α pathway. Implication of the Pgc-1α pathway is consistent with PPARγ transactivation by PFOA and reveals new understanding and possibly new critical targets for assessing or averting the associated metabolic disease.

Evolutionary roots of iodine and thyroid hormones in cell-cell signaling

In vertebrates, thyroid hormones (THs, thyroxine, and triiodothyronine) are critical cell signaling molecules. THs regulate and coordinate physiology within and between cells, tissues, and whole organisms, in addition to controlling embryonic growth and development, via dose-dependent regulatory effects on essential genes. While invertebrates and plants do not have thyroid glands, many utilize THs for development, while others store iodine as TH derivatives or TH precursor molecules (iodotyrosines)-or produce similar hormones that act in analogous ways. Such common developmental roles for iodotyrosines across kingdoms suggest that a common endocrine signaling mechanism may account for coordinated evolutionary change in all multi-cellular organisms. Here, I expand my earlier hypothesis for the role of THs in vertebrate evolution by proposing a critical evolutionary role for iodine, the essential ingredient in all iodotyrosines and THs. Iodine is known to be crucial for life in many unicellular organisms (including evolutionarily ancient cyanobacteria), in part, because it acts as a powerful antioxidant. I propose that during the last 3-4 billion years, the ease with which various iodine species become volatile, react with simple organic compounds, and catalyze biochemical reactions explains why iodine became an essential constituent of life and the Earth's atmosphere-and a potential marker for the origins of life. From an initial role as membrane antioxidant and biochemical catalyst, spontaneous coupling of iodine with tyrosine appears to have created a versatile, highly reactive and mobile molecule, which over time became integrated into the machinery of energy production, gene function, and DNA replication in mitochondria. Iodotyrosines later coupled together to form THs, the ubiquitous cell-signaling molecules used by all vertebrates. Thus, due to their evolutionary history, THs, and their derivative and precursors molecules not only became essential for communicating within and between cells, tissues and organs, and for coordinating development and whole-body physiology in vertebrates, but they can also be shared between organisms from different kingdoms.

The Porcine Mitochondrial Transcription Factor a Gene: Molecular Characterization, Radiation Hybrid Mapping and Genetic Diversity among 12 Pig Breeds

Problem statement: Mitochondrial transcription factor A (TFAM) is a nucleus-encoded protein that is a key activator of mitochondrial transcription as well as a major participant in mitochondrial genome replication. Genomic characterization of the porcine TFAM gene is, therefore, necessary to determine its involvement in regulation of fat depots and meat quality traits in pigs. Approach: Genomic DNA sequence was determined using a comparative in silico annotation approach. RT-PCR was used for analysis of alternative splicing. Genome location was determined using Radiation Hybrid (RH) mapping. Genetic marker was identified by sequencing and genotyped by the PCR-RFLP method with SacI. GENEPOP version 3.3 software was used for statistic analysis. Results: We determined both full-length cDNA and genomic DNA sequences of the porcine TFAM gene. Gene expression analysis revealed an alternative 5’ splice site, which excludes exon 4 of the pig gene. We assigned this gene to porcine chromosome 14 (SSC14). A G/A substitution was detected in intron 1 of porcine TFAM gene and genotyped on a total of 252 animals, including 165 from seven Chinese and 87 from five Western pig breeds. The Bayesian analysis via MCMC (Markov chain Monte Carlo) revealed that these two groups of pigs were well separated at this locus during the breed history; 95% of the posterior difference of TFAM allelic frequency between these two pig groups was greater than zero. Conclusion/Recommendations: All these data provided basic genomic information needed for further functional characterization of the porcine TFAM gene. Because marked differences in fat and lean tissue deposition exist between Western and Chinese pig breeds, the G557A mutation in the TFAM gene deserves further evaluation to determine its phenotypic effect on fattening and carcass traits in commercial pig populations.

Loss of mitochondrial DNA under genotoxic stress conditions in the absence of the yeast DNA helicase Pif1p occurs independently of the DNA helicase Rrm3p

How the cellular amount of mitochondrial DNA (mtDNA) is regulated under normal conditions and in the presence of genotoxic stress is less understood. We demonstrate that the inefficient mtDNA replication process of mutant yeast cells lacking the PIF1 DNA helicase is partly rescued in the absence of the DNA helicase RRM3. The rescue effect is likely due to the increase in the deoxynucleoside triphosphates (dNTPs) pool caused by the lack of RRM3. In contrast, the Pif1p-dependent mtDNA breakage in the presence and absence of genotoxic stress is not suppressed if RRM3 is lacking suggesting that this phenotype is likely independent of the dNTP pool. Pif1 protein (Pif1p) was found to stimulate the incorporation of dNTPs into newly synthesised mtDNA of gradient-purified mitochondria. We propose that Pif1p that acts likely as a DNA helicase in mitochondria affects mtDNA replication directly. Possible roles of Pif1p include the resolution of secondary DNA and/or DNA/RNA structures, the temporarily displacement of tightly bound mtDNA-binding proteins, or the stabilization of the mitochondrial replication complex during mtDNA replication.

Removal of Oxidative DNA Damage via FEN1-Dependent Long-Patch Base Excision Repair in Human Cell Mitochondria

Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision repair (SP-BER) replacing a single nucleotide. However, certain oxidative lesions cannot be processed by SP-BER. Here we report that 2-deoxyribonolactone (dL), a major type of oxidized abasic site, inhibits replication by mitochondrial DNA (mtDNA) polymerase gamma and interferes with SP-BER by covalently trapping polymerase gamma during attempted dL excision. However, repair of dL was detected in human mitochondrial extracts, and we show that this repair is via long-patch BER (LP-BER) dependent on flap endonuclease 1 (FEN1), not previously known to be present in mitochondria. FEN1 was retained in protease-treated mitochondria and detected in mitochondrial nucleoids that contain known mitochondrial replication and transcription proteins. Results of immunofluorescence and subcellular fractionation studies were also consistent with the presence of FEN1 in the mitochondria of intact cells. Immunodepletion experiments showed that the LP-BER activity of mitochondrial extracts was strongly diminished in parallel with the removal of FEN1, although some activity remained, suggesting the presence of an additional flap-removing enzyme. Biological evidence for a FEN1 role in repairing mitochondrial oxidative DNA damage was provided by RNA interference experiments, with the extent of damage greater and the recovery slower in FEN1-depleted cells than in control cells. The mitochondrial LP-BER pathway likely plays important roles in repairing dL lesions and other oxidative lesions and perhaps in normal mtDNA replication.

Mitochondrial biogenesis in the axons of vertebrate peripheral neurons

Mitochondria are widely distributed via regulated transport in neurons, but their sites of biogenesis remain uncertain. Most mitochondrial proteins are encoded in the nuclear genome, and evidence has suggested that mitochondrial DNA (mtDNA) replication occurs mainly or entirely in the cell body. However, it has also become clear that nuclear-encoded mitochondrial proteins can be translated in the axon and that components of the mitochondrial replication machinery reside there as well. We assessed directly whether mtDNA replication can occur in the axons of chick peripheral neurons labeled with 5-bromo-2'-deoxyuridine (BrdU). In axons that were physically separated from the cell body or had disrupted organelle transport between the cell bodies and axons, a significant fraction of mtDNA synthesis continued. We also detected the mitochondrial fission protein Drp1 in neurons by immunofluorescence or expression of GFP-Drp1. Its presence and distribution on the majority of axonal mitochondria indicated that a substantial number had undergone recent division in the axon. Because the morphology of mitochondria is maintained by the balance of fission and fusion events, we either inhibited Drp1 expression by RNAi or overexpressed the fusion protein Mfn1. Both methods resulted in significantly longer mitochondria in axons, including many at a great distance from the cell body. These data indicate that mitochondria can replicate their DNA, divide, and fuse locally within the axon; thus, the biogenesis of mitochondria is not limited to the cell body.

Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae

DNA polymerase gamma, a mitochondrial replication enzyme of yeasts and animals, is not present in photosynthetic eukaryotes. Recently, DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases (POPs). However, POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon merolae contains two genes encoding proteins related to Escherichia coli DNA polymerase I (PolA and PolB). Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs, which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA, PolB, and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids, whereas PolB was present in both plastids and mitochondria. The expression of PolB was regulated by the cell cycle. The available results suggest that PolB is involved in the replication of plastids and mitochondria.

Transcriptional Paradigms in Mammalian Mitochondrial Biogenesis and Function

Mitochondria contain their own genetic system and undergo a unique mode of cytoplasmic inheritance. Each organelle has multiple copies of a covalently closed circular DNA genome (mtDNA). The entire protein coding capacity of mtDNA is devoted to the synthesis of 13 essential subunits of the inner membrane complexes of the respiratory apparatus. Thus the majority of respiratory proteins and all of the other gene products necessary for the myriad mitochondrial functions are derived from nuclear genes. Transcription of mtDNA requires a small number of nucleus-encoded proteins including a single RNA polymerase (POLRMT), auxiliary factors necessary for promoter recognition (TFB1M, TFB2M) and activation (Tfam), and a termination factor (mTERF). This relatively simple system can account for the bidirectional transcription of mtDNA from divergent promoters and key termination events controlling the rRNA/mRNA ratio. Nucleomitochondrial interactions depend on the interplay between transcription factors (NRF-1, NRF-2, PPARalpha, ERRalpha, Sp1, and others) and members of the PGC-1 family of regulated coactivators (PGC-1alpha, PGC-1beta, and PRC). The transcription factors target genes that specify the respiratory chain, the mitochondrial transcription, translation and replication machinery, and protein import and assembly apparatus among others. These factors are in turn activated directly or indirectly by PGC-1 family coactivators whose differential expression is controlled by an array of environmental signals including temperature, energy deprivation, and availability of nutrients and growth factors. These transcriptional paradigms provide a basic framework for understanding the integration of mitochondrial biogenesis and function with signaling events that dictate cell- and tissue-specific energetic properties.

Global Identification of Myc Target Genes Reveals Its Direct Role in Mitochondrial Biogenesis and Its E-Box Usage In Vivo

The Myc oncoprotein is a transcription factor involved in a variety of human cancers. Overexpression of Myc is associated with malignant transformation. In normal cells, Myc is induced by mitotic signals, and in turn, it regulates the expression of downstream target genes. Although diverse roles of Myc have been predicted from many previous studies, detailed functions of Myc targets are still unclear. By combining chromatin immunoprecipitation (ChIP) and promoter microarrays, we identified a total of 1469 Myc direct target genes, the majority of which are novel, in HeLa cells and human primary fibroblasts. We observed dramatic changes of Myc occupancy at its target promoters in foreskin fibroblasts in response to serum stimulation. Among the targets of Myc, 107 were nuclear encoded genes involved in mitochondrial biogenesis. Genes with important roles in mitochondrial replication and biogenesis, such as POLG, POLG2, and NRF1 were identified as direct targets of Myc, confirming a direct role for Myc in regulating mitochondrial biogenesis. Analysis of target promoter sequences revealed a strong preference for Myc occupancy at promoters containing one of several described consensus sequences, CACGTG, in vivo. This study thus sheds light on the transcriptional regulatory networks mediated by Myc in vivo.

Inverted Replication of Vertebrate Mitochondria

After analyzing the base composition asymmetry of coding regions in vertebrate mitochondria, we identified 2 fishes, Albula glossodonta and Bathygadus antrodes, with inverted compositional patterns. Both species appear to have an unusual control region (CR), and in B. antrodes, it has switched from the light strand to the heavy strand. To our knowledge, this is the first report in vertebrates of inverted mitochondrial replication, caused by an inversion of the CR. These findings support the strand-asymmetric model of mtDNA replication and suggest that vertebrate mtDNA can tolerate globally reversed mutational pressures. In addition, we propose that nucleotide bias is not strand specific but that it depends on the location of the CR.

Molecular control of mitochondrial function in developing rhesus monkey oocytes and preimplantation-stage embryos

The mitochondrion undergoes significant functional and structural changes, as well as an increase in number, during preimplantation embryonic development. The mitochondrion generates ATP and regulates a range of cellular processes, such as signal transduction and apoptosis. Therefore, mitochondria contribute to overall oocyte quality and embryo developmental competence. The present study identified, for the first time, the detailed temporal expression of mRNAs related to mitochondrial biogenesis in rhesus monkey oocytes and embryos. Persistent expression of maternally encoded mRNAs was observed, in combination with transcriptional activation and mRNA accumulation at the eight-cell stage, around the time of embryonic genome activation. The expression of these transcripts was significantly altered in oocytes and embryos with reduced developmental potential. In these embryos, most maternally encoded transcripts were precociously depleted. Embryo culture and specific culture media affected the expression of some of these transcripts, including a deficiency in the expression of key transcriptional regulators. Several genes involved in regulating mitochondrial transcription and replication are similarly affected by in vitro conditions and their downregulation may be instrumental in maintaining the mRNA profiles of mitochondrially encoded genes observed in the present study. These data support the hypothesis that the molecular control of mitochondrial biogenesis, and therefore mitochondrial function, is impaired in in vitro-cultured embryos. These results highlight the need for additional studies in human and non-human primate model species to determine how mitochondrial biogenesis can be altered by oocyte and embryo manipulation protocols and whether this affects physiological function in progeny.

Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells

Stem cells are a key target of environmental toxicants, but little is known about their toxicological responses. We aimed at developing an in-vitro model based on adult human stem cells to identify biomarkers of heavy metal exposure. To this end we investigated the responses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI]) and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood were exposed for 48 h to 0.1 μM and 10 μM Cr(VI) or Cd. Cultures treated with 10 μM Cr(VI) or Cd showed marked cell loss. Ultrastructural analysis of surviving cells revealed prominent autophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated with mitochondrial damage and replication, dilatation of the rough endoplasmic reticulum and Golgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did not show the morphologic hallmarks of apoptosis. Treatment with 0.1 μM Cr(VI) or Cd did not result in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulum and evidence of mitochondrial damage. We conclude that autophagy is implicated in the response of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd. Autophagy, which mediates cell survival and death under stress, deserves further evaluation to be established as biomarker of metal exposure.

Fidelity and Processivity of Reverse Transcription by the Human Mitochondrial DNA Polymerase

We have characterized, by transient-state kinetic methods, the polymerase and exonuclease activities of the human mitochondrial DNA polymerase (pol gamma) during reverse transcription, employing a synthetic oligonucleotide consisting of a DNA primer and an RNA template. In comparison with the kinetic parameters observed with a DNA template, the rate of correct deoxynucleotide incorporation was reduced 25-fold (5.5+/-0.2 s(-1)), whereas the dissociation constant (Kd) for nucleotide binding was increased 4-fold (12+/-1 microm). In addition, discrimination against mismatches was reduced approximately 20-fold to only 15,000 on average. The proofreading exonuclease favored the removal of an incorrect nucleotide (0.0021+/-0.0002 s(-1) for correct versus 0.034+/-0.004 s(-1) for incorrect), and the partitioning between incorporation beyond a mismatch (5.5x10(-5)+/-0.4x10(-5) s(-1)), and exonuclease removal of that mismatch favors removal of the mismatch. These data suggest that the "reverse transcriptase activity" of mitochondrial polymerase could be physiologically relevant. However, the enzyme stalls and is unable to efficiently incorporate beyond a single nucleotide with an RNA template. Additionally, we present a refined method for calculating net discrimination, which more accurately describes the contributions of correct and incorrect incorporation. The biological and biotechnological significance of these results are discussed.

Mitochondrial disorders

Mitochondrial disorders are increasingly acknowledged as a major category in clinical neurology. In this review we highlight the most recent advances in the field, including the characterization of new disease genes, new physiopathological insights, and the role of mitochondrial dysfunction in neurodegeneration. Substantial progress has been made on the genetic basis and pathogenic mechanisms in disorders associated with altered mitochondrial DNA stability and expression. These defects include a wide spectrum of neurological conditions caused by genetic abnormalities of the mitochondrial replication and translation machineries, and of the metabolic pathways controlling the nucleotide supply to organelles, cells and tissues. Another relevant contribution has been given to the molecular dissection of coenzyme Q deficiency, a clinically heterogeneous, potentially treatable condition, thanks to the biochemical and genetic characterization of the first defects in coenzyme Q biosynthesis. Finally, the genetic determinants controlling the penetrance of mitochondrial disorders, as well as the role of mitochondrial dysfunction in neurodegenerative conditions such as Parkinson's and Huntington's diseases, have been investigated in both patients and animal models. The dual genetic contribution controlling mitochondrial biogenesis, and the intricacy and universality of the metabolic pathways operating in the mitochondrion explain the complexity of what is now known as 'mitochondrial medicine'.

Dynamin, a GTPase involved in the initial stages of endocytosis.

Dynamin is a high molecular mass (100 kDa) GTPase which binds to and co-purifies with microtubules. Molecular cloning of rat brain dynamin has revealed the three well-established consensus sequence elements for GTP binding within the N-terminal third of the protein, as well as sequence similarity within this region to the interferon-inducible antiviral Mx proteins, the product of the yeast membrane sorting gene VPS1, and the product of the yeast mitochondrial replication gene MGM1. More extensive sequence similarity between rat dynamin and the product of the Drosophila gene shibire, which is involved in endocytosis, has also been found. In in vitro assays microtubules strongly stimulate the dynamin GTPase. This effect can be reversed by removal of the dynamin C-terminus using papain, which abolishes microtubule binding. Overexpression of mutant forms of dynamin in vivo using Cos-7 cells inhibits transferrin uptake and alters the distribution of clathrin and of alpha-adaptin, but not gamma-adaptin. Deletion of the C-terminus of mutant forms of dynamin abolishes these effects. Together these results suggest a critical role for dynamin in the early stages of endocytosis. It is uncertain whether microtubules interact with dynamin in vivo or whether the in vitro effects of microtubules mimic the effects of other regulatory elements in vivo.