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S A Kovalenko

Publications and source records attributed to S A Kovalenko.

13 recordsLinked to original sources
PubMed2000

Tissue mitochondrial DNA changes. A stochastic system.

Several lines of evidence support the view that the bioenergetic function of the mitochondria in postmitotic tissue deteriorates during normal aging. Skeletal muscle is one such tissue that undergoes age-related fiber loss and atrophy and an age-associated rise in the number of cytochrome c oxidase (COX) deficient fibers. With such metabolic pressure placed on skeletal muscle it would be an obvious advantage to supplement the cellular requirement for energy by up-regulating glycolysis, and alternative pathway for energy synthesis. Analysis of rat skeletal muscle utilizing antibodies directed against key enzymes involved in glycolysis has provided evidence of an age-associated increase in the enzymes involved in glycolysis. Fructose-6-phosphate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase protein levels appeared to increase in the soleus, gracilis, and quadriceps muscle from aged rats. The increase in the level of these proteins appeared to correlate to a corresponding decrease in the amount of cytochrome c oxidase protein measured in the same tissue. Together these results are interpreted to represent a general upregulation of glycolysis that occurs in response to the age-associated decrease in mitochondrial energy capacity. Mitochondrial DNA (mtDNA) damage and mutations may accumulate with advancing age until they reach a threshold level were they impinge on the bioenergy capacity of the cell or tissue. Evidence indicates that mtDNA from the skeletal muscle of both aged rats and humans not only undergoes changes at the nucleotide sequence level (mutations and DNA damage), but also undergoes modifications at the tertiary level to generate unique age-related conformational mtDNA species. One particular age-related conformational form was only detected in aged rat tissues with high demands on respiration, specifically in heart, kidney, soleus muscle, and, to a lesser extent, the quadriceps muscle. The age-related form was not detected in gracilis muscle which is predominantly dependent upon glycolysis with regard to its energy requirements. Finally, a comprehensive hypothesis is presented that features the stochastic nature of the mitochondrial system. The basis of the hypothesis is that a dynamic relationship exists between endogenous mutagen production, DNA repair, mtDNA turnover, and nuclear control of mtDNA copy number and that age-associated changes in the dynamics of this relationship lead to a loss of functional full-length mtDNA that eventually leads to bioenergy decline.

Aging↗
PubMed1998

Tissue-specific distribution of multiple mitochondrial DNA rearrangements during human aging.

Mitochondria, according to the free radical theory of aging, are the major source of reactive oxygen species (ROS). The results, presented in this paper, question the role of reactive oxygen species in contributing significantly to the extent of mitochondrial bioenergy degradation of the tissues, which can be correlated with mtDNA rearrangements. We report here that mtDNA rearrangements, including deletions and duplications, in tissues from human aged subjects, occur in levels ranging from very low in liver, to considerable in cardiac muscle, to almost total in skeletal muscle. The extent of mtDNA rearrangements is correlated at both the individual tissue and cell level with cytochrome oxidase (COX) activity as the exemplifier of cellular bioenergy capacity. Thus, the ROS proposal in its simplest form as it affects mtDNA and mitochondrial electron transport system is not supported by the available data.

Aged↗
PubMed1998

An age-associated correlation between cellular bioenergy decline and mtDNA rearrangements in human skeletal muscle.

Post-mitotic tissues such as skeletal muscle develop a tissue bioenergy mosaic during the process of normal aging that eventually culminates into a bioenergetically diverse tissue containing cells ranging in their oxidative phosphorylation capacity from normal to grossly defective. The mosaic is postulated to develop continuously from birth with the relative proportions of cytochrome c oxidase (COX) proficient (positive) and COX deficient (negative) muscle fibers differing dramatically as a function of age. Generally, young individuals only display the rare fiber deficient in COX activity while aged individuals show a significantly higher proportion of negative fibers. There appears to be a random element governing which cells will be affected. Consequently, adjacent cells within a given tissue may exhibit vastly differing COX activities. Multiple mitochondrial DNA (mtDNA) deletions also appear to accumulate in skeletal muscle, similarly displaying a dramatic disparity as a function of age. Our previous findings have indicated that the accumulation of multiple mtDNA deletions, along with a concurrent decrease in wild-type mtDNA, strongly correlates with the age-associated decrease in COX activity observed in skeletal muscle. Although no definitive associations were established at the cellular level, an important prediction arose from this study. Cells that accumulate large numbers of mitochondrial mutations and have reduced levels of full-length mtDNA would be expected to be severely affected and show reduced COX activity as a consequence. Cells that accumulate fewer mutations or retain adequate amounts of wild-type mtDNA would be predicted to be less affected or even retain normal oxidative metabolism. In order to establish a link associating COX activity to the status of mtDNA within individual fibers, we developed single cell extra-long PCR (XL-PCR). The procedure was used to assess the relative concentration of full-length mtDNA with respect to any mtDNA deletions detected in individual human skeletal muscle fibers of 'pre-established' COX activity. Single cell XL-PCR analysis of COX positive fibers dissected from a 5-year old and 90-year old individual showed that 80% or more of the fibers contained full length mtDNA and few, if any, mtDNA rearrangements. COX deficient or COX intermediate fibers taken from the same individuals, by contrast, depicted a heterogeneous population of rearranged mtDNA species with no detectable full-length mtDNA. The data presented here indicates that COX deficient muscle fibers extracted from individuals, regardless of age, were accompanied by extensive mtDNA rearrangements and reduced levels of full-length mtDNA. This provides compelling evidence linking mtDNA mutations to COX activity decline in skeletal muscle and has important implications when considering the molecular basis of the aging process.

Aged↗
PubMed1998

The age-associated decrease in the amount of amplifiable full-length mitochondrial DNA in human skeletal muscle.

There has been a continuous evolution in our concept [1] that mtDNA undergoes a range of mutations with age and that such alterations lead to a decline in mitochondrial bioenergy capacity. Here we report that a wide range of deletion mutations accumulate with age and the amount of full-length mtDNA (FLmtDNA) amplifiable by extra-long PCR (XL-PCR) markedly decreases with age. An analysis of single human quadriceps muscle fibres reveals a close correlation between the decrease in FLmtDNA and the decline in cytochrome c oxidase activity, an exemplifier of mitochondrial bioenergy. However, Southern blotting analysis of unamplified genomic DNA shows that there is little decrease in FLmtDNA in aged quadriceps. The results are interpreted to indicate that while there is little change in the total mtDNA with age, nonetheless a significant proportion of this mtDNA is extensively damaged such that it cannot be amplified by XL-PCR. The amplifiable FLmtDNA, which putatively represents the functional component of the mtDNA, decreases markedly with age.

Adolescent↗
PubMed1997

Deltoid human muscle mtDNA is extensively rearranged in old age subjects.

Extra long PCR analysis of mitochondrial DNA (mtDNA) isolated from skeletal muscle of humans of different ages revealed three phenomena: (i) the amount of normal length mtDNA (16.5 kb) was progressively reduced with age, such that the cells of old age individuals (about 90 years) contained little, or undetectable amounts of normal length mtDNA; (ii) the total amount of mtDNA did not appear to be greatly decreased rather the extent of mtDNA deletions greatly increased; (iii) in old age subjects, considerable amounts of over-sized mtDNA (more than 16.5 kb) was observed. Enzyme histochemical analysis of cytochrome-c oxidase (COX) activity in the muscle tissue of all subjects evidenced a cellular bioenergy mosaic with cells ranging from high to zero detectable enzyme activity in the muscle samples. The frequency of COX deficient muscle fibres was highly dependent on the age of the subject. We have found that the extent of the mtDNA mutational changes strongly correlate with the observed progressive decrease in COX activity. Therefore, it was suggested that the total extent of mtDNA mutation is very large in old age subjects and is sufficient to account for the decline in cellular COX activity with age and for a progressive decrease of overall mitochondrial bioenergetic capacity.

Adult↗
PubMed1997

Method for in situ investigation of mitochondrial DNA deletions.

A number of mitochondrial DNA (mtDNA) deletions have been recently identified in the tissues of patients with mitochondrial diseases and in elderly individuals. To investigate the distribution of mutant mitochondrial genomes within any particular tissue, we have developed a sensitive method based on indirect in situ PCR. Our experiments have shown that the new method had the advantage of selectively amplifying only mtDNA bearing the 4,977 bp deletion. We show that this method is more sensitive than in situ hybridization for detecting the 4977 bp mtDNA deletion while using only a low number of PCR cycles that minimize damage to tissue architecture. By using this method, we have demonstrated that the mutation does not occur uniformly among the cells of a given tissue/organ. This technique will be useful studying the distribution/localization of mtDNA mutations in individual cells of tissues and when combined with enzyme histochemical procedures in adjacent sections will enable the correlation between mtDNA mutations and bioenergy defects in single cells.

Adult↗
PubMed1996

Accumulation of deletions and point mutations in mitochondrial genome in degenerative diseases.

Accumulation of various mutations in the mitochondrial genome is proposed as an important contributor to aging and degenerative diseases. Extensive fragmentation of mtDNA was detected in association with increased 8-hydroxydeoxyguanosine content in the heart mitochondrial DNA (mtDNA) from a patient with premature aging and mitochondrial cardiomyopathy, who carried a mutation within the mitochondrial tRNA(Asp) gene. This result suggests that damage to mtDNA by hydroxyl radical and accumulation of deleted mtDNA can be accelerated by a specific mitochondrial genotype. Similarly, extensive fragmentation of mtDNA was also detected in cultured cells exposed to a high oxygen concentration atmosphere, implying that mtDNA is vulnerable to reactive oxygen species. To clarify the role of point mutations accumulated in mtDNA, we examined the sequence heterogeneity of mtDNA in the skeletal muscle of a MELAS patient who carried a mutation within the mitochondrial tRNA(leu)(UUR) gene. The analysis revealed that the frequency of mutant clones in the MELAS muscle was significantly higher than those in an age-matched control muscle and a control placenta. Some of these nucleotide substitutions were missense and nonsense mutations, which potentially have deleterious effects on the mitochondrial function. The frequency of nucleotide substitutions in the striatum of three patients with Parkinson's disease was also significantly higher than that in control tissues. We also observed increased protein modification by 4-hydroxy-2-nonenal, a lipid peroxidation by-product, in Parkinson's disease. These results suggests that a vicious cycle contributes to the progression of degenerative process. In this cycle, first a primary mitochondrial mutation(s) induces a mitochondrial respiratory defect, which increases the leakage of reactive oxygen species (ROS) from the respiratory chain. Then the ROS would trigger accumulation of secondary mtDNA mutations in postmitotic cells, leading to further aggravation of mitochondrial respiratory defects and increased production of ROS and lipid peroxides from mitochondria, and thus resulting in degeneration of cellular components.

Aged↗
PubMed1996

Accumulation of somatic nucleotide substitutions in mitochondrial DNA associated with the 3243 A-to-G tRNA(leu)(UUR) mutation in encephalomyopathy and cardiomyopathy.

To understand the pathogenesis of mitochondrial encephalomyopathy and cardiomyopathy, we analyzed the sequence heterogeneity of the skeletal muscle mitochondrial DNA from a patient with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS). A mtDNA segment of 347 bp amplified from the total DNA was cloned into a vector. Analysis of 60 independent clones (20,800 bp in total) revealed the 3243 A-->G transition in all the sequenced clones and additional nucleotide substitutions at 5 sites in 10 clones. The frequency of mutant clones (10/60) in the MELAS patient was significantly higher [chi2 = 10.909, P < 0.05] than that in an age-matched skeletal muscle control (0/60) as well as in a normal placenta (2/60). These results support our hypothesis that secondary somatic mtDNA mutations can be initiated by the 3243 A-->G mutation and that the accumulation of somatic mutation in individuals with deleterious inherited mitochondrial genotype can contribute to the progressive mitochondrial dysfunction in MELAS.

Adult↗
PubMed1995

Mitochondrial DNA mutations in cardiomyopathy: combination of replacements yielding cysteine residues and tRNA mutations.

Mutations occur in mitochondrial DNA (mtDNA) in a strand-asymmetric manner. The suppressed usage of cysteine residues in the H-strand-encoded subunits can be ascribed to the mutational instability of the codon for cysteine. The usage of cysteine was suppressed even in the L-strand-encoded ND6 subunit in which the codon for cysteine was stable. Survey of the entire sequences of mtDNA from 43 individuals revealed three amino acid replacements creating cysteine residues. A patient with fatal infantile cardiomyopathy carried a mutation causing a Tyr-->Cys replacement along with three tRNA mutations. A patient with hypertrophic cardiomyopathy carried two mutations causing a Ser-->Cys replacement and a Tyr-->Cys replacement besides two tRNA mutations. The gain of cysteine residues might accelerate the inactivation of the subunits either by reactive oxygen species or by lipid-peroxidation products, and this gain, possibly in association with tRNA mutations, can be a genetic risk factor for degenerative diseases.

Adult↗
PubMed1994

A common hot spot for somatic and germline mutations in rat mitochondrial DNA: analysis by fluorescence-based SSCP.

To estimate the degree of sequence heterogeneity of mtDNA, we have developed an efficient system for mutant detection using fluorescence-based single strand conformational polymorphism (F-SSCP). The F-SSCP analysis 200 clones from Wistar rat and sequencing of 2 clones from each 40 inbred Wistar rats at the age of 7 weeks detected no sequence differences, suggesting that the mtDNA sequence heterogeneity is low in young rat hearts. One of the 387 clones isolated from a Donryu rat showed a different mobility in the F-SSCP from other clones. Sequencing of the mutant clone revealed double contiguous TT-->CC transitions at nucleotide positions 15443 and 15444, compared with the normal Donryu clone. The nucleotide at 15444 was different between the Wistar and Donryu rats, indicating that this is the site of germline mutation. These results suggest that the somatic and germline mutations can occur at a common hot spot.

Animals↗