[DNA methylation level and telomere length as a basis for the biological aging clock model construction].

Aging is a process that depends on a variety of both external and internal factors. The biological age of a person determines body deterioration and the risk of age-related diseases. Currently, as indicators of biological age are considered different characteristics including average length of telomeres in cells and the level DNA methylation. We propose to combine the two approaches to create a model to assess the biological age of the person. Application of qPCR to determina the length of telomeres and MS-HRM for analysis of DNA methylation will help us to determine the parameters of interest quickly when using a minimum set of equipment.

[Telomere length, telomerase activity, stress and aging].

The review is dedicated to analysis of data available at present time concerning possible influence of stress upon telomere lengths and telomerase activity, as well as various ways of counteracting it. Present-day telomerase theory of aging gains a new impetus, shedding light upon the influence of psychological state of humans and their ability to counteract stress, upon the process of aging. It also tends to regard telomere shortening and the decrease in the activity of telomerase as a marker of level of the ability to adapt to both inner and outer influences. Both aging and age-dependent diseases are proved to be substantially retarded not only by the administration of drugs, but also by psychological means, which forms a good way towards healthy longevity. With complete understanding of the impossibility to prevent or even to slow down natural senescence itself, these methods allow to remove causes, which accelerate senescence, and to increase the average human longevity.

[Violation of the homologous chromosomes approchement as the answer to the small doses of roentgen irradiation].

Any violation of the homologous chromosome DNA repair leads to the genome instability, characteristic for hereditary syndromes and for aging cells. Using low doses of ionizing radiation (3-10 cGy) we have found any transference of the homologous centromere loci of the chromosome 1 (1q12) from the periphery to the cen- tre of the nucleus in the lymphocytes of young healthy donors. The same effect was found after any influence of RNA-polymerase inhibitor a-amanitine. Some changes in the chromatin structure during aging (70-80 years old patients) result in the difficulties in chromosome displacement, accompanied with any trouble in the appro- achement of the homologous chromosome loci as an answer to low doses of radiation.

[Effects of iron ion additional introduction in radiation therapy of tumor-bearing animals].

The gender differences of the transplantable glioma-35 growth were revealed in tumor-bearing rats. The effect of mineral iron containing water (ICW) as drinking water was studied during radiation therapy (RT) in tumor bearing male rats. It was shown that ICW used separately had a weak anti cancer effect. The ICW use after irradiation caused no significant changes in hematological reactions as compared with RT only. The mineral water administration before irradiation resulted in the reduction of the supercoiled DNA index on the 1st and 21st day after irradiation, as well as in monocytopeny and a definite decrease in the tumor volume on the 21st day of the experiment as compared with control, which is associated with the ferroptosis effect of ICW. It was established that the reaction peculiarities of the blood DNA structure index in irradiated rats at the early stage of glioma growth may serve an express marker of the efficiency of RT.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 5. SkQ1 prolongs lifespan and prevents development of traits of senescence.

Very low (nano- and subnanomolar) concentrations of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) were found to prolong lifespan of a fungus (Podospora anserina), a crustacean (Ceriodaphnia affinis), an insect (Drosophila melanogaster), and a mammal (mouse). In the latter case, median lifespan is doubled if animals live in a non-sterile vivarium. The lifespan increase is accompanied by rectangularization of the survival curves (an increase in survival is much larger at early than at late ages) and disappearance of typical traits of senescence or retardation of their development. Data summarized here and in the preceding papers of this series suggest that mitochondria-targeted antioxidant SkQ1 is competent in slowing down execution of an aging program responsible for development of age-related senescence.

Replicative mosaicism might explain the seeming contradictions in the telomere theory of aging.

All living organisms are regarded as a mosaic of cells with different replicative histories explaining all the contradictions in the telomere theory of aging. Exhausted proliferative potential of cells in some areas of the organ tissue might be sufficient to promote one of the age-dependent diseases. Thus a combination of these disorders, gradually increasing with age, is aging. Nobody dies because of the age. Nothing other than the age-related diseases occur with age, and if we separate these diseases, we will get not just a healthy old man, but according to Dr Hayflick's new hypothesis (Exp. Gerontol., 33 (1998) 639) a healthy young man, if not a newborn child.

Decreased number of simultaneously operating adjacent clusters of replicons in some human strains with and without X-irradiation.

Several parameters of DNA replicons and replicon clusters have been examined using DNA fiber autoradiography in normal vs. mutant human cell lines showing increased chromosomal sensitivity to ionizing radiation as well as radioresistant DNA synthesis. Rates of synthesis of individual replicons for unirradiated ataxia telangiectasia (AT) AT5MO, basal cell naevus syndrome (BCNS) BCN1SP and Down's Syndrome LCH944 appeared to be in the range seen with two normal fibroblast lines. With the longer labelling times (60-165 min), the average track lengths were longer in normal fibroblasts than mutant cell; after 5 Gy of radiation, normal and mutant cells had similar track lengths for all labelling times (10-165 min), as well as unchanged rates of replicon synthesis. These observations led to the determination of 'chain length', which measures simultaneously active adjacent replicon clusters. The main finding is that 'chain length' in mutant lines was significantly lower than that in the normal fibroblasts; upon 5 Gy irradiation, the values in normal cells were reduced about two-fold while the values for mutant cells remained about the same as controls. Thus, the experiments suggest that in unirradiated mutant cells DNA replication is delayed in a comparable manner as that induced by ionizing radiation in normal cells. A possible relation of the data to the chromosomal radiosensitivity and radioresistant DNA synthesis in the mutant lines is discussed.

Phylogenetic position of mammoth and Steller's sea cow within Tethytheria demonstrated by mitochondrial DNA sequences.

Here we report DNA sequences from mitochondrial cytochrome b gene segments (1,005 base pairs per species) for the extinct woolly mammoth (Mammuthus primigenius) and Steller's sea cow (Hydrodamalis gigas) and the extant Asian elephant (Elephas maximus), the Western Indian manatee (Trichechus manatus), and the hyrax (Procavia capensis). These molecular data have allowed us to construct the phylogeny for the Tethytheria. Our molecular data resolve the trichotomy between the two species of living elephants and the mammoth and confirm that the mammoth was more closely related to the Asian elephant than to the African elephant. Our data also suggest that the sea cow-dugong divergence was likely as ancient as the dugong-manatee split, and it appears to have been much earlier (22 million years ago) than had been previously estimated (4-8 million years ago) by immunological comparison.

Slower synthesis of individual replicons and adjacent replicon clusters in a radiosensitive xeroderma pigmentosum strain with and without X-irradiation.

Two replication parameters, synthesis of individual replicons and adjacent replicon clusters, were measured using DNA fiber autoradiography in a radiosensitive form of group C xeroderma pigmentosum (XP) XP2SP, in group C XP4SP, in group A Cockayne syndrome (CS) CS1SP and two normal human fibroblast strains. The novel observation here is that in non-irradiated XP2SP cells synthesis of individual replicons was significantly retarded as compared with all other cell lines tested and remained unchanged after 5-Gy X-rays. Also the number of simultaneously operating adjacent replicon clusters was uniquely reduced in only non-irradiated XP2SP cells and remained unaltered after 5-Gy irradiation. While the normal, XP4SP and CS1SP cells are radiosensitive to reduction in this replication parameter to a low level seen in both non-irradiated and 5-Gy irradiated XP2SP cells. Thus, non-irradiated XP2SP cells mimic irradiated normal, XP4SP and CS1SP cells. A possible relation of the above abnormalities in individual replicons and adjacent replicon clusters to a high incidence of spontaneous sister-chromatid exchanges and X-irradiation-induced chromosomal aberrations in XP2SP cells is discussed.

Sister chromatid exchanges and inhibition of DNA synthesis in irradiated human cells.

X-ray irradiation inhibits DNA synthesis and enhances the frequency of sister chromatid exchanges (SCE) in normal human lymphocytes. On the contrary, cells from patients with Down's syndrome, Xeroderma pigmentosum (form II) and progeria, characterized by radioresistant DNA synthesis, do not show such an increase in SCE frequency. We suggest that radiation-induced SCE frequency is a result of inhibition of DNA replication, rather than a direct damage of chromosomes by ionizing radiation. It is in agreement with Painter's /13/ hypothesis according to which SCE are formed due to asynchronous completion of replication in contiguous replicon clusters. So, probability of SCE formation is the more the lower is rate of replication. Thus, the extent of radiation damage cannot be measured directly by the SCE frequency.

[Disruption of DNA replication in non-irradiated cells in basal cell nevus syndrome and the effect of ionizing radiation]. / Narushenie replikatsii DNK v neobluchennykh kletkakh pri sindrome bazal'nokletochnogo nevusa i deistvie ioniziruiushchei radiatsii.

Analysis of DNA fiber autoradiograms from basal cell nevus syndrome (BCNS) skin fibroblasts has revealed for the first time a new defect in DNA replication earlier unknown in other chromosomal instability syndromes, that involves a significantly decreased rate of DNA-chain growth in unirradiated cells. Here we present evidence that the defect may be due to a marked reduction in number of simultaneously operating groups of replicons compared to that in normal cells, the rate of fork movement and the fusion of neighbouring units in the group remaining unchanged. Radioresistant DNA synthesis was observed in the BCNS cells. The exposure of cells derived from normal donor to gamma-rays at a dose of 5 Gy reduces the number of simultaneously operating groups of replicons to the level occurring in unirradiated BCNS cells, the rate of folk movement being unchanged in both cell types. However, the incidence of fusion between neighbouring units within the group is lower in the cells exposed to gamma-rays, due perhaps to a radiation-induced lesion in the group. Thus, ionizing radiation reduces the rate of DNA synthesis to the same level, however from different initial levels. Our data suggest that the phenomenon of radioresistant DNA synthesis may be explained by the presence of the initial defect in DNA replication in BCNS or any other chromosomal instability disorders.

Sister-chromatid exchanges in a special form of xeroderma pigmentosum (form II).

The frequency of sister-chromatid exchanges (SCE) was studied in peripheral blood lymphocytes from a xeroderma pigmentosum (form II, XPII) patient. The cells were irradiated with UV or X-rays. In some experiments novobiocin (NB), inhibitor of topoisomerase II, or caffeine (CA), inhibitor of DNA repair were added to the cultures. The level of spontaneous SCE in the patient's lymphocytes was found to be significantly increased in comparison to that in the cells from normal donors. The inhibitors and UV-light caused a rise in the frequency of SCE in the cells taken from normal donors and except for NB, in the lymphocytes from the patient XPII. X-Rays did not increase SCE frequency in normal lymphocytes and lowered it in the patient's cells. SCE frequency rose when inhibitors of DNA replication and repair were used in combination with mutagens.

Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation.

The rate of DNA synthesis after gamma-irradiation was studied either by analysis of the steady-state distribution of daughter [3H]DNA in alkaline sucrose gradients or by direct assay of the amount of [3H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of gamma-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations.