Palmiro Cantatore

Evolutionary analysis of cytochrome b sequences in some perciformes: Evidence for a slower rate of evolution than in mammals

To obtain information relative to the phylogenesis and microevolutionary rate of fish mitochondrial DNA, the nucleotide sequence of cytochrome b gene in seven fish species belonging to the order of Perciformes was determined. Sequence analysis showed that fish mitochondrial DNA has a nucleotide compositional bias similar to that of sharks but lower compared to mammals and birds. Quantitative evolutionary analysis, carried out by using a markovian stochastic model, clarifies some phylogenetic relationships within the Perciformes order, particularly in the Scombridae family, and between Perciformes, Gadiformes, Cypriniformes, and Acipenseriformes. The molecular clock of mitochondrial DNA was calibrated with the nucleotide substitution rate of cytochrome b gene in five shark species having divergence times inferred from paleontological estimates. The results of such analysis showed that Acipenseriformes diverged from Perciformes by about 200 MY, that the Perciformes common ancestor dates back to 150 MY, and that fish mitochondrial DNA has a nucleotide substitution rate three to five times lower than that of mammals.

Mitochondrial DNA copy number and mitochondrial DNA deletion in adult and senescent rats.

In order to understand the cause of the reduced mitochondrial DNA transcription in heart and brain of senescent rat previously reported, we focused our attention on the content and structure of rat mitochondrial DNA in adult and senescent rats. The estimate of the mtDNA copy number in liver, heart and brain of adult and senescent rats showed that in all organs examined the senescent individuals have a mtDNA content higher than the adult counterparts. The analysis of mtDNA structural changes involved the search for point mutations and large deletions. As for the first case, the determination of the nucleotide sequence of many independent clones containing two mtDNA restriction fragments isolated from rat cerebral hemispheres did not show any sequence difference between adult and senescent individuals. However, analysis of mtDNA deletions by the polymerase chain reaction in liver and brain of adult and senescent rats identified a small population of mtDNA molecules harboring a deletion of 4834 bp. The estimate of the proportion of deleted molecules in the liver showed that they represent 0.02% and 0.0005% of total mtDNA in senescent and adult rat liver respectively. Therefore, a mtDNA deletion also accumulates in the rat during aging. This result supports the hypothesis of the accumulation of deleted mtDNA molecules in aging. However, the low percentage of deleted mtDNA molecules already found and the reversibility of the reduced mitochondrial DNA transcription in senescent rat raise doubts on the primary role of the irreversibly damaged mtDNA molecules in aging. Deleted mtDNA molecules along with changes caused by lipid peroxidation of mitochondrial membranes might contribute to the overall decline of mitochondrial function.

Age-related mitochondrial genotypic and phenotypic alterations in human skeletal muscle.

To have a clearer picture of how mitochondrial damages are associated to aging, a comprehensive study of phenotypic and genotypic alterations was carried out, analyzing with histochemical and molecular biology techniques the same skeletal muscle specimens of a large number of healthy subjects from 13 to 92 years old. Histochemical data showed that ragged red fibers (RRF) appear at about 40 years of age and are mostly cytochrome c oxidase (COX)-positive, whereas they are almost all COX-negative thereafter. Molecular analyses showed that the 4977 bp deletion of mitochondrial DNA (mtDNA(4977)) and the 7436 bp deletion of mtDNA (mtDNA(7436)) are already present in individuals younger than 40 years of age, but their occurrence does not change with age. After 40 years of age the number of mtDNA deleted species, as revealed by Long Extension PCR (LX-PCR), increases, the 10422 bp deletion of mtDNA (mtDNA(10422)) appears, although with a very low frequency of occurrence, and mtDNA content is more than doubled. Furthermore, mtDNA(4977) level directly correlates with that of COX-negative fibers in the same analyzed subjects. These data clearly show that, after 40 years of age, the phenotypic and genotypic mitochondrial alterations here studied appear in human skeletal muscle and that they are closely related.

Efficient mitochondrial biogenesis drives incomplete penetrance in Leber’s hereditary optic neuropathy

The mechanisms of incomplete penetrance in Leber’s hereditary optic neuropathy are elusive. Giordano et al. show that mitochondrial DNA content and mitochondrial mass are both increased in tissues and cells from unaffected mutation carriers relative to affected relatives and control individuals. Upregulation of mitochondrial biogenesis may represent a therapeutic target.

The MTERF family proteins: mitochondrial transcription regulators and beyond.

The MTERF family is a wide protein family, identified in Metazoa and plants, which consists of 4 subfamilies named MTERF1-4. Proteins belonging to this family are localized in mitochondria and show a modular architecture based on repetitions of a 30 amino acid module, the mTERF motif, containing leucine zipper-like heptads. The MTERF family includes the characterized transcription termination factors human mTERF, sea urchin mtDBP and Drosophila DmTTF. In vitro and in vivo studies show that these factors play different roles which are not restricted to transcription termination, but concern also transcription initiation and the control of mtDNA replication. The multiplicity of functions could be related to the differences in the gene organization of the mitochondrial genomes. Studies on the function of human and Drosophila MTERF3 factor showed that the protein acts as negative regulator of mitochondrial transcription, possibly in cooperation with other still unknown factors. The complete elucidation of the role of the MTERF family members will contribute to the unraveling of the molecular mechanisms of mtDNA transcription and replication.

REDUCED SYNTHESIS OF MTRNA IN ISOLATED MITOCHONDRIA OF SENESCENT RAT BRAIN

A system for studying RNA synthesis in isolated mitochondria from rat brain was set up to investigate the mechanisms responsible for the age-dependent reduction of mtRNA content. In the presence of an appropriate incubation buffer both synaptic and non-synaptic mitochondria from cerebral hemispheres were able to synthesize and process mtRNA in a way quantitatively and qualitatively similar to the in vivo transcription. The comparison of the electrophoretic pattern of mtRNAs synthesized by adult and senescent rat showed, in the senescent rat, a 50% reduction in the mtRNA synthesis rate relative to the adult value. This indicates that the age-dependent decrease of the mtRNA content is linked to a lower efficiency of the mt transcription.

Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in the brain of aged subjects but not Alzheimer’s disease patients

The levels of mitochondrial DNA 4977 bp deletion (mtDNA4977) and mitochondrial DNA 8′‐hydroxy‐2′‐deoxyguanosine (OH8dG) were determined in the same samples from two brain areas of healthy subjects and Alzheimers disease (AD) patients. A positive correlation between the age‐related increases of mtDNA4977 and of OH8dG levels was found in the brain of healthy individuals. On the contrary, in both brain areas of AD patients, mtDNA4977 levels were very low in the presence of high OH8dG amounts. These results might be explained assuming that the increase of OH8dG above a threshold level, as in AD patients, implies consequences for mtDNA replication and neuronal cell survival.—Lezza, A. M. S., Mecocci, P., Cormio, A., Real, M. F., Cherubini, A., Cantatore, P., Senin, U., Gadaleta, M. N. Mitochondrial DNA 4977 bp deletion and OH8dG levels correlate in the brain of aged subjects but not Alzheimers disease patients. FASEB J. 13, 1083–1088 (1999)

Increased expression of mitochondrial transcription factor A and nuclear respiratory factor-1 in skeletal muscle from aged human subjects.

The expression of two factors involved in the nuclear–mitochondrial crosstalk, namely the mitochondrial transcription factor A (TFAM) and the nuclear respiratory factor‐1 (NRF‐1), was studied in human skeletal muscle biopsies of young and aged subjects. Aged subjects presented a 2.6‐fold and an 11‐fold increase of the levels of TFAM protein and TFAM mRNA, respectively. The increased expression of TFAM was associated to the doubling of NRF‐1 DNA‐binding affinity and to a 6‐fold increase of NRF‐1 mRNA level. The upregulation of TFAM and NRF‐1, in aged skeletal muscle, appears involved in the pathway leading to the age‐related increase of mitochondrial DNA content.

Organization, structure, and evolution of mammalian mitochondrial genes.

Publisher Summary This chapter presents an up-to-date account of the organization, structure, and evolution of mammalian mitochondrial genes, with the belief that the knowledge of these features should supply valuable information to understand the interrelationships between the mitochondrion and the rest of the cell. The concerted effort of several laboratories has produced a great deal of information on the structure of mitochondrial genomes of several mammalian species. Mitochondria1 DNA ( MtDNA) sequence of man, cow, and mouse as well as a large part of rat sequence have been determined and the transcription products of these DNAs have been identified and mapped. Mammalian MtDNA is a circular closed double-stranded molecule, containing about 16,500 base pairs. This gene organization is it extremely compact with the exception of the region containing the origin of replication of the mtDNA heavy strand (D-loop region), the heavy (H)-strand is completely filled with discrete products. The mammalian transcripts are colinear with the DNA, showing a relevant difference in the gene organization as compared to lower eukaryote mtDNA such as yeast and N. crassa —that is,the lack of introns.

Accumulation of overoxidized Peroxiredoxin III in aged rat liver mitochondria.

Overoxidation and subsequent inactivation of Peroxiredoxin III (PrxIII), a mitochondrial H(2)O(2) scavenging enzyme, have been reported in oxidative stress conditions. No data are available in the literature about the presence of overoxidized forms of PrxIII in aged tissues. Liver mitochondria from 12-month-old rats and 28-month-old rats were here analyzed by two-dimensional gel electrophoresis. A spot corresponding to the native form of PrxIII was present in adult and old rats with the same volume, whereas an additional, more acidic spot, of the same molecular weight of the native form, accumulated only in old rats. The acidic spot was identified, by MALDI-MS analysis, as a form of PrxIII bearing the cysteine of the catalytic site overoxidized to sulphonic acid. This modified PrxIII form corresponds to the irreversibly inactivated enzyme, here reported, for the first time, in aging. Three groups of 28-month-old rats treated with acetyl-l-carnitine were also examined. Reduced accumulation of the overoxidized PrxIII form was found in all ALCAR-treated groups.