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Evolutionary Cytogerontology Sector, School of Biology, Lomonosov Moscow State University, Moscow, Russia khokhlov@mail.bio.msu.ru The considerably increased recently interest in experimental-gerontological research has led to a paradoxical situation: although more and more papers in the field appear, only a small part of them is really devoted to the study of mechanisms of aging. In my opinion, it is determined, inter alia, by the following circumstances: 1) As a rule, the classical definition of aging as a set of age-related changes, leading to an increase in the probability of death of organism is ignored. 2) The focus is placed upon achieving a longer life span, although it often has nothing to do with the impact on the aging process (in particular, quite successfully, you can increase life span of nonaging organisms; at the same time, the presence of aging does not necessarily indicate a low longevity). 3) Animals with certain abnormalities (like genetic disorders), increasing the probability of their death, are used as control, so any remedy against these pathologies lead to prolongation of life. 4) Too much importance is attached to an increase in average life span, which is largely determined by factors in no way related to aging. 5) Increasingly, gerontological experiments are conducted on model systems that allow obtaining only indirect information about the mechanisms of aging, the interpretation of which depends strongly on the fundamental concept to which the specific researchers adhere.

Unfortunately, the above also applies to cytogerontological studies that became extremely popular in recent decades. In particular, many of the conclusions reached earlier on the basis of the results of experiments conducted on Hayflick's model (aging in vitro), were subsequently found to be wrong. In addition, our cytogerontological studies of various anti-aging factors with the help of the "stationary phase aging" model, the cell kinetics model and assessment of colony-forming ability, have shown that in very many cases the factors studied have no beneficial effect on the viability of cultured cells, although they prolong life in experimental animals and increase the well-being of humans. This allowed us to assume that, in many cases, the anti-aging agent action appears only at the organism level, and is not limited to just improving the viability of some of its constituent cells.


Lemeshko Victor V.

Escuela de Fisica, Facultad de Ciencias, Universidad Nacional de Colombia, sede Medelln, Calle 59-A, No 63-20, Medellin, Colombia vvasilie@unal.edu.co Apoptosis is defined as programmed cell death, and mitochondria, particularly their cytochrome c, play a crucial role in this process. Earlier, we discovered two populations of rat liver mitochondria with significantly different outer membrane integrity. During growth and development of the organism, the population of mitochondria with the fragile outer membrane replaced the other mitochondrial population with significantly more resistant outer membrane [Lemeshko, Biofizika, 1982, 37:837-840]. These results allowed us to suggest that the animal aging might be genetically programmed at the level of the outer membrane of mitochondria, through replacing of one population of mitochondria by the other, although both types of these organelles synthesize ATP well, as it was demonstrated in other our works. It means that the outer membrane permeabilization by the same internal and/or external environmental pro-apoptotic factors might be more probable in aged than in young organisms.

The fundamental role of mitochondria in cell death has been finally recognized in 1998 [Susin et al., Mitochondria as regulator of apoptosis: doubt no more. Biochim Biophys Acta, 1998, 1366:151-165]. The young population of mitochondria, having the resistant outer membrane, seems to be needed to resist relatively high intensity of the free radical lipid peroxidation of biomembranes in young animals that we considered as important physiological factor allowing fast remodeling of biomembranes in young animals, allowing their growth and development. Although oxidative stress and reactive oxygen substances are widely recognized as pro-aging factors, our results clearly demonstrated that the cell antioxidant enzyme system has a low activity in young animals and significantly increases with age of rats up to half of the lifespan, thus leading to a decrease of free radical processes at this age period. Some subsequent decrease in the activity of the antioxidant enzyme system in aged animals, accompanied with relatively low rate of the membrane structure recovery, might be the fundamental basis for the development of degenerative diseases and even death events, as a result of increased population of aged mitochondria having fragile outer membrane. All these mean that not directly the death, but the probability of its realization under external environmental conditions is genetically programmed. A somewhat similar situation takes place at the cellular level that relates to the cytochrome c-mediated cell death. The release of cytochrome c from mitochondria under various pro-apoptotic factors, for example, does not yet mean cell death: as long as cytoplasmic reducing factors maintain cytochrome c in the reduced state, this hemoprotein is not yet pro-apoptotic. We recently demonstrated that only oxidized, but not reduced form of cytochrome c is capable to directly induce mitochondrial aggregation [Lemeshko, Mol.Cell. Biochem., 2012, 360:111-119], the process known as one of the attributes of apoptosis. In conclusion, we hypothesize that the probability of death of multicellular organisms is significantly contributed by genetically programmed structural and functional changes of various cells, like a change in mitochondrial populations, which increase the probability of multiple apoptotic processes.



Roshina N. V., Symonenko A. V., Pasyukova E. G.

Institute of Molecular Genetics of RAS, Moscow, Russia egpas@rambler.ru Earlier, we demonstrated that several genes participating in regulation of the nervous system development are involved in lifespan control. An insertion of the P{GT1} vector 300 bp downstream of the structural part of escargot (esg) that encodes an RNA polymerase II transcription factor and insertions of P-based vectors in the structural part of aPRC that encodes atypical protein kinase C were associated with increase in male and female lifespan.

Both esg and aPKC are involved in regulation of a crucial step in the nervous system development, asymmetric neuroblast division (AND). We suggested that other genes interacting with esg and aPKC during AND could be important for lifespan control. inscrutable (insc) is a gene essential for AND. We demonstrated that an insertion of the P{EPgy2} vector in the structural part of insc prolonged female lifespan.

Analysis of lifespan of heterozygous esg and insc double mutants allowed us to predict that these genes interact in the course of lifespan determination. Decrease of esg transcription was shown to be associated with lifespan increase in mutant esg females and males. Mutant insc females with increased lifespan also demonstrated decrease in esg transcript amount. This result confirmed both interaction between esg and insc and association of prolonged lifespan with low esg expression.

aPKC is directly phosphorylated by GSK3 (glycogen synthase kinase 3) encoded by shaggy (sgg), which further provides AND. We demonstrated that several insertions of P-based vectors in the structural part of sgg were associated with alterations of male and female lifespan. Altogether, our recent findings indicate that genes affecting AND during early steps of development could influence longevity of Drosophila adults.



Shilovsky G.A.1, Shram S.I.2, Khokhlov A.N.1 Evolutionary Cytogerontology Sector, School of Biology, Moscow State University, Moscow, Russia Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia gregory_sh@list.ru Various harmful genome modifications (mutations, unrepairable DNA damage, etc.) are known to accumulate with age. This leads to impairment of the genome functioning. The accumulation is due both to increased production of reactive oxygen species and reduction in antioxidant defense and DNA repair efficacy. At present, however, an important role in regulating of the genome stability is given also to epigenetic factors. In this regard, posttranslational modifications of chromatin-associated proteins are of special interest for molecular gerontology.

The significance of this process is already shown, for example, for histone deacetylation. However, the role of poly(ADP-ribosyl)ation is less clear. This is one of the earliest cell responses to DNA damage catalyzed by a family of transferases poly(ADP-ribose) polymerases (PARP). The most studied is the principal member of the family PARP-1 (EC Poly(ADP-ribosyl)ation is accompanied by a number of cellular processes, including DNA repair, replication and recombination, as well as apoptosis and necrosis. To understand PARP role in aging and lifespan determination, the data on changes of PARP activity and PARP-1 gene expression with age and in aging-associated diseases, with special attention given to the use of various molecular-genetic approaches (knockdown, knockout, etc.), are reviewed. Analysis of other researchers results and our own data demonstrates that PARP activity paradoxically decreases with age while the level of PARP-1 stays the same and the level of the PARP-activating DNA breaks increases. This might serve as a predictor of age-related pathologies and genomic instability, as well as a perspective marker of aging. Changes in the size and number of poly(ADP-ribose) chains as well as in the proportion and pattern of poly(ADP-ribosyl)ated nuclear proteins leading to impairment of their functioning are suggested as the possible reasons for the unusual change of PARP activity mentioned.



Vorobyova A.K.1, Alinkina E.S.1, Misharina T.A.1, Fatkullina L.D.1, Burlakova E.B.1, Khokhlov A.N.2 Laboratory of Physico-Chemical Fundamentals of Biological System Regulation, N.M. Emanuel Institute of Biochemical Physics of RAS and 2Evolutionary Cytogerontology Sector, School of Biology, Lomonosov Moscow State University, Moscow, Russia ak.vorobyova@gmail.com Carvacrol-bearing essential oils (EO) are well known biologically active preparations widely used to improve health, mental abilities, and well-being. Recently we found that one of them, the savory EO, had a positive effect on the average life-span of AKR mice with the high incidence of spontaneous leukemia. Besides, we have shown that one more carvacrol-bearing preparation, the oregano EO, obtained from Orignum vulgre (its composition is very similar to the composition of the savory EO), increases the average life-span of long-living BALB/c mice. To clarify the possible cytological mechanisms underlying the effects we had studied the oregano EO in experiments on transformed cultured Chinese hamster cells. In the preliminary investigations we evaluated various concentrations of the oil in terms of its cytotoxic or mitogenic effects by analyzing the cell culture density on the 4th day of growth. The concentrations in the growth medium studied (on carvacrol basis) ranged from 110-15 to 510-4 M. As a result the concentrations of 2.510-5 and 2.510-4 M were chosen for the further cytogerontological experiments because the former seemed to be 100 % non-toxic and the latter induced just about 50 % reducing in the final cell density. The preparation at 2.510-5 M was shown to have no effect neither on colony-forming ability of the cells nor on saturation density (an index of cell culture biological age) or stationary phase aging of the culture (aging-like degradation of cells in the stationary phase of growth).
In contrast, oregano EO at 2.510-4 M abruptly diminished colonyforming ability of the cells and influenced as a pro-aging factor on the saturation density and the death rate in the stationary phase modifying respectively the culture survival curve. Basing on our conception of aging and the data obtained we assumed that the beneficial effect of oregano EO on the mice life-span could be realized at the organismic level only but not related to any anti-aging activity manifesting at the cellular level and improving cell viability.




Yablonskaya O.I.1, Ryndina T.S.2, Voeikov V.L.1, Khokhlov A.N.2 Department of Bioorganic Chemistry and 2Evolutionary Cytogerontology Sector, School of Biology, Lomonosov Moscow State University, Moscow, Russia yablonskaya@mail.bio.msu.ru Hydrated fullerene (HyFn) is the molecule of C60-fullerene (C60) encased in a stable shell of water molecules, providing hydrophilic properties to this complex and its solubility in aqueous solutions. HyFn, as well as chemically modified fullerenes and fullerenes solubilized in water in other ways, is known to have a broad spectrum of biological activities both in vivo and in vitro with no signs of toxicity. HyFn and various chemically modified fullerenes in aqueous environment exhibit a peculiar chemical activity on the one hand, they can be regarded as strong antioxidants, on the other hand as electron donors for oxygen, i.e. prooxidants. Given that C60 is not very active chemically and that in HyFn it is surrounded by a water shell, it could be assumed that the entire pattern of HyFn activity is due to unique properties of the water shell formed around C60.

There are also some evidences of influence of HyFn at ultra-low concentrations on biochemical processes and free-radical reactions occurring in vitro. Previously, we have shown the effect of HyFn at ultra-low concentrations on redox processes in human whole blood as well as in aqueous bicarbonate solutions. In the present study we investigated effect of HyFn aqueous solution on the growth and "stationary phase aging" (accumulation of "age" changes in cultured cells during cell proliferation slowing down within a single passage and subsequent "aging" in the stationary phase of growth) of transformed B11-dii FAF28 Chinese hamster cells. The final calculated concentration of HyFn in the growth medium after serial dilutions of its original solution was equivalent to 10-19 M. It is paradoxical, but, in contrast to the known data about absence of HyFn cytotoxicity at higher concentrations, in our experiments it inhibited cell proliferation and accelerated the process of "stationary phase aging" of the cell culture. Moreover, addition of HyFn aqueous solution at this calculated concentration to the cells that had already reached the stationary phase of growth caused a rapid (within no more than 24 h) death of a significant part of the cell population. Perhaps, the observed features of HyFn at ultra-low concentration are determined by some special properties of the water surrounding C60, namely, its ability to serve as an electron donor and acceptor regulating redox processes in aqueous systems, especially those in which oxygen is involved. At the moment we cannot to rule out the possibility that HyFn at ultra-low concentrations affect transformed cells only. Therefore in our further studies we plan to carry out the similar experiments on normal fibroblasts possessing limited mitotic potential. Probably, the anti-aging effect of fullerenes revealed by other researchers in experimental animals is related to their antitumor and antibacterial activities.



Bal Ra., Trk G.a, Tuzcu M.a, Yilmaz O.a, Ozerkan I.a, Kuloglu T.a, Gr S.a, NedzvetskyV.b, Tykhomyrov A.c, Andrievsky G.c, Baydas G.d, Naziroglu M.e a Firat University, Elazig, Turkey; rbal@firat.edu.tr; b National University of Dniepropetrovsk, Dniepropetrovsk, Ukraine; nedzvetskyvictor@gmail.com; c Institute of Physiologically Active Compounds LLC, Kharkov, Ukraine; yard@kharkov.ua; d Bingol University, Bingol, Turkey;

e Suleyman Demirel University, Isparta, Turkey Diabetes mellitus (DM) is considered to be a premature aging process and characterized by producing male sexual dysfunction. Searching for the agents that could alleviate diabetes-induced impact on reproductive system is yet the important area of inquiry. The present study was performed to evaluate whether antioxidant hydrated C60 fullerene (C60HyFn) alleviate testicular dysfunction induced by streptozotocyn (STZ)-diabetes in rats. Sexually mature Wistar male albino rats were taken for experiment and divided into four groups as follows (n = 6 per group): (1) control group (untreated animals), (2) C60HyFn-treated nondiabetic group, (3) STZ-diabetic group, and (4) C60HyFn-treated diabetic group. Diabetes in animals of 3-rd and 4-th groups was induced by a single intraperitoneal injection of a buffered solution of STZ at a dose of 50 mg/kg body weight. Once sugar levels achieved consistent value after STZ injection (more than 300 mg/dl), rats in the 2-nd and 4-th groups were treated with C60HyFn (in the form of drinking water) at the dose of 4 g/kg daily for 5 weeks. In diabetic rats, levels of serum testosterone, testicular reduced glutathione (GSH) and alpha-tocopherol were significantly reduced and testicular lipid peroxidation level was increased (p 0.001). Treatment of diabetic rats with C60HyFn resulted in significant corrective effects on these parameters towards the control levels.

Furthermore, C60HyFn treatment in diabetic and nondiabetic rats resulted in considerable elevations of some important polyunsaturated fatty acids. However, administration of C60HyFn to diabetic animals did not make any significant change in blood glucose level. In diabetic rats, relative weights of right cauda epididymis, seminal vesicles, prostate, sperm motility and epididymal sperm concentration were significantly less than those of control group, but which were restored in the fourth group treated with C60HyFn (p 0.001). Marked histopathological changes including degeneration, desquamation, disorganisation and reduction in germinal cells, interstitial oedema and congestion were evident in the testis of diabetic rats, but C60HyFn treatment resulted in recovery of histopathological changes. In conclusion, we have presented for the first time substantial evidence that administration of C60HyFn significantly reduces diabetes-induced oxidative stress and associated complications such as testicular dysfunction and spermatogenic disruption.



Ushakova G.A.1, Kovalchuk Y.P.1, Kovalenko T.N.2, Osadchenko I.A.2, Skibo G.G.2, Pierzynowski S.G.3,4 Dnepropetrovsk National University named by Oles Gonchar, Gagarin ave. 72, 49050, Dnepropetrovsk, Ukraine;

Bogomoletz Institute of Physiology, Bogomoletz str.4, 01024, Kiev, Ukraine;

SGPlus, Vimpelgatan 21, S-211 14 Malmo, Sweden;

Lund University, Helgonavgen 3b, S-223 62 Lund, Sweden ushakova_g@ukr.net Aging is associated with many physiological alterations such as changes in metabolism and food intake, and brain dysfunction may represent a crucial cause linking homeostatic and cognitive dysfunction. A loss of neuronal components and coupling between components of neuronal networks is characteristic of aging. Normal brain aging is associated with development of reactive astrogliosis and deficits in learning and memory. The nutrition plays an important supportive role in the management of the critically ill neurological patient, especially for old persons. Many agedependent diseases are caused by free radical-mediated neurotoxicity induced by inhibition of mitochondrial dehydrogenases (specially, alpha-ketoglutarate dehydrogenase), followed by a decrease in neuronal mitochondrial transmembrane potential and ATP, prior to neuronal death.

The main purpose of the study was to investigate the glial reaction in the old gerbil brain after long-lasting dietary effect of 2-oxoglutarate as a food compounds.

For long-lasting feeding (during 6 month) we used 36 Mongolian gerbils, which were randomly selected into 3 groups at start of experiment: 1 adult (6 month of age), 2 old (18 month of age) with standard diet, 3 old (18 month of age) with diet including 1 % Ca AKG+1 % Na2AKG. Animals were decapitated at the end of experiment under weak anesthesia, three brain areas were isolated: cerebellum, thalamus and hippocampus, which were subsequently used to obtain cytozol and cytoskeleton protein fractions. The level of S100b (calcium-binding protein) and GFAP (glial fibrillary acid protein) was determined by solid-phase competitive inhibitory ELISA.

Statistical processing of results was carried out using SPSS 10.0 for Windows, the difference was considered reliable at p 0.05.

The experimental data indicate a stable level of astrocyte specific proteins in thalamus and cerebellum of gerbils aged 6 months and 2 years. A significant increase in the S100b level in old gerbils (2,10,6) g/ml compared with adults (0,60,09) g/ml was determined in the hippocampus indicating the development of reactive astrogliosis. Dietary treatment with 2-oxoglutarate 2 % in dry feed for 6 months inhibited the hyperactivation of astrocytes in old animals. The level of S100b protein in these animals was (1,22 0,18) g/ml. The same alteration was shown for GFAP distribution in hippocampus.

Analysis of the data indicates the absence of probable changes in metabolism of astrocyte specific proteins in thalamus and cerebellum in old gerbils, but also established a violation of integrative brain functions, information homeostasis in securing and optimizing by the hippocampus. Long-lasting dietary treatment with 2-oxoglutarate (main component of glutamateglutamine cycle of astrocytes) prevents the development of reactive astrogliosis in the hippocampus relating to the processes of aging.


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