The Field of Biological Aging: Past, Present and Future

The Field of Biological Aging: Past, Present and Future Editor

Abdullah Olgun Erzincan Mil. Hospital, Biochemistry Lab.24000, Erzincan, Turkey

Transworld Research Network, T.C. 37/661 (2), Fort P.O., Trivandrum-695 023 Kerala, India

Published by Transworld Research Network 2011; Rights Reserved Transworld Research Network T.C. 37/661(2), Fort P.O., Trivandrum-695 023, Kerala, India Editor Abdullah Olgun Managing Editor S.G. Pandalai Publication Manager A. Gayathri Transworld Research Network and the Editor assume no responsibility for the opinions and statements advanced by contributors ISBN: 978-81-7895-513-1

Preface Aging and age related pathologies are among the main medical problems of modern societies. Although aging is generally seen as an inevitable normal developmental period, I believe that its acceptence as a disease -even though it might not- will have many pragmatical consequences and break the mental barriers against studying it.

There are many theories tested in the field of biogerontology. Free radical theory is the most studied one. But in the last few years there has been many controversial studies concerning the role of free radicals in aging.

Decades long studies to find strategies aiming to postpone aging resulted only with a few successes like caloric restriction and rapamycin in model animals. Therefore we likely need a paradigm shift in the future. Therefore, in this book extreme plant longevity/plant aging and traditional medicine were emphasized as new areas to focus on in biogerontological studies.

Interestingly they seem neglected so far. Current and past achievements of the field were also tried to be covered widely.

I would like to thank all of the contributors for their very kind efforts that made this book a reality.

I am grateful to Transworld Research Network team for kindly giving me the opportunity to edit this book and their professional work during the publication process.

Abdullah Olgun, MD., Assoc. Prof.

Contents

Chapter 1 Biological principles of aging and approaches for interventions Suresh I. S. Rattan

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Chapter 2 Studies of in vitro cellular senescence Olivia M. Pereira-Smith and Kaoru Tominaga

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Chapter 3 Protein oxidation and repair mechanisms in aging Ayse Banu Demir and Ahmet Koc

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Chapter 4 Living long or dying young in plants and animals: Ecological patterns and evolutionary processes Renee M. Borges

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Chapter 5 Long living plants as longevity models and sources of anti-aging medicines Abdullah Olgun

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Chapter 6 Plant seed: A relevant model to study aging processes Erwann Arc, Laurent OgĂŠ, Philippe Grappin and LoĂŻc Rajjou

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Chapter 7 Gerontology in Russia: Past, present and future Vladimir N. Anisimov and Olga N. Mikhailova Chapter 8 A short look at aging, anti-aging, geriatry and death in Turkish history before the 19th century Nil Sar覺

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The Field of Biological Aging: Past, Present and Future, 2011: 1-15 ISBN: 978-81-7895-513-1 Editor: Abdullah Olgun

1. Biological principles of aging and approaches for interventions Suresh I. S. Rattan Laboratory of Cellular Ageing, Department of Molecular Biology, Aarhus University Gustav Wieds Vej 10; DK8000 Aarhus-C; Denmark

Abstract. Aging can be understood at various levels, from evolutionary and biological to psychological and sociological levels. At the biological and molecular levels aging is characterized by the stochastic occurrence and accumulation of molecular damage. Damages in the maintenance and repair pathways comprising homeodynamic space lead to age-related failure of homeodynamics, increased molecular heterogeneity, altered cellular functioning, reduced stress tolerance, increased probability of diseases and ultimate death. Novel approaches for testing and developing effective means of intervention, prevention and modulation of aging incorporate means to minimize the occurrence and accumulation of molecular damage. Mild stress-induced hormesis by physical, biological and nutritional methods, including hormetins, is a promising strategy for achieving healthy aging and for preventing age-related diseases.

Introduction Two of the pioneers of modern biogerontology, Leonard Hayflick and Robin Holliday, have declared that aging is no longer an unsolved problem in Correspondence/Reprint request: Dr. Suresh Rattan, Laboratory of Cellular Ageing, Department of Molecular Biology, Aarhus University, Gustav Wieds Vej 10; DK8000 Aarhus-C; Denmark. Email: rattan@mb.au.dk

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Table 1. Biological principles of aging and longevity derived from modern biogerontological research.

1.

2.

3.

4.

Evolutionary life history principle: Aging is an emergent phenomenon seen primarily in protected environments, which allow survival beyond the natural lifespan of a species, termed ‘essential lifespan’ (ELS), [3-5].

Non-genetic principle: There is no fixed and rigid genetic program, which determines the exact duration of survival of an organism, and there are no real gerontogenes whose sole function is to cause aging and to determine precisely the lifespan of an organism.

Differential principle: The progression and rate of aging is different in different species, organisms within a species, organs and tissues within an organism, cell types within a tissue, sub-cellular compartments within a cell type, and macromolecules within a cell.

Molecular mechanistic principle: Aging is characterized by a stochastic occurrence, accumulation and heterogeneity of damage in macro-molecules, leading to the shrinkage of the homeodynamic space and the failure of maintenance and repair pathways.

biology [1, 2]. The bold assertion by Hayflick and Holliday underlines the fact that biological basis of aging is well understood and a distinctive framework has been established, based on which general principles of aging and longevity can be formulated, and can be the basis for developing interventions towards achieving a healthy old age. These biological principles of aging and longevity are summarized in Table 1.

Thus, aging is an emergent, epigenetic and a meta-phenomenon, which is not controlled by a single mechanism. Although individually no tissue, organ or system becomes functionally exhausted even in very old organisms, it is their combined interaction and interdependence that determines the survival of the whole. The evidence that genes have a limited (about 25%) influence upon lifespan in human beings has mainly come from the studies performed on centenarians and their siblings, twins and long living families. The value of the genetic determinant of longevity was calculated from studies on Danish twins, and it was shown that the heritability of longevity in men and women was 0.26 and 0.23, respectively [6]. A combination of genes, milieu and chance determine the course and consequences of aging and the duration of survival of an individual [7].

Homeodynamic space and its shrinkage Survival and longevity are a function of the ability of various maintenance and repair mechanisms to keep up with damage and wear-and-tear. All living

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systems have the intrinsic ability to respond, to counteract and to adapt to the external and internal sources of disturbance. The traditional conceptual model to describe this property is homeostasis, which has dominated biology, physiology and medicine since 1930s. However, advances made in our understanding of the processes of biological growth, development, maturation, reproduction, and finally, of aging, senescence and death have led to the realization that homeostasis model as an explanation is seriously incomplete. The main reason for the incompleteness of the homeostasis model is its defining principle of “stability through constancy”, which does not take into account the new themes, such as cybernetics, control theory, catastrophe theory, chaos theory, information and interaction networks, which comprise and underline the modern biology of complexity [8]. Since 1990s, the term homeodynamics is being increasingly used to account for the fact that the internal milieu of complex biological systems is not permanently fixed, is not at equilibrium, and is a dynamic regulation and interaction among various levels of organization [9].

Aging, senescence and death are the final manifestations of unsuccessful homeostasis or failure of homeodynamics [10, 11]. A wide range of molecular, cellular and physiological pathways of repair are well known, and these range from multiple pathways of nuclear and mitochondrial DNA repair to free radical counteracting mechanisms, protein turnover and repair, detoxification mechanisms, and other processes including immune- and stress-responses. All these processes involve numerous genes whose products and their interactions give rise to a “homeodynamic space” or the “buffering capacity”, which is the ultimate determinant of an individual’s chance and ability to survive and maintain a healthy state [10, 11]. A progressive shrinking of the homeodynamic space is the hallmark of aging and the cause of origin of age-related diseases.

In a normal, healthy and young individual, the complex network of maintenance and repair systems (MARS) constitute a functional homeodynamic space. Since no MARS can be 100% perfect 100% of the time, there is a probability of imperfect homeodynamics giving rise to a zone of vulnerability, manifested in age-independent diseases and mortality.

However, a progressive accumulation of molecular damage and its effects on the interacting molecular networks leads to the reduction in the functional homeodynamic space, and effectively increases the vulnerability zone, and thus allows for the occurrence and emergence of age-related diseases.

Alzheimer’s disease, cancer, cataract, diabetes-2, osteoporosis, Parkinson’s disease, sarcopenia and other age-related diseases are the result of reduced homeodynamic space of the individual.

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Molecular theories of aging At the molecular level, aging is best characterized by the accumulation of molecular damage. There are three major sources of damages within a cell: (1) Reactive oxygen species (ROS) and free radicals (FR) formed due to external inducers of damage (for example ultra-violet rays), and as a consequence of cellular metabolism involving oxygen, metals and other metabolites; (2) Nutritional glucose and its metabolites, and their biochemical interactions with ROS; and (3) Spontaneous errors in biochemical processes, such as DNA duplication, transcription, post-transcriptional processing, translation, and posttranslational modifications.

The so-called mechanistic theories of biological aging have often focused on a single category of inducers of molecular damage as an explanation for possible mechanisms of aging. Of these, two theories, which have been the basis of most of the experimental biogerontology research, are the free radical- and the protein error-theory of aging. Although neither of them can be considered to be the complete theory of biological aging, their contributions in providing a solid scientific footing to experimental aging research and anti-aging interventions cannot be overestimated.

Free radical theory of aging (FRTA) FRTA, proposed in 1954, arose from a consideration of the aging phenomenon from the premise that a single common biochemical process may be responsible for the aging and death of all living beings (for an update, see [12]). There is abundant evidence to show that a variety of ROS and other FR are indeed involved in the occurrence of molecular damage, which can lead to structural and functional disorders, diseases and death. The chemistry and biochemistry of FR is very well worked out, and the cellular and organismic consequences are well documented [13]. However, the main criticisms raised against this theory are with respect to its lack of incorporation of the essential and beneficial role of FR in the normal functioning and survival of biological systems [14, 15]. Additionally, FRTA presents FR as the universal cause of damage without taking into account the differences in the wide range of FR-counteracting mechanisms in different species. Furthermore, a large body of data showing the contrary and/or lack of predictable and expected beneficial results of anti-oxidant and FR-

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scavenging therapies have restricted the FRTA to being only a partial explanation of some of the observed changes during aging [16-18].

Protein error theory of aging (PETA) The history of PETA, also known as the error catastrophe theory, is full of controversy and premature declaration of its demise [19-21]. Since the spontaneous error frequency in protein translation is generally several orders of magnitude higher than that in DNA replication and RNA transcription, the role of protein errors and their feedback in biochemical pathways has been considered to be a crucial one with respect to aging. Several attempts have been made to determine the accuracy of translation in cell-free extracts, and most of the studies show that there is an age-related increase in the misincorporation of nucleotides and amino acids [19-21]. It has also been shown that there is an age-related accumulation of aberrant DNA polymerases and other components of the transcriptional and translational machinery [19-24].

Further evidence in support of PETA comes from experiments which showed that an induction and increase in protein errors can accelerate aging in human cells and bacteria [19-21, 25, 26]. Similarly, an increase in the accuracy of protein synthesis can slow aging and increase the lifespan in fungi [27-29]. Therefore, it is not ruled out that several kinds of errors in various components of protein synthetic machinery, including tRNA charging, and in mitochondria do have long-term effects on cellular stability and survival [30-33]. However, almost all these methods have relied on indirect in vitro assays, and so far direct, realistic and accurate estimates of age-related changes in errors in cytoplasmic and mitochondrial proteins, and their biological relevance, have not been made. Similarly, applying methods such as two-dimensional gel electrophoresis, which can resolve only some kinds of mis-incorporations, have so far remained insensitive and inconclusive [19-21]. It will be necessary to combine several methods, such as electrophoresis, mass-spectrometry, protein-protein interactions and antibody-based detection of molecular heterogeneity to find out the extent of protein errors and their biological role in aging.

From FRTA and PETA to higher order theories Both the FRTA and PETA provide molecular mechanisms for the occurrence of molecular damage. Additionally, nutritional components, specially the sugars and metal-based micronutrients can induce, enhance and amplify the molecular damage either independently or in combination with other inducers of damage. The biological consequences of increased levels of

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molecular damage are wide ranging and include altered gene expression, genomic instability, mutations, molecular heterogeneity, loss of cell division potential, cell death, impaired intercellular communication, tissue disorganization, organ dysfunctions, and increased vulnerability to stress and other sources of disturbance. Historically, each of these biological consequences has been used as the basis of putting forward other theories of aging, such as replicative senescence theory, neuroendocrine theory, pineal gland theory, immunological theory and many more [5, 15, 34].

Genetics, post-genetics and epigenetics of aging Since all molecular processes in a living system are based in and regulated by genes, an attractive research strategy has been to discover genes for aging, termed gerontogenes [35-37]. However, the evolutionary explanation for the origin of aging, and limited lifespan discussed above, have generally ruled out the notion of any specific genetic program involving specific gerontogenes. But a lack of specific gerontogenes with the sole purpose of causing aging and terminating the lifespan of an individual does not imply that genes do not or cannot influence survival, longevity and the rate of aging.

There is ample evidence from studies performed on yeast and other fungi, nematodes, insects, rodents and humans that mutations in various genes can either prolong or shorten the lifespan, and some of these are also the cause of premature aging syndromes in human beings [38-40].

Additionally, genetic linkage studies for longevity in mice have identified major histocompatibility complex regions and quantitative trait loci on several chromosomes as putative genes for aging. In gene association studies with human centenarians, certain alleles of HLA locus on chromosome 6, regions of chromosome 4, different alleles of APO-E and APO-B, and DD genotype of angiotensin converting enzyme (ACE) have been linked to exceptional longevity. Similarly, several other studies have reported an association between human longevity and single nucleotide polymorphisms (SNP) in a variety of genes in various biological pathways, including heat shock response, mitochondrial functions, immune response, cholesterol metabolism and others [40-46].

An analysis of the various functions of the genes associated with aging and longevity shows that these genes cover a wide range of biochemical pathways, such as energy metabolism, kinases, kinase receptors, transcription factors, DNA helicases, membrane glucosidases, GTP-binding protein coupled receptors, chaperones, and cell cycle check point pathways [40, 46].

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What is clear from the identification of the genes influencing aging and longevity is that whatever their normal function and mechanism of action may be, these gerontogenes did not evolve to cause and accumulate molecular damage, to cause functional disorders, and to terminate the life of the organism.

Most of these genes have well defined roles in normal metabolism, in intra- and inter-cellular signaling, and in maintenance and repair functions including stress response. It is the damage-induced changes in the regulation, structure and/or activity of their gene products, which result in their altered biological role with age. Therefore, such genes have been termed “virtual gerontogenes” [36, 47]. Furthermore, a lack of evolutionary selection of virtual gerontogenes has given rise to the notion of post-genetics or “postreproductive genetics” as an explanation for different biological roles played at different ages by the same genetic variants [48].

Epigenetics of aging Although genes are the foundation of life, genes in themselves are non functional entities. It is the wide variety of gene products, including coding and non-coding RNAs, proteins and other macromolecules, which constitute the biochemical and biophysical milieu for life to exist. “Epigenetics” is the most commonly used term to account for and to explain the consequences of the intracellular and extracellular milieu, which establish and influence the structural and functional stability of genes. These epigenetic effects and alterations generally remain uninherited from one generation to the next, but have strong deterministic effects on the health, survival and aging of the individual.

So far, there is only scanty information available about the involvement in aging of various intracellular epigenetic markers such as methylated cytosines, oxidatively modified nucleotides, alternatively spliced RNAs, and post-translationally modified proteins, including protein folding [49]. The full spectrum of epigenetics of aging is yet to be unraveled and at present is one of the most attractive and challenging areas of research in biogerontology [50, 51]. A major reason for apparent difficulties in fully understanding the epigenetics of aging is the existence of several orders higher complexity and diversity of the constituting components, such as physical, chemical, biological and environmental factors, including psychological factors in human beings. Furthermore, in order to understand how various conditions influence, regulate and modulate the actions, interactions and networks of gene products during aging will require new intellectual and technical tools,

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such as systems analysis, bioinformatics, and functional genomics involving simultaneous multiple analyses.

From understanding to intervention As a biomedical issue, the biological process of ageing underlies all major human diseases. Although the optimal treatment of each and every disease, irrespective of age, is a social and moral necessity, preventing the onset of age-related diseases by intervening in the basic process of ageing is the best solution for improving the quality of human life in old age.

According to the three principles of ageing and longevity described above, having the bodies that we have developed after millions of years of evolution, occurrence of ageing in the period beyond ELS, and the onset of one or more diseases before eventual death, appear to be the normal sequence of events.

This viewpoint makes modulation of ageing by prevention very much different from the treatment of a specific disease.

Scientific and rational anti-ageing strategies aim to slow down ageing, to prevent or delay the physiological decline, and to regain lost functional abilities. In order to modulate ageing for achieving healthy old age and for extending lifespan, three main conditions need to be fulfilled, as represented by the equation E = GMC2, where genes and milieu are the critical factors [7].

Gene therapy for aging One of the earliest experimental studies which demonstrated that an induced mutation in a single gene can increase the lifespan of an organism was the discovery of the so-called age-1 mutant in the nematode Caenorhabditis elegans [52, 53]. Since then hundreds of putative gerontogenes or longevity genes have been reported in C. elegans, Drosophila and rodents, which when mutated result in the extension of average and maximum lifespan of the organism. The methods used for the identification of such genes include induction of mutations and deletions by irradiation and chemical mutagens, alterations in gene expression by knockout, homologus recombination, or by gene addition, and reduction in gene expression by RNAi-induced abrogation of translation (for the latest information on such genes, refer to various online databases, such as: http://genomics.senescence.info/genes/longevity.html, http://wormbase.org/db/misc/site_map?format=searches, http://sageke.sciencemag.org/index.dtl It is important to realize that in almost all such cases longevity extension had occurred when one or multiple interventions resulted in the reduction or

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total inhibition of the activity of one or more genes [54]. Some of the main pathways whose “loss of function� is shown to associate with extended period of survival are: (i) energy generation and utilization in mitochondrial respiratory chain; (ii) nutrition and hormonal sensing and signalling including insulin/insulin-like growth factor-1 and its target forkhead transcription factor FOXO, transcriptional silencing by sirtuin-mediated histone deacetylase; and (iii) translational interference through target of rapamycin (TOR). Similarly, there are other examples which show that several mutant mice strains with defects in growth hormone (GH) pathways including deficiencies of GH levels and GH receptor have extended lifespans. Application of RNAi technology will further identify numerous genes whose normal levels of activities are lifespan restricting [54].

In contrast to the above, studies have also been performed in which the effects of adding one or multiple copies of various genes leading to the increased expression of their gene products has resulted in the extension of lifespan. Some such transgenic manipulations in model systems include the addition of gene(s) for one of the protein elongation factors, antioxidant genes superoxide dismutase and catalase, sirtuin, forkhead trascription factor FOXO, heat shock proteins (Hsp), heat shock factor (HSF), protein repair methyltransferase and klotho, which is an inhibitor of insulin and IGF1 signalling [54].

Although theses studies have demonstrated longevity-extending effects of various genes in controlled laboratory conditions, there is very little information available on the basic process of ageing in terms of the rate and extent of occurrence and accumulation of macromolecular damage and its physiological consequences in these animals. There is also almost no information available as to what is the physiological price paid for inactivating such genes whose normal function is a part of the general metabolism and signaling. There is some evidence that laboratory-protected longevity mutants in C. elegans have reduced Darwinian fitness when competing with the wild type worms under nutritionally challenging conditions [55-58]. Similarly, klotho-induced insulin resistance and the paradox of the insulin/IGF-1 signalling pathways in longevity extension seriously question the practicality of such gene manipulations in humans [58-60].

Another system in which genetic interventions have been tried as potential anti-ageing therapies is the Hayflick system of limited proliferative lifespan of normal diploid differentiated cells in culture [61]. Almost all the genetic interventions by transient or permanent transfection and ectopic expression of various genes on this model system have focused on extending the replicative lifespan of cells by bypassing the cell cycle check-points [62, 63]. One of the most widely used genetic interventions in extending

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indefinitely the replicative lifespan of normal cells has been the ectopic expression of telomerase in a wide variety of cells [64, 65]. However, continuous proliferation by such genetically modified non-ageing cells often leads to their genomic instability, transformation and cancer-forming activity [66, 67]. In the case of animals, whereas telomerase negative mice show reduced lifespan and some other abnormalities after six-generations, [68] overexpression of telomerase in the skin increases myc-induced hyperplasia [69] without any extension of lifespan.

Considering that the molecular cause of ageing is the progressive accumulation of macromolecular damage and increased molecular heterogeneity [70], there are at least three major targets for anti-ageing genetic interventions: (1) increasing the repair of damaged macromolecules, for example DNA repair pathways; (2) increasing the removal of damaged macromolecules, for example proteasomal and lysosomal pathways; and (3) decreasing the source of damaging agents, for example reactive oxygen species, other free radicals, and reactive sugar metabolites. Whereas the first two targets basically imply achieving genetic enhancement or genetic improvement, the third target requires resetting the metabolic pathways.

All of the above targets for anti-ageing interventions involve hundreds of genes and gene products, whose expression and action are evolutionarily highly regulated in a cell-type-specific manner. Although there are several approaches in development for gene-based enhancement of physical strength, endurance, appearance and memory, there are serious technical limitations and ethical and safety concerns that remain to be resolved. Preventing or treating one or more age-related diseases by gene therapy, including stem cells, are at best the piecemeal treatments which often are temporary or become unsuccessful since these are overshadowed by the systemic ageing of the whole body. Ideally, gene therapy for the process of ageing requires a significant and “intelligent” redesigning already at the level of the zygote for better maintenance and survival of the body without having to trade-off with growth, development and reproduction. Chances of such an “intelligently redesigned” and directed evolution to succeed in competition with the Darwinian natural selection from much larger random variations and combinations are practically none.

Manipulating the milieu The second parameter M in the equation E = GMC2 represents milieu – the environment in which living systems operate and survive. The milieu in which genes and gene products function ranges from the intracellular

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molecular and ionic milieu to all other levels of organization including cellular, physiological, psychological, and societal. Almost all the ongoing work on aging modulation and intervention at present is aimed at modifying the milieu by either replenishing those enzymes, hormones and other molecules, such as antioxidants and micronutrients, whose levels are reported to decrease during aging. Although some of these approaches have been shown to have some clinical benefits in the treatment of some diseases in the elderly, none of these really modulate the aging process itself.

A critical component of the homeodynamic property of living systems is their capacity to respond to stress. In this context, the term “stress� is defined as a signal generated by any physical, chemical or biological factor (stressor), which in a living system initiates a series of events in order to counteract, adapt and survive. A successful and over-compensatory response to low doses of stressors improves the overall homeodynamics of cells and organisms. This approach for the strengthening of homeodynmaics through mild stress is known as hormesis [71]. Hormesis in ageing is defined as the life supporting beneficial effects resulting from the cellular responses to single or multiple rounds of mild stress. Various mild stresses that have been reported to delay ageing and prolong longevity in cells and animals include temperature shock, irradiation, heavy metals, pro-oxidants, acetaldehyde, alcohols, hypergravity, exercise and food restriction [72]. All such compounds which bring about biologically beneficial effects by causing mild stress and thus stimulating defense pathways are termed as hormetins [71].

Components of various medicinal plants, such as Aswagandha, used frequently in the Indian Ayurvedic system of medicine for potential antiaging effects, appear to be potential hormetins.

Aging modulatory effects of hormesis have been reported for various human cell types in vitro. For example, using a regimen of repeated mild heat shock at 41°C, for 1 hr twice a week, given to cultured normal human skin fibroblasts, keratinocytes, endothelial cells, and telomerase-immortalised bone marrow mesenchymal stem cells, a variety of hormetic effects have been reported. These effects include slowing down of cellular aging, some extension of replicative lifespan, maintenance of youthful morphology, reduction in the levels of oxidatively- and glycoxidatively-damgaed proteins, stimulation of proteasomal activities, increased levels of chaperones, enhancement of stress tolerance, and improvement in differentiation, wound healing and angiogenesis [73]. Other hormetic conditions, which have been shown to have some anti-aging effects in human cells, are irradiation, mechanical stretching and electromagnetic field shock [74-76]. Thus, the proof of the principle regarding the applicability of hormesis as a modulator of aging in human cells is well demonstrated. However, further short term

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and long term studies, using a wide variety of human cell types, and a combination of stressors, are required in order to establish the universality of the phenomenological and mechanistic aspects of this issue.

However, not all pathways of stress response respond to every stressor, and although there may be some overlap, generally these pathways are quite specific. The specificity of the response is mostly determined by the nature of the damage induced by the stressor and the variety of downstream effectors involved. Yet, the major pathways of stress response can be used as the screening platform for discovering, testing and monitoring the effects of novel hormetins. Such hormetins may be categorized as: (1) physical hormetins, such as exercise, heat and radiation; (2) biological and nutritional hormetins, such as infections, micronutrients, spices and other sources; and (3) psychological hormetins, such as mental challenge and focused attention or meditation. Hormesis may also be an explanation for the health beneficial effects of numerous other foods and food components, such as garlic, Gingko, and other fruits and vegetables [77-81]. Understanding the hormetic and interactive mode of action of natural and processed foods is a challenging field of research, and has great potential in developing nutritional and other life style modifications for aging intervention and therapies. For example, it may be possible to develop multi-hormetin formulations as anti-aging drugs and nutriceuticals whose mode of action is through hormetic pathways by mild stress-induced stimulation of homeodynamic processes.

Finally, while the G and M components of the E = GMC2 formula for eternal life are being taken care of by various experimental approaches, the third factor C represents chance, which is the probability of stochastic events leading to a cascade of error-catastrophe in complex interacting systems.

Recent developments in our understanding of complex networks at all levels of organisation from molecular to societal and global networks have highlighted the vulnerability of all strong and weak links, and has reasserted the significance of chance events which are not amenable to regulation and manipulation. In the context of modulating aging, repeated mild stressinduced hormesis increases the boundaries of the homeodynamic space thus giving cells and organisms wider margins for metabolic fluctuation and adaptation. Slowing down the shrinkage of the homeodynamic space hormetically will reduce the probability of occurrence and emergence of various diseases in old age, and thus extend the health-span.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

The Field of Biological Aging: Past, Present and Future, 2011: 17-37 ISBN: 978-81-7895-513-1 Editor: Abdullah Olgun

2. Studies of in vitro cellular senescence Olivia M. Pereira-Smith and Kaoru Tominaga

Sam and Ann Barshop Institute for Longevity and Aging Studies and Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA

Abstract. We have attempted to survey the literature on cellular senescence since its inception as a model system for aging in vitro and in vivo. We have presented data that are not known and their impact not recognized by individuals new to the field, because of PUBMED as a source of information. The area of cell senescence has come a long way from the initial proposal by Hayflick. It is clear that there is a strong genetic component to the program, and rather than random damage it is epigenetic events that are thought to be involved in the entry to senescence. We have referenced Bruce Howard in our Concluding Remarks, as he was the first individual to propose an epigenetic aspect to cellular senescence and this hypothesis, made many years ago, is now coming to fruition. It behooves all scientists to pay attention to previous literature when they first enter a field, and thereby not address old questions as new ones.

Introduction Aging is a complex process involving changes in cells and tissues thereby affecting organ function and the appearance of the aged phenotype.

Correspondence/Reprint request: Prof. Olivia M. Pereira-Smith, Department of Cellular and Structural Biology, Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, STCBM Building 15355 Lambda Drive, San Antonio, TX 78245-3207, USA. E-mail: smitho@uthscsa.edu

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To understand human aging the best approach would be through a longitudinal study such as the one being conducted at the Shock Center on Aging at Baltimore. However, studies with human subjects are complicated by the genetic heterogeneity of the population under study and therefore interpretation of results, besides being very expensive. It is for these reasons that scientists have looked for alternate model systems that now include cells, yeast, nematodes, flies, rodents, and monkeys. Each has made contributions to an understanding of important genes and gene pathways involved in aging related processes, as well as other areas of research.

The model system of cell senescence was introduced by Hayflick in 1961 [1, 2]. He described the fact that normal cells undergo limited replication before entering an irreversible non-dividing state, that he termed senescence.

His report was met with resistance because Alexis Carrel, a prominent biologist, had been touting the fact that cells grown in culture were able to divide indefinitely, using a chick heart cell system. This was most likely an artifact of cell culture conditions as a preparation of chick embryo extract was used to maintain the cultures [2]. However, since the first publication by Hayflick, the phenomenon of cellular or replicative senescence has been reproduced in many laboratories and observed with many cell types, including fibroblasts, endothelial cells, T lymphocytes, adrenocortical cells, melanocytes, keratinocytes, and breast epithelial cells. It is now accepted as a model for aging at the cellular level.

Early studies In the initial period following the description of the phenomenon of replicative senescence, a number of studies comparing young with senescent cells were performed and a series of differences described [3]. A key observation made by Dell’Orco et al. [4] was that cellular senescence was directly related to the number of population doublings (PD) achieved and not a consequence of time in culture. This was demonstrated by arresting normal human fibroblasts in the quiescent, non-reversible, non-dividing state by removal of growth factors, maintaining the cells in this non-dividing state for varying periods of time and then returning them to serial subculture. All cultures achieved close to the same final number of PD irrespective of the time in growth arrest. This result was also demonstrated using chick embryo fibroblasts [5].

Another key observation was the demonstration of clonal heterogeneity in cultures of normal human fibroblasts. The number of PD that the individual cells in the culture can undergo is highly variable, with as many as 50% of the cells capable of very limited division potential [6]. This

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heterogeneity develops very rapidly as even a clone of cells, when analyzed for the division potential of individual cells, has developed extensive heterogeneity within the 20 PD that it takes to obtain 1 million cells from a single cell [7, 8]. Analysis of the doubling potential of the two daughter cells derived from a single cell division revealed that they could have very different division potentials, with the difference increasing as the culture neared the end of in vitro lifespan [7]. These data have been analyzed extensively by mathematical modeling and led to the thinking that there is an internal “clock� mechanism that defines the number of PD a culture can undergo, but superimposed on it are stochastic events that lead to a sudden loss of proliferative ability [9].

Genetic analyses provide evidence for cellular senescence as a tumor suppressor mechanism Cell fusion studies of young and senescent cells demonstrated that hybrids were similar to the senescent population indicating the phenotype of cell senescence was dominant [10]. Furthermore, fusion of various normal human cells (fibroblasts, T cells and endothelial cells) with immortal human cells produced hybrids that regained growth control indicating immortality was recessive and most likely due to loss of cell senescence related genes/gene pathways [11-13]. These data also supported the idea of cell senescence as a tumor suppression mechanism. It should be emphasized that unlike stress induced senescence the hybrids achieved various numbers of population doublings before they stopped dividing, and did not enter immediate growth arrest. Four such genes/gene pathways were identified by fusion of immortal cell lines with each other and their assignment to four distinct complementation groups for indefinite division [14, 15]. Micro cell mediated chromosome transfer of normal human chromosomes to cell lines assigned to groups B (chromosome 4) [16], C (chromosome 1) [17] and D (chromosome 7) [18] indicated a very specific senescence induction effect by the respective chromosome on cell lines assigned to one group, and more importantly, no effect on cell lines assigned to the other complementation groups. The chromosome 4 story has been continued and resulted in the identification of a novel family of transcription regulators [19], that are also involved in chromatin modification and epigenetic effects, discussed in more detail below.

Studies of heterokaryons and cybrids, rather than hybrids, revealed a contribution of both cytoplasm and nucleus to involvement in the cell senescence program [20-22].

All these data provided strong evidence for a genetic component to a program leading to cell senescence.

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Cell cycle genes and cell senescence Since cellular senescence is a terminally non-dividing state, loss of response of genes involved in cell cycle is an expected outcome. Thus, in senescent human diploid fibroblasts c-fos [23], cdc 2, cyclin A and cyclin B [24] are not expressed in response to serum mitogens and most likely contribute to the non-dividing state. In contrast, quiescent young cells, in which expression of these genes are low, respond by expressing all these genes following mitogen stimulation, and re-entering the cell cycle.

Similarly, senescent human fibroblasts were found to not phosphorylate Rb in response to serum stimulation [25]. In 1991 Noda et al. [26] cloned three novel inhibitors of DNA synthesis from senescent cells by an expression screen, including SDI 1 (senescent derived inhibitor 1), which was later found to be the cyclin dependent kinase inhibitor p21/Cip 1/ Waf 1 [27-30]. p21 expression remains high in senescent cells, though levels decrease in young, quiescent cells following serum stimulation [26]. It is considered a key negative regulator for senescence in fibroblast and adrenal cells [26, 31, 32]. p16 (another cdk inhibitor) has been found to accumulate in some fibroblast cells held in the senescent state, and is most likely, in this case, serving as a back up negative cell cycle regulatory mechanism to p21 [33, 34]. However, such changes do not occur in a similar manner in all cell types.

For example, fibroblasts derived from individuals with Werner’s Syndrome respond to mitogen stimulation by increasing expression of c-fos, however, other regulatory processes must be in place which causes these cells to senesce very rapidly [35]. In the case of melanocytes the key negative regulator of cell cycle and maintenance of senescence is p16 [36].

However, despite the fact that different cell types utilize different genes/pathways to arrive at senescence, the final phenotype remains the same- a terminal loss of proliferation and an inability to successfully complete another cell cycle. Viral proteins such as SV40 T antigen or papilloma virus E6 and E7can induce senescent cells to enter DNA synthesis as measured by BrdU uptake, but the cells never complete mitosis [37, 38].

This suggests senescent cells have yet another mechanism to hold cells in growth arrest, and this involves the G2/M transition.

Telomeres, replicative and stress induced premature senescence (sips) The first report, that average telomere length declined over the in vitro lifespan of fibroblast cultures appeared in 1990 [39]. This result was then

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extended to other cell types in culture, and in vivo [40]. Though purely correlative these data led to the hypothesis that the shortened telomere was sensed as DNA damage by the cell resulting in activation of the check point of p53 and consequently p21 expression [41]. Another possibility raised was that telomere shortening resulted in silencing of genes near the telomere, as had been observed in yeast [42]. However, neither has been demonstrated to be the case in mammalian cells and it remains largely hypothetical at this point. One aspect of telomere shortening that may be valid is that it is the internal “clock” of normal human cells that defines the number of PD a culture can achieve. Telomerase, the enzyme that maintains telomere length in most cancer derived cell lines and tumors was thought to be expressed only in normal germ line cells. However, other studies have shown that it is transiently expressed in normal T cells as they respond to mitogenic signals and in hematopoietic cells [43, 44]. Additionally, normal human fibroblast cells could be immortalized by the catalytic subunit of telomerase, hTERT, only when they were pre-senescent and in fact, only a sub population was immortalized [45], similar to that observed following the introduction of viral genes such as SV40 T antigen [46]. This indicates that the negative regulatory, dominant genes of senescence are not easily over-ridden.

Furthermore, tumor cells do not always utilize telomerase to acquire the immortal phenotype, but instead choose the alternative lengthening of telomeres (ALT) pathway [47, 48]. It is of interest that analysis of complementing hybrids from fusions of cell lines from the different complementation groups, as well as microcell fusion products, often expressed high levels of telomerase as they ceased proliferation (PereiraSmith, unpublished data) [49]. These data suggest that an over simplified telomere length loss based hypothesis, with no clear mechanism of action between telomere shortening and entry into senescence, is premature. There is now some addendum to the hypothesis suggesting capping and uncapping at the telomere and t-loop formation are the critical events that can cause sudden exit from the cell cycle [50], but what this does not address is the numerous changes in gene expression that occur as cells enter senescence, including the secretory ”inflammatory” repertoire of genes [51-53]. The latter have been suggested to have a negative aspect on surrounding tissue, making it more prone to tumorigenesis in vivo, but as recently discussed [54] it is possible that this provokes clearance of senescent cells by the innate immune system. A more reasonable explanation for the numerous changes in gene expression observed would be epigenetic events that affect chromatin and thereby gene expression, though the details remain to be determined.

Another new development has been the induction of “senescence” by various forms of stress, rather than subculturing cell cultures routinely until

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they cease proliferating, in the classic Hayflick method of replicative senescence. Treatments with agents such as hydrogen peroxide, ceramide and bleomycin, or non-physiologically high over expression of oncogenes, results in cell withdrawal from proliferation and expression of senescence associated beta-galactosidase activity with changes in morphology [55]. The similarity to replicative senescence is not totally clear and indeed microarray analyses have indicated that the gene expression changes are not the same. However, relevant to the discussion on telomeres, is that these induced forms of growth arrest occur independent of telomere shortening. There are arguments regarding the greater importance of such a phenotype as most likely to be the predominant withdrawal from the cell cycle in vivo than replicative senescence. This is valid because cells in vivo are exposed to many stresses and the over expression of oncogenes, which could lead to tumor formation, resulting in cell senescence, would serve as a tumor suppressing mechanism.

In the case of HIV infection and T lymphocyes, it is clear that replicative senescence occurs because of over proliferation of T cells [56]. However, if one considers the “stem/progenitor “cell compartment of various tissues, these can also contribute to functional loss with aging observed in vivo as a result of deficits in proliferation, and indeed there are studies describing such changes in various stem cell compartments that are impacted by aging.

Epigenetics and cellular senescence DNA methylation About 3-6% of DNA in normal cells is methylated at cytosine residues [57-59]. This methylation is involved in gene silencing of satellite DNAs and imprinted genes. It is known that DNA methylation decreases globally during cellular senescence and organismal aging [60]. Global hypomethylation of the genome during aging may contribute to the dysregulation of tissue homeostasis or cell growth control. DNA methyltransferase activity in normal human fibroblasts decreases during replicative senescence and this correlates well with global hypomethylation of the genome. Inhibition of DNA methyltransferase by 5-aza-2-deoxycytidine (CdR) in young normal human fibroblasts results in growth suppression through induction of expression of p21, the cyclin kinase inhibitor which is an important regulator of cellular senescence [61]. Interestingly, CdR treatment cannot induce growth suppression in p21 deficient fibroblasts. Moreover, telomerase expression in normal human fibroblasts stabilizes DNA methyltransferase activity [62]. This suggests that global hypomethylation of the genome contributes to the induction of cellular senescence through p21 expression,

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although the detailed molecular mechanisms remain unknown. Interestingly, disruption of PASG (proliferation associated SNF-2-like gene) in mouse, which facilitates DNA methylation in mice, also causes global DNA hypomethylation and a premature aging phenotype [63]. PASG mutant mice and fibroblasts derived from their tissues show increased expression of senescence-associated genes such as p16 and p21. This suggests proper maintenance of DNA methylation is important for aging and longevity and changes in epigenetic patterns contribute to aging at the cellular and organismal level.

In addition to global DNA hypomethylation, DNA hypermethylation of some specific genes during aging have been described [64].

Hypermethylation occurs at the CpG island in the promoters of the estrogen receptor (ER) [65], myogenic differentiation antigen (MyoD), tumorsuppressor candidate 33 (N33) [66], and insulin-like growth factor II (IGF2) [67]. Although the physiological meaning of this hypermethylation is unclear, it might be related to declining homeostasis in tissues.

Senescence-associated heterochromatin foci (SAHF) formation A dramatic reorganization of chromatin structure occurs in cells as they enter senescence after either excessive rounds of division or over-expression of the activated Ras oncogene. Many cells acquire condensed chromatin spots, which are specialized facultative heterochromatin foci, in the nucleus.

These are referred to as senescence-associated heterochromatin foci (SAHF) and it is thought that formation of these foci results in silencing of proliferation related genes and thereby the growth arrest phenotype of senescent cells [68-70]. SAHF is enriched for markers of heterochromatin, such as heterochromatin protein 1 (HP1) and trimethylated histone H3 at lysine 9 (H3K9), which is recognized by HP1 [71]. Euchromatic markers, acetylated H3K9 and trimethylated H3K4, are excluded from these foci.

SAHF formation is largely dependent on the Rb/p16 pathway and knockdown of either gene results in reduced SAHF formation induced by over-expression of oncogenic Ras [72]. The hypothesis is that the active, hypophosphorylated form of Rb binds to E2F transcription factor sites in promoters of E2F target genes related to cell proliferation and inhibits transcription. Rb does this by recruiting the histone H3K9 methyltransferase SUV39H1. HP1 then binds to methylated histone H3K9, and accelerates SAHF formation. p16 activates Rb through inhibition of Cdk4 and 6, the kinases required for Rb inactivation. In fact, heterochromatin markers do accumulate on the promoters of some cell cycle related genes during cellular senescence.

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SAHF are also characterized by their depletion of linker histone H1 and enrichment in the HMGA proteins and the histone variant macroH2A. HMGA1 and HMGA2 belong to a family of non-histone chromatin proteins, the high-mobility group (HMG) family, and were identified through biochemical analysis of chromatin-associated proteins in senescent cells [73].

HMGA proteins are essential structural components of SAHF and involved in stable senescent arrest. Interestingly, linker histone H1 competes for DNA binding with HMGA proteins and is absent from chromatin in senescent cells [74]. HMGA proteins are related to cell proliferation and cell transformation.

Both genes are expressed in highly proliferating tissues in embryos and their expression is induced by serum stimulation in cells and down regulated following differentiation. Furthermore, HMGA transgenic mice develop tumors and gene amplification of HMGA is found in many human cancers.

From these observations, it is surprising that HMGA proteins are enriched in SAHF and contribute to senescence-associated cell growth arrest and suppression of transformation in fibroblasts. However, there are some reports suggesting that HMGA play a role as tumor suppressors. It could well be that the activity of these proteins vary depending on the other protein partners present in the nuclear protein complexes with which they associate.

MacroH2A is a member of a family of histone variants with three members, macroH2A1.1, macroH2A1.2, and macroH2A2. MacroH2A is an unusual histone variant in that it contains an N-terminal histone H2-like domain and a C-terminal large non-histone region known as the “macro domain�. Active gene promoters are depleted of macroH2A1, whereas the inactive X chromosome shows preferential enrichment of this protein along the chromosome. Additionally, macroH2A-containing chromatin is resistant to ATP-dependent chromatin remodeling proteins activity in vitro. These data suggest that macroH2A contributes to gene silencing. In fact, macroH2A1 deficient mice show increased expression of some genes, which are normally inactivated by enrichment of macroH2A1. Deposition of macroH2A in SAHF may therefore contribute to gene silencing by these foci [75]. The factors responsible for incorporation of macroH2A in SAHF are unknown, although ATP-dependent chromatin remodeling factors similar to other H2A variants may contribute to this process.

In addition to macroH2A, Adams and co-workers have found that the histone chaperones, Asf1a and HIRA, trigger chromosome condensation during SAHF formation in human fibroblasts [75, 76]. Ectopic expression of Asf1a or HIRA in primary human fibroblasts accelerates SAHF formation whereas shRNA-mediated knockdown of Asf1a inhibits SAHF formation triggered by oncogenic Ras. Formation of SAHF formation also requires interaction between Asf1a and histone H3.

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Polycomb Group proteins and silencing of p16INK4A/ARF allele Polycomb Group (PcG) proteins were originally identified in Dorosophila melanogaster as transcriptional repressors that participate in cell fate decision by repressing Hox gene expression in a body segment specific manner during embryogenesis [77]. It is now recognized that PcG proteins are a large family of chromatin associated proteins, highly conserved evolutionally from plants to humans, and involved in many cell memory processes [78-80]. PcG is comprised of structurally and functionally divergent proteins and forms multiple protein complexes of two basic types, Polycomb repressive complex 1 and 2 (PRC1 and PRC2). These complexes, in a coordinated manner, modify histone tails post-translationally and cause transcriptional repression of target genes by changing higher order chromatin structure. The PRC2 complex has three core components and is involved in the trimethylation of histone H3 on lysine 27 (H3K27me3), which is catalyzed by Ezh1 or 2 in the complex. The PRC1 complex is composed of four components; Polycomb (Pc), Posterior sex comb (Psc), Polyhomeotic (Ph), and Sex comb extra (Sce), in a stoichiometric manner. The chromodomain in the Pc proteins recognizes H3K27me3 which is catalyzed

by PRC2 and Ring1A or 1B protein in the Sce proteins catalyzes the monoubiquitination of histone H2A on lysine 119, thereby causing chromatin compaction and shut down of transcription of target genes. Multiple orthologs of PRC1 components exist and a combinational diversity of PRC1 complexes is expected and observed in mammalian systems. There are five Pc proteins (CBX2, CBX4, CBX6, CBX7 and CBX8), six Psc proteins (Bmi1, Mel18, MBLR, NSPC1, RNF159 and RNF3), three Ph proteins (HPH1, HPH2 and HPH3) and two Sce proteins (Ring1A and Ring1B).

Bmi1 was originally identified as a proto-oncogene that cooperates with c-Myc to promote the generation of mouse lymphoma. Loss of function of Bmi1 in mice results in neurological defects and severe proliferative defects in lymphoid cells [81]. Bmi1 deficient primary mouse embryonic fibroblasts (MEFs) are impaired in progression into the S phase of the cell cycle and undergo premature senescence because the expression of the tumor suppressors p16Ink4a and p19Arf, which are encoded by Ink4a locus [82], is increased [83]. This was the first evidence that PcG silences p16Ink4a and p19Arf expression and inhibits induction of cellular senescence. In normal human fibroblasts, Bmi1 has been shown to be downregulated during cellular senescence whereas over-expression of Bmi1 extends replicative lifespan consistent with suppression of p16Ink4a expression [84].

CBX7, was identified by a genetic screen for cDNAs that bypass replicative senescence of normal human prostate epithelial cells, and

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overproduction of CBX7 extends lifespan by downregulating expression p16Ink4a and Arf [85]. CBX7 expression is downregulated during replicative senescence and knockdown of CBX7 expression by shRNA induces senescence through Ink4a/Arf. This CBX7 function occurs independent of Bmi1, demonstrating the functional redundancy of PcG in mammalian systems. CBX8 is also involved in regulation of cell proliferation through direct binding to the Ink4a/Arf locus [86]. Ectopic expression of CBX8 leads to repression of the Ink4a/Arf locus and bypass of senescence. Mel18 null MEFs undergo premature senescence through the induction of ARF/p53/Ink4a. The Arf/p53 pathway seems to be more important in this case because deletion of either gene abrogates senescence in Mel18 null MEFs, despite the fact that Ink4a is still upregulated in these cells [87]. PcG and E2F3b seem to cooperatively repress Arf expression on the promoter.

Thus PcG can silence the Ink4a/Arf locus by multiple mechanisms [88].

There are several reports that indicate the importance of the PRC2 complex in repression of the Ink4a/Arf locus. Ezh2, which is a catalytic subunit of the PRC2 complex for H3K27me2/3, is downregulated in stress induced and replicative senescent cells at the Ink4a/Arf locus [89, 90]. This coincides with decreased levels of H3K27me3, displacement of Bmi1, and transcriptional activation of this locus. The H3K27me3 demethylase JMJD3/KDM6B acts in an opposing manner. JMJD3 is induced upon activation of the Ras signaling pathway, recruited to the Ink4a/Arf locus, and contributes to the transcriptional activation of Ink4a. Inhibition of JMJD3 expression in MEFs results in suppression of Ink4a and Arf expression and immortalization [91, 92]. Thus, the Ink4a/Arf locus is controlled by PcG complexes, to either repress or induce cellular senescence in response to intra- and extra-cellular signaling.

Histone acetyl transferase (HAT) and deacetylase (HDAC) complexes in regulation of cell senescence The MORF4/MRG novel transcriptional regulatory family was identified from studies of cell senescence [93]. As indicated in the section on Genetic analyses a normal human chromosome 4 was identified as having a senescence inducing effect on immortal human cell lines assigned to complementation group B [16]. A fragment of chromosome 4 was then isolated and found to have senescing inducing activity similar to the intact chromosome. This fragment was less than 1MB of DNA and had been transferred into the mouse A9 cell line. Alu PCR probes to this region of human DNA were generated and used on BAC filters from Human Genome systems and a BAC contig constructed [94]. The BACs were then used to

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probe cDNA filters and 7 cDNAs identified in the region. One was expressed only in placenta and eliminated from further analyses. The smallest genomic equivalent of the remaining cDNAs were then transfected into HeLa or T98G immortal cell lines assigning to complementation group B, and one was identified as inducing senescence in these cells but not cell lines assigned to the other complementation groups [19]. Sequencing of the genomic DNA and the cDNA identified on filters, revealed they were related but different genes.

MORF4 (mortality factor on human chromosome 4) was a truncated version of MRG15 (MORF4 related gene on human chromosome 15) and subsequently a total of 7 family members were identified and cloned. Of these, 3 were found to be expressed: MORF4, MRG15 and MRGX. They have a large region of common predicted protein sequence and have now been found to interact with many similar proteins in various nuclear protein complexes, including histone acetyl transferases and deacetylases. MRG15 is unique in that it has a chromodomain, similar to the MSL3 protein in Drosophila, and is highly conserved from yeast to humans [95-98]. MRGX evolved later and is only expressed in vertebrates [96, 99].

Unlike MORF4, MRG15 and X have proven to be positive regulators of cell proliferation [100], leading to the hypothesis that MORF4 acts by competing with these proteins in their complexes to disrupt/dysregulate their activity and inhibit proliferation, though action of MORF4 via its own unique complexes cannot be ruled out. The positive versus negative effects on proliferation of these proteins appears to be dependent on interaction with HATs or HDACs, respectively. Both MRG15 and X have been found in the Tip60 HAT [101-107] as well as mSin3/HDAC complexes [103, 106, 108, 109]. Analyses of neural stem/progenitor cells derived from whole brain of MRG15 null versus wild-type embryos indicate proliferative deficits as well as decreased ability to differentiate into neurons but not glial cells [110].

Relevant to some thoughts on cell senescence, besides a role in cell proliferation MRG15 has been shown to be required for repair of double strand break repair in Drosophila and MEFs derived from MRG15 null versus wild-type embryos [111, 112].

Both MRG15 and MRGX expression levels decline about 3 fold at the protein level in senescent versus young cells. Reintroduction of these genes into pre-senescent cells using adenovirus vectors increases BrdU incorporation and conversely, knock down by shRNAs in young cells causes decreased BrdU incorporation, consistent with their role in positive regulation of cell proliferation. Analyses with MRG15 null MEFs indicated they entered senescence before their wild-type counterparts and that this was accompanied by up regulation of p21 [100].

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Studies with MORF4 have been more difficult because it is so similar to MRG15 and because even modest over-expression is toxic to cells. We have generated an antibody specific to MORF4 but it does not have high affinity for the protein. However, we have developed a tetracycline inducible system using HeLa cells and reproduced the growth inhibitory effect of the protein, similar to what we had observed with an intact chromosome 4 and a fragment of the chromosome [113]. What this system allowed us to determine was that MORF4 was a very unstable protein, with a half life of less than 1 hour, and that it was degraded by the proteasome/ubiquitin pathway. This is most likely why we have had problems with detection of the endogenous protein.

However, consistent with our hypothesis regarding the action of MORF4 we have shown that a chromodomain minus mutant of MRG15 disrupts the MAF2 complex that involves MRG15 and the HAT hMOF [114].

Acquisition of better reagents and the use of the inducible system may help us eventually understand how and when MORF4 induces senescence in only immortal cell lines assigned to complementation group B. The inhibitor of growth (ING) family of proteins consists of five members with various isoforms and has features remarkably similar to the MORF/MRG gene family and raises the question of overlap in function in different cells and tissues [115, 116]. The major difference is that the ING proteins in cell senescence are different isoforms resulting from splicing, whereas the MORF/MRG genes are unique. Additionally, MRG15 has a chromodomain, whereas the primary motif of the ING1a and b proteins, that have been implicated in senescence are a plant homeodomain (PHD) finger region. However, interestingly the different families are involved in recognition of methylated histones, H3K4me3 in the case of the ING proteins and H3K36me2/3 by the MRG proteins. The ING1a isoform appears to be associated with cell senescence as RNA and protein levels increase in senescent versus young cells, whereas ING1b levels decrease [117]. ING1a also associates with an increased amount of HDAC1 in senescent cells though overall levels of HDAC1 are unchanged. Studies with the PCNA promoter demonstrated increased presence of ING1a protein and Rb at the promoter whereas H3 acetylation decreased. ING1a does not appear to act via p21 but rather affects the Rb, p16 pathway and promotes SAHF formation.

It is of interest that these gene families appear to utilize different pathways, ie p21 versus p16, to affect cell senescence. It may well be that they work in concert in all cells or that one or the other is involved in gene regulation in a specific cell type or tissue. In collaboration with N. Timchenko (Baylor College of Medicine) we analyzed nuclear extracts from young and old liver by size fractionation HPLC. The interesting result was a shift of MRG15 to higher molecular weight complexes with Rb and

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HDAC1 and 2 in old liver. MRG15 protein levels of MRG15 also declined with age in vivo (unpublished results).

The sirtuins are yet another family of deacetylases. They have been implicated in affecting lifespan in yeast and flies. Knockout of the various family members in mice have produced varied phenotypes and there is no clear relationship to cellular senescence. We therefore refer the reader to a recent review [118].

Concluding remarks All of the data we have compiled here demonstrate a genetic component to cellular senescence that is impacted by epigenetic mechanisms. Bruce Howard was the first to propose the epigenetic aspect of cell aging [119]. He developed a high throughput screening to demonstrate this, using cells derived from individuals of different age [120, 121]. The tools to perform epigenetic/chromatin modification analyses have been slow in coming and as they are developed we can move forward scientifically. The complexity of the system also confuses interpretation and results. However, one conclusion we can firmly draw is that in the almost 50 years since Hayflick reported the phenomenon of cell senescence, the field has moved forward rapidly to defining many of the basic molecular mechanisms. It has contributed not only to aging related science, but also to an understanding of cell cycle and transcriptional regulation, as well as the basic biology of DNA repair and tumor suppression/cancer: broad contributions that have been significant for cell biologists.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

The Field of Biological Aging: Past, Present and Future, 2011: 39-59 ISBN: 978-81-7895-513-1 Editor: Abdullah Olgun

3. Protein oxidation and repair mechanisms in aging Ayse Banu Demir and Ahmet Koc Izmir Institute of Technology, Department of Molecular Biology and Genetics, 35430, Urla, Izmir, Turkey

Abstract. The aerobic metabolism by-products have important effects on the redox homeostasis in a cell via the reactions they participate. Macromolecular damages are the most detrimental results of the oxidative stress. Protein oxidation is one these damages and is known to be involved in the aging process as well as many other diseases such as Alzheimer’s and Parkinson’s disease. Oxidative invasions on proteins result in either reversible or irreversible changes. However, both eukaryotic and prokaryotic cells have evolved repair mechanisms for these changes. For the reversible changes, different enzymatic repair systems cause proteins to switch into their normal functional forms and the irreversible changes are eliminated via different degradation pathways, which include proteosomes or proteases. In this part, these repair mechanisms of the oxidized proteins and their role in the aging process will be focused on in detail.

Introduction Aerobic cells, at a resting state, convert approximately 2% of the oxygen they consume into reactive oxygen species (ROS) [1, 2]. This amount is, Correspondence/Reprint request: Dr. Ayse Banu Demir, Izmir Institute of Technology, Department of Molecular Biology and Genetics, 35430, Urla, Izmir, Turkey. E-mail: aysedemir@iyte.edu.tr

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most likely, much higher in a cell that is in its active state [3]. Reactive oxygen species may act both as signaling molecules for the defense mechanisms against oxidative stress [2, 4, 5] up to a certain threshold level, and as mediators of the oxidative stress over this threshold level. Oxidative stress that results from the insufficient free radical defense systems of the cells is harmful, since it leads to subsequent macromolecular damages due to an excess of free radicals [6].

Proteins are among the mostly affected macromolecules from the oxidative stress probably due to their amount in a cell. The oxidative invasions on proteins lead to certain damages such as polypeptide chain fragmentation, aggregation or cross-linking, site-specific amino acid modifications, and alterations in protein surface properties [for detailed reviews see [7],[8],[86]]. Unless these modifications are repaired, they can lead to further cellular pathologies. Therefore, certain repair mechanisms have been evolved by the cells to prevent the alterations in cell functions.

Degradation systems, like proteosomes and lysosomal proteases, have been evolved to eliminate the irreversible changes on proteins (i.e. protein carbonylation, etc.) and enzymatic repair systems, involving the thioredoxin and the glutaredoxin systems, methionine sulfoxide reductases and sulfiredoxins, have been evolved for the repair of the reversible changes (i.e. formation of disulfide bridges, methionine sulfoxide reduction, cysteinsulfinic acids, etc).

Protein repair mechanisms are of great importance since oxidized proteins are involved in the aging process as well as in certain neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and arteriosclerosis. In this review, the protein repair mechanisms of the cells and their role in physiological aging process will be discussed.

Repair mechanisms of the irreversible protein modifications Various amino acid side chains and peptidic backbone are the main targets of oxidation by the free radicals, which are the by-products of the aerobic metabolism [9-11]. The free radical oxidation mainly results in loss of protein function [6, 12], which in turn can be threatening for the cell; especially in the case of proteins that are vital for the cell survival. Therefore, it is important for a cell to repair these oxidative modifications for a proper cell function and if cannot repair, to degrade and get rid of these nonfunctional proteins.

Among the irreversible protein modifications, the formation of carbonyls and hydroxyls take up an important place. Some of the irreversible

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modifications can be summarized as follows: Oxidation of poly-unsaturated fatty acids lead to aldehyde formation, which is one of the mostly seen carbonyl derivatives upon protein oxidation; Aromatic and aliphatic amino acid oxidation lead to hydroxyl derivatives and keto acid formation, respectively. Oxidative polypeptide chain fragmentation that results in ketoacyl (carbonyl) formation, nitration process and formation of crosslinked protein aggregates via covalent bond formation are also irreversible protein modifications upon oxidation [reviewed in [7, 8]]. Degradation of the irreversibly oxidized proteins is the only way to get rid of them and different degradation pathways exist that target both extracellular and intracellular oxidized proteins.

The extracellular oxidized proteins are assumed to be taken up into the cells and degraded by lysosomes, or degraded directly via extracellular proteases. However, it is not clear whether the selective degradation of these extracellular oxidized proteins is via lysosomes or proteosomes.

For the intracellular protein degradation, proteolytic systems with a wide range of substrate spectrum make up the heart of the degradation process.

These systems include the proteosomal and the lysosomal degradations, as well as the calcium-dependent calpains. The peptidases that are involved in cleavage of smaller peptides and proteases that target specific substrates or motifs, such as processing proteases, proteases involved in several hormone regulations, and caspases of the proteolytic apoptotic cascade, also function as part of the intracellular protein degradation system [86].

Calpains are cysteine proteases that are dependent on cytosolic Ca2+ levels [13]. These proteases are in close proximity to the cytoskeleton and function in degradation of cytoskeletal proteins. They also play role in degradation of protein kinases, phosphatases, transport and regulatory proteins [14, 15]. They are activated upon the increased Ca2+ levels subsequent to the oxidative stress, however information about these enzymes is limited.

Mitochondrial oxidized proteins are another class of oxidized proteins and they are degraded in mitochondria. Mitochondria have their own proteolytic systems which include Lon proteases, Clp-like proteases and AAA-proteases [16-18]. Since mitochondria are major source of the reactive oxygen species, they are also the major targets for the oxidative damage.

Therefore, it is not surprising that cells have evolved a special repair system for the mitochondria.

Since proteosomal, lysosomal and mitochondrial degradation systems are the major repair systems in the cells, these three systems will be focused on in more details.

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The proteosomal degradation system Upon oxidation, the hydrophobicity of most of the proteins increases via the exposure of the core hydrophobic residues to the surface of the proteins [19, 20]. Aromatic and hydrophobic amino acids are the preferred targets of the proteasome. Therefore, most of the oxidized proteins and the aggregates of the intracellular oxidized proteins are degraded via the proteosomal system that is located in the cytosol, nucleus, endoplasmic reticulum and the cell membrane [19, 21]. The system is composed of a core 20S proteasome, which is a multicatalytic protease [86], and several regulatory units such as 19S and 11S regulators [19, 21, 22].

The 20S proteasome contains several active centers. It is composed of homologous α- and homologous β-subunits, which form the outer and the inner ring structures of the cylindrical shape of the system, respectively (Fig. 1). The oxidized proteins are cleaved from their carboxyl side; basic amino acids via trypsin-like activity, hydrophobic amino acids via chymotrypsin-like activity and acidic amino acids via peptidylglutamylpeptide hydrolase activity [19].

C L O S E D F O R M

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Figure 1. Proteosomal degradation system.

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The barrel shaped 20S proteasome degrades the oxidized proteins in an ATP and ubiquitin-independent manner [19, 21, 22]. It is mostly in its closed form [23], and it combines with the regulatory subunits (either with 19S or 11S) to induce the open form of the Îą-subunits [23, 24] and recognize its specific targets. When 20S core proteosome combines with the 19S regulatory unit, it forms the 26S proteosomal complex that recognizes the ubiquitin chains on proteins and degrades these ubiquitinated proteins in an ATP-stimulatable manner by removing the polyubiquitin chain and subsequently degrading the protein [22]. The 19S regulator is the activator of the 26S proteosomal system since it contains ATP hydrolases, ubiquitin chain recognizing proteins and enzymes that play role in deubiquitination [22]. In vitro studies showed that the recognition of the oxidized proteins by the 26S proteosome is limited when compared with the 20S core proteosome [22], since the function of the 26S complex is dependent on ubiquitin and stimulable by ATP. Upon combination with the 11S regulatory unit, the 20S core proteosome forms the proteosomal system that plays role in antigen processing via regulating the length of the peptides of MHC class I complex [22, 23, 25].

11S regulator unit is also thought to allow the 11S/20S complex to degrade only the peptides [23, 26].

So the great portion of the intracellular oxidized proteins is degraded by the proteosomal system; especially the proteins that are short-lived and quickly-degradable [27, 28]. The 20S proteasome is the mostly used proteosomal system, especially in mammalian cells [86], where it is followed up by the 26S and 11S/20S proteosomal complex functions.

The lysosomal/autophagic degradation system Lysosomes are the organelles that are capable of degrading any macromolecules due to the function of hydrolases that lay inside it. The degradation process that takes place inside the lysosomes is also referred as autophagy [29]. Both autophagocytosed intracellular and phagocytosed extracellular proteins are thought to be degraded by the lysosomal system [30].

There are three autophagic pathways that are well characterized in the lysosomal degradation system; which are macroautophagy, microautophagy, and chaperon-mediated autophagy [29].

Macroautophagy Macroautophagy plays role mainly in the degradation of long-lived soluble proteins (slowly-degradable) as well as short-lived proteins [27-29]

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and complete organelles under stress conditions; especially under the Endoplasmic reticulum (ER) stress [27, 31]. During macroautophagy, inclusion bodies, which include the oxidized target proteins, are engulfed in a double layered membrane that is formed from the cytosol. This resultant membrane structure is called as autophagosome. Upon fusion with the lysosomes, autophagosomes function as degradation machines since the necessary enzymes for degradation are supplied by the lysosomes (Fig. 2).

Both ubiquitinated protein aggregates and misfolded proteins can be degraded via the macroautophagic pathway [32]. Even this pathway is accepted as a non-specific degradation for long years, recent data show that some specificity may play role in macroautophagy event [27, 33].

Microautophagy Microautophagy is the lysosomal degradation pathway that does not require any intermediate autophagosome formation (Fig. 3) [34, 35]. Whole regions of the cytosol with the target protein are engulfed into the lysosome and degraded once the vesicle is inside the lysosome [29, 35].

Microautophagy is also used in peroxisome degradation, which is also known as micropexophagy [29, 36].

Macroautophagy: OX

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Figure 2. Macroautophagy pathway.

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No autophagas ome formation

OX

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L Y S O S O ME Figure 3. Microautophagy pathway.

Chaperone-mediated autophagy (CMA) Approximately 30% of the cytosolic proteins are degraded via the chaperone-mediated autophagy (CMA) [37, 38]. The oxidized target proteins are directly translocated across the lysosomal membrane, without any vesicle or autophagosome formation [29, 39]. Instead, a special sequence motif, which is a KFERQ-like motif, is necessary to target the proteins to the lysosomes [29, 37, 39]. This motif is the first player in the CMA degradation pathway.

KFERQ-like motif can be any motif that includes a Q on either side of the sequence motif which is made of four amino acids, that should include a basic (K, R), an acidic (D, E), a bulky hydrophobic (F, I, L, V), and a repeated basic or bulky hydrophobic (K, R, F, I, L, V) amino acid residues [37, 40].

The second player for the degradation is the chaperone protein Hsc70, which is the constitutively expressed form of the heat-shock protein of 70 kDa (Hsp70). Hsc70, together with its regulator proteins Hsp90, Hsp40, Hsc70 interacting protein (hip), and hsc70-hsp90 organizing protein (hop), forms the chaperone complex that recognizes the KFERQ-like motif on target proteins (Fig. 4) [37, 41]. The resultant chaperone complex targets the oxidized proteins towards the lysosome. Here, a third player takes place, which is the lysosome-associated membrane protein type 2A (LAMP-2A) receptor. LAMP-2A is a single transmembrane protein [37, 42] that interacts

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Ayse Banu Demir & Ahmet Koc K F E R Q‐like motif

C haperone c omplex Hs c 70

L AMP ‐2A rec eptor H

sc 70

L Y S O S O ME Figure 4. CMA pathway.

with the cytosolic chaperone complex to internalize the oxidized cytosolic proteins. LAMP2A is one of the three alternatively spliced forms of the LAMP2 protein and among all these three isoforms, which are LAMP2A, LAMP2B, and LAMP2C, only LAMP2A is the receptor for the CMA and the functions of the other isoforms are not known yet [37, 43].

The target proteins are then translocated across the LAMP2A receptor with the help of the lysosomal Hsc70. It has been suggested that lysosomal Hsc70 functions as pulling down the targeted proteins into the lysosome [37, 44]. Once the substrate is inside the lysosome, it is degraded.

How do cells choose the degradation pathway that will be used: Proteosomal degradation, macroautophagy or chaperone-mediated autophagy?

How it is decided whether the oxidized proteins are going to be degraded via the proteosomal system, macroautophagy or chaperone-mediated autophagy? It has been shown that ‘short-lived’ and ‘long-lived’ proteins are degraded mainly via the proteosomal and the lysosomal pathways, respectively. However, it is also shown that this is not a must and vice versa can also take place. The choice of the degradation pathway for the oxidized proteins is thought to be due to a coordination event between the proteosomal and lysosomal systems. Three different possibilities were suggested [27]. The

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first possibility is the proteosomal capacity excision; in which the misfolded soluble proteins are primarily directed to the proteosomal system, however, the lysosomal pathway is chosen once the proteosome capacity is exceeded.

The second possibility is a limit of the endoplasmic reticulum (ER) stress levels. Under a certain threshold level, the proteosomal pathway is activated but once the ER stress levels pass over this threshold level, the lysosomal pathway starts to act. The third possibility is thought to be the misfolded protein organization. The monomeric soluble forms of the oxidized proteins are easily degraded by the proteosomal system, whereas the aggregates formed upon oxidation may not be degradable via proteasome and be targeted to the lysosomal degradation. So insoluble protein aggregates are chosen to be degraded via the autophagocytosis and the soluble forms are targeted to the proteosomal system.

Once the lysosomal pathway is chosen, the decision of ‘macroautophagy or chaperone-mediated autophagy’ is given by the presence of the KFERQlike motif on proteins. If the protein carries this sequence, CMA pathway is activated and if not macroautophagy takes place. Since both pathways are active under stress conditions, such as nutritional stress, and act as sequential events, the presence of a cross talk between these pathways is thought to exist [45]. Recent studies have shown that a coordinative activation takes place between these two pathways [45]. Once the CMA pathway is impaired, the macroautophagic degradation of proteins is shown to be increased and vice versa is also observed. Even the compensation is not totally complete, there is a cross-talk between CMA and macroautophagic degradation pathways [45].

The mitochondrial degradation system Mitochondria are referred as the energy sources of the cell since the energy production in the form of ATP takes place in this organelle via the aerobic metabolism. They also constitute an important role in a cell as being the production sites of the reactive oxygen species (ROS), which are the major players of the macromolecular damages.

Since ROS are produced primarily in the mitochondria, the macromolecules inside this organelle are also the primary targets of ROS. So mitochondria have evolved special enzymatic defense mechanisms against these molecular damages, such as the antioxidant defense system and enzymatic repair systems. However, there takes place some irreversible protein modifications that cannot be repaired and have to be degraded.

Mitochondria also include their own proteolytic systems for the degradation of the irreversible protein modifications [46].

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There are three different degradation mechanisms that play role in mammalian mitochondrial degradation process. These are LON-proteases, Clp-like proteases and AAA-proteases and all function in an ATP- dependent manner [18]; the former being a homo-oligomeric complex encoded by the nuclear genome and functions in the mitochondrial matrix [47], whereas the latter two proteases are hetero-oligomeric complexes that are located in the matrix and the inner mitochondrial membrane, respectively [18]. Lonproteases are the types of AAA+ family of proteins and are the best characterized degradation system in mammalian cell mitochondria up to today; so it will be focused in more detailed.

Lon protease (also known as protease La), is an ATP dependent protease that is first discovered in E. coli lon gene mutants and is shown to be essential for the cell homeostasis via eliminating the damaged or abnormal polypeptides as well as some regulatory proteins [48, 49]. Lon protease plays an important role in maintaining the mitochondrial integrity since some studies showed that the downregulation of the human Lon protease resulted in loss of function and disruption of mitochondrial structure [18, 50]. This protein is highly conserved among archaea, eubacteria, and eukaryotic mitochondria. The protein sequence of E. coli Lon protein shows 50% sequence similarity with the archaebacterium Thermoplasma acidophilum and 25% sequence similarity with yeast Saccharomyces cerevisiae and Homo sapiens [48].

There are three domains of the Lon protease. The N-terminal domain that interacts with the proteins and functions in oligomerization, the AAA+ module (also referred as the ATPase domain or the A domain) that functions in ATP binding and ATP hydrolysis and the P domain that has the proteolytic activity [18, 51]. The two types of Lon protease, which are LonA and LonB, only differ in their N-terminal domains, in which the former contains a long N-terminal domain while the latter has an intermembrane domain [47, 52].

LonB is mainly found in archaebacteria, whereas LonA is found in a wide range of organisms [47].

The AAA+ module of the Lon protease is necessary for the target recognition [16]. The Walkertype motif of the A domain is involved in nucleotide binding and a subsequent ATP hydrolysis [47, 53]. The Nterminus of the Lon protease is its site for protein interaction and interaction with protein targets takes place by binding of the hydrophobic amino acids of target proteins to the N-terminus site of the Lon preotease [47, 54]. Once the target protein is recognized and bound to the N-terminus, it is translocated through the Lon complex via ATP hydrolysis and undergoes proteolytic cleavage in the P domain of the complex [47, 54].

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Lon protease is important for the mitochondrial homeostasis. It was shown that Lon can bind to mtDNA in its ADP binding form. Under stress conditions, upon the formation of damaged proteins, Lon-DNA interaction is weakened and Lon recognizes the damaged proteins as its substrates and degrades them via ATP hydrolysis. When the cellular stress levels are reduced under the threshold level, Lon is returned into its ADP bound form and interacts with the DNA again.

Three reasons were hypothesized for the foundation of Lon protease bound to mtDNA. The first hypothesis is to keep Lon away from native functional proteins in the mitochondrial matrix to prevent their excessive degradation. Second is to keep Lon protease close enough to its mitochondrial nucleoid region protein substrates. Finally, the third hypothesis is the contribution of Lon protease to mitochondrial transcription or replication by binding to the D-loop which is the promoter region of the mitochondrial DNA replication and transcription [47].

Clp protease is another type of protease found in E. Coli and is conserved between prokaryotes and eukaryotes through the evolution. It has two major subunits; ClpA subunit has the ATPase activity while the ClpP subunit has the proteolytic activity that is only functional in the presence of ClpA function [55]. There are other energy-dependent proteases that have been detected in mammalian mitochondria, plant chloroplasts, and other variety of eukaryotic cells [55, 56]. These are referred as Clp-like proteases, however, their exact functions are still to be determined [18].

AAA proteases are a group of proteins that are also a subfamily of the AAA+ family of proteins [53, 57]. These proteins contain an ‘α-β-α’ core domain that is a nucleotide-binding domain, a Walker A (GX4GK[S/T]) motif, which is a highly conserved nucleotide-phosphate binding motif, and a Walker B motif (xxxx[D/E]), where x represents the hydrophobic residues [57, 58]. These proteins function as hetero-oligomers in hexameric arrangement [18, 57, 59]. AAA-proteases also function in an ATP hydrolysis manner and are important for the mitochondrial genome stability maintenance and degradation of the misfolded and damaged proteins [18, 53].

Both Clp-like proteases and AAA-proteases seem to have a chaperone activity [60, 61], however, the data about the functions of these proteins are limited [57, 60].

Repair mechanisms of the reversible protein modifications (enzymatic repair systems) The oxidized proteins are mainly degraded by the proteosomal system, since these modifications are mostly irreversible. However, there exist some

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protein modifications that can be repaired enzymatically instead of being eliminated via protein degradation. Formation of disulfide bridges, mixed disulfides, cysteine sulfenic and sulfinic acids, and methionine sulfoxides are examples of such modifications that are repaired via the enzymatic systems in the cells [62].

The sulfur containing amino acids, methionine and cysteine, are most prone to attack by the reactive oxygen species [11, 63, 64]. There are four main enzymatic repair mechanisms in cells for the repair of these two amino acids; Thioredoxin / Thioredoxin reductase system, Glutaredoxin / Glutathione / Glutathione reductase system, Methionine sulfoxide reductase (Msr) system and Sulfiredoxins and sestrins.

Thioredoxin / Thioredoxin reductase repair system is used for the sulfenic-acid reduction and Glutathione / Glutathione reductase repair system is used for the repair of the low molecular weight mixed disulfides, such as glutathione, while for the disulfide bridge and mixed-disulfide repair both systems can be used [11, 62]. These two systems can also be referred as the ‘Thiol repair systems’.

Methionine sulfoxide reductases, sulfiredoxin and sestrins are used as the direct enzymatic repair systems; as the former directly act on oxidized methionine and the latter two directly on cysteine sulfinic acids [11, 65, 66].

Thiol repair system (repair of the oxidized cysteine) There are two different repair systems for the oxidatively modified cysteine residues: Thioredoxin / Thioredoxin reductase system and Glutaredoxin / Glutathione / Glutathione reductase system [11, 62]. Both systems are found in the cytosol and the mitochondria of the cell and they act not only as repair systems but also as redox signaling molecules to protect the cell against oxidative stress [11, 64].

Both thioredoxin and glutaredoxin belong to the thiol/disulfide oxidoreductase family of proteins and both are small ubiquitous proteins [11].

They consist of a redox active center with two cysteines, which upon oxidation can form a disulfide bridge. Thioredoxin reductase and Glutathione, subsequently reduce the oxidized thioredoxin and glutaredoxin, respectively, in a NADPH-dependent manner [11]; since the reduction of the oxidized thioredoxin and glutaredoxin is necessary for their further function.

In the Thioredoxin system, thioredoxin with two –SH groups, transfers the hydrogen atom to the protein containing the disulfide (S-S) bridge. The protein is then reduced to its normal form with a -SH group and thioredoxin is oxidized with a subsequent disulfide bridge formation on it. For the reduction of the oxidized thioredoxin, thioredoxin reductase transfers the

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electrons from NADPH by the help of the flavin and thioredoxin function is restored [67, 68].

In the Glutathione system, glutaredoxin reduces the oxidized protein and then two glutathione molecules transfer the hydrogen atom from NADPH and reduce the oxidized glutaredoxin non-enzymatically. The oxidized glutathione is further reduced by the action of the glutathione reductase.

Instead of glutathione reductase, another protein called thiol transferase can also act for the reduction of disulfide bridges [69]. The GSH-dependent cytosolic thiol transferase dethiolates the protein-S-S-glutathione and restores the free thiol groups which are necessary for the normal protein function.

This enzyme has also shown to be highly resistant to oxidation via hydrogen peroxide, whereas glutathione reductase is severely inactivated with hydrogen peroxide [70].

Direct enzymatic repair The sulfur containing amino acids, methionine and cysteine, are the mostly oxidized amino acids by the reactive species. However, they are the only amino acids that can gain their function back via enzymatic repair systems. There are two main direct enzymatic repair systems which are Methionine sulfoxide reductase system and Sulfiredoxins and sestrins.

Methionine sulfoxide reductases (The Msr System) Methionine can be oxidized to methionine sulfoxide and further to methionine sulfone [62]. While methionine is oxidized to methionine sulfoxide, two different diastereomeric forms are formed, which are S- methionine sulfoxide (Met-S(O) ) or R- methionine sulfoxide (Met-R(O)).

The system that restores the methionine function is called the Msr system. The Msr system is composed of two different enzymes, MsrA and MsrB, each catalyze the reduction of S- and R- diastereoisomers of methionine sulfoxide, respectively [62, 71]. MsrA and MsrB, are subsequently reduced by thioredoxin/ thioredoxin reductase system in vivo [62, 72].

There is only one MsrA gene in the human genome, which is located on chromosome 8 [62, 71]. There are three conserved cysteine residues in MsrA, which are Cys-72, Cys-218, and Cys-227. These residues play a central role in the functional mechanism of MsrA protein. Cys72 is especially important for the catalytic activity of the MsrA [62, 73]. The sulfur atom on Met-S(O) is attacked by the Cys72 and the internal rearrangements result in the formation of a cysteine-sulfenic acid intermediate and the release of the

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reduced methionine. The cysteine-sulfenic acid intermediate is then reduced by the disulfide formation first between Cys72 and Cys218, and then between Cys218 and Cys227 [73]. Cys72 is further released and the disulfide bond between Cys218 and Cys227 is reduced with the thioredoxin/ thioredoxin reductase system [62, 72].

There are three MsrB genes in the human genome, each located on chromosome 10 [62]. MsrB1 is located in the cytosol and the nucleus and has a selenocysteine in its active site; MsrB2 is located in the mitochondria, and MsrB3A and MsrB3B are located in the Endoplasmic Reticulum (ER) and in the mitochondria, respectively [11, 74]. MsrB enzymes are zinc- containing proteins. The mechanism of action of all MsrB enzymes are similar to each other and they function via the same pathway as MsrA do; in which SO2H and disulfide bond is formed respectively and then reduction with thioredoxin/ thioredoxin reductase pathway takes place [11, 75].

Sulfiredoxins and sestrins (repair of peroxiredoxins) Peroxiredoxins are ubiquitous peroxidases that reduce hydroperoxides [76]. They control cytokine-induced peroxide levels and regulate the signal transduction in mammalian cells. Peroxiredoxins (Prx) are divided into three groups: typical 2-Cys peroxiredoxins, atypical 2-Cys peroxiredoxins and 1Cys peroxiredoxins. All proxiredoxins exist as homodimers and contain a conserved cysteine residue in their N-terminal region [77]. They only differ in that typical 2-Cys peroxiredoxins, which are Prx I, II, III and IV, have an additional conserved cysteine residue in their C terminal region, whereas, atypical 2-Cys (Prx V) and 1-Cys peroxiredoxins do not [76].

Typical 2-Cys peroxiredoxins have both a peroxidatic cysteine residue (Cys-SPH) and a resolving cysteine residue (Cys-SRH). During oxidation, the peroxidatic cysteine residue attacks the O-O bond of ROOH and sulfenic acid (Cys-SPOH) is formed. The resolving cysteine residue further reacts with the sulfenic acid and produces a disulfide bond. The sulfenic acid may also react with a second peroxide molecule and form a sulfinic acid (Cys-SPO2H) [78, 79]. For a long time, the sulfinic acid products were thought as not to be repaired. However, two newly discovered enzymes were shown to repair the sulfinic acid forms of peroxiredoxins. Sulfiredoxins and sestrins are cysteine sulfinic acid reductases [78, 79] and these enzymes selectively reduce the oxidized typical 2-Cys peroxiredoxins in eukaryotes.

Sulfiredoxins were first discovered in the budding yeast Saccharomyces cerevisiae [21].For the peroxidase reduction by sulfiredoxins; the conserved cysteine of sulfiredoxins, ATP hydrolysis, Mg2+ and a thiol as an electron

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donor, are needed [76]. The function of sulfiredoxins is specific to typical 2Cys peroxiredoxins only [77].

Sestrins are a group of proteins whose activities can be modulated by the tumor suppressor p53 [77]. They also act to reduce the oxidized peroxiredoxins, however their mechanism of action is not clear yet.

Impairment of protein repair mechanisms and aging Aging is a complex process that is generally characterized by accumulation of some metabolic products due to excessive molecular oxidation and to the decline in physiological functions of an organism throughout its life. Protein repair systems are shown to be impaired with age upon excessive oxidation of repair system proteins.

The accumulation of oxidized and ubiquitinated proteins and decrease in protein turnover are implications of age related impairment of the proteosomal system [23]. The ubiquitinating enzyme (E1, E2, and E3) activities were shown not to be changed during aging [23]. Therefore, the accumulation of ubiquitin-protein complexes reflects a defect in the proteosomal system and the proteosome activity is shown to decrease with age in variety of tissues [23, 80, 81]. The defect in the proteosomal system is not only at the protein level but also at the gene level, since the genes that encode 20S and 26S proteosomes were shown to be down-regulated with age [82].

The lysosomal repair system also undergoes a variety of changes as the cells age [29]. The lysosomal volume increases, lysosomal stability decreases, hydrolase activity changes and the lysosomal pH regulation is impaired [29]. These changes are due to the decreased degradation rates of the oxidized proteins. Macroautophagy and chaperone-mediated autophagy are shown to be altered during aging [23, 29]. In chaperone-mediated autophagy process, the lysosomal CMA receptor levels were shown to be decreased with age and this can be explained as the reason of the decrease in CMA function [29, 83].

Excessive protein oxidation, accumulation of protein aggregates upon decreased proteosomal function and defective autophagy, were shown to be related not only with aging but also some neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Hungtington’s diseases, as well as cancer, arteriosclerosis, myopathies, diabetes and others [23, 83].

Mitochondrial degradation systems are also affected by the aging process. The Msr system activity is shown to be decreased during aging [11, 84]. The Lon protease activity is shown to be declined in the liver and the mouse skeletal muscle, it is not changed in the heart mitochondrial matrix [60, 85].

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The balance between protein oxidation and protein repair plays an important role for the cellular homeostasis. Increased oxidation of proteins, decreased activity of the protein repair systems or both events, lead to cellular alterations and aging is one of those outcomes of the altered cellular function.

Therefore, it is important to understand the molecular mechanisms behind the protein repair systems and their relation with other cellular mechanisms to enlighten the molecular mechanism of the aging process.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

The Field of Biological Aging: Past, Present and Future, 2011: 61-82 ISBN: 978-81-7895-513-1 Editor: Abdullah Olgun

4. Living long or dying young in plants and animals: Ecological patterns and evolutionary processes Renee M. Borges Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560 012, India

Abstract. Plants and animals are similar and different in many ways and comparing them provides an opportunity to examine whether ecological constraints affect senescence and longevity patterns similarly in them. This paper compares clonal versus non-clonal organisms, and social versus solitary taxa, since a survey of longevity patterns in such organisms would span life forms with varied life history traits. Co-evolving longevities in interacting organisms are discussed. Strategies such as dormancy in plants and diapause in animals that may contribute to prolonging total lifespan from embryo to adult are reviewed. Longevity patterns that are peculiar to plants and animals with special ecologies, e.g. deep sea forms, or special traits such as chemical weapons, are explored.

Biomechanical and physical constraints on longevity are also examined. This review therefore attempts to provide an evolutionary and ecological framework using which longevity and ageing can be understood across organisms. It also suggests exciting and fruitful new areas for research in the ecology and evolution of ageing. *This paper is dedicated to the great evolutionary biologist G C Williams (1926−2010) who contributed significantly to ideas on the evolution of senescence.

Correspondence/Reprint request: Dr. Renee M. Borges, Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560 012, India. E-mail: renee@ces.iisc.ernet.in

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Introduction In Hindu mythology, gods and demons battled for amrit or the nectar of life which conferred immortality. This amrit was obtained by the churning of the oceans with the help of the great snake Vasuki, and fell into the hands of demons. The powerful god Vishnu, by devious means that involved distracting and beguiling the demons by transforming himself into beautiful Mohini, regained amrit for the gods, making them alone immortal. The sweet-tasting amrit is analogous to ambrosia or the nectar of the gods in Greek mythology, a drink which also conferred immortality and which was supposed to have been brought by doves to the gods residing in Olympus.

This cross-cultural similarity in immortality myths and the differences in detail are symbolic of the great similarities and differences in why and how plants and animals live long or die young.

Plants and clonal animals rarely get cancer, while non-clonal animals do; naked mole rats and queen bees live long while other rats and worker bees die young; trees age slowly while shrubs age fast; fish and tortoises are long lived as are flying squirrels compared to flightless birds; ramets die but genets of clonal organisms may be immortal. How can this variation in longevity be explained, and are there differences between patterns of longevity in organisms within the plant and animal kingdoms?

The geneticist Theodosius Dobzhansky has been quoted countless times for his aphorism “Nothing in biology makes sense except in the light of evolution�, and indeed this saying could not be more relevant for a review on longevity since ageing and longevity can only be viewed within the framework of evolution for any meaningful understanding of these phenomena. The study of longevity and of ageing is now rich with both evolutionary traditions as well as functional genomics and has entered the realm of what may be called evolutionary gerontology [1].

This paper evolved from an earlier mini-review [2] which emphasized the inherent plasticity that must underlie longevity in plants and animals.

Since traditional boundaries between the study of plants and animals are in the process of fast dissolving, it is hoped that integrated reviews, such as this one, which attempt to move seamlessly between the kingdoms, will assist in providing an overall evolutionary framework to examine longevity in life forms with varied ecologies. In this paper, the major focus is on comparing longevity in clonal versus non-clonal organisms and in social versus solitary species, since these dichotomies cover the vast majority of growth and life history strategies of life forms on earth. Potentially co-evolving longevities of interacting species such as mutualistic or parasitic interactants will also be

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discussed. The impact on longevity of the differential ability of organisms to regenerate will also be addressed, as also the issue of dormancy and diapause.

Proximate factors influencing longevity will be examined in some cases.

Before examining longevity patterns and their possible causes, it is necessary to acknowledge the difficulties inherent in defining mortality [3] as well as the need to distinguish between the mortality and immortality of individuals and of lineages. It is also important to differentiate between organisms with determinate versus indeterminate growth, between clonal and non-clonal organisms, and between solitary, eusocial and colonial species.

This is because growth and life history parameters have fundamental consequences for the longevity of individuals within species [4].

Definitions Evolutionary biologists define senescence as the reduction in reproductive output and the increase in the probability of mortality with age [5] while senescence for a physiologist or cell biologist is the decline in cellular or physiological function with age [6].

Determinate growth is when an organism’s growth trajectory is set early on in the ontogeny of the organism so that the organism cannot change this trajectory after an initial growth period; thus the organism reaches a fixed size [7]. Indeterminate growth is when an organism can continue to grow throughout its life and can also show considerable plasticity in growth pattern based on temporal variation in resource availability which may even result in body shrinkage [7]. Indeterminate growth that exhibits plasticity as well as an asymptotic function with age is often displayed by soft-bodied marine, freshwater and terrestrial invertebrates as well as some fish [7]; the asymptotic size can increase or decrease over an order of magnitude depending on habitat conditions and resource availability. Indeterminate growth is regarded as the major factor delaying senescence in fish [8]. Plastic and exponential indeterminate growth is exhibited by clonal or colonial organisms with a modular architecture [7]. Here each module contributes to the energy acquisition of the whole colony or clone. Plastic and attenuating indeterminate growth occurs when growth slows down with an increase in size [7]. This is the pattern of growth observed in trees [9]. The only real difference between determinate and indeterminate growth is that in the latter there is no genetically fixed upper size limit, and the organism can change its size throughout its life in response to changing environments.

The problem of defining the individual [10] is a particularly acute one in understanding patterns of senescence and thereby longevity [11]. In

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non-clonal plants, the zygote develops into a single rooted unit (the genet), while in clonal plants, the zygote (genet) is duplicated asexually via multiple rooted units (clones) that may continue to be connected to the genet via root sprouts or by rooted stems such as stolons or rhizomes. The genetically defined individual (the genet) that is the product of sexual reproduction and that can reproduce itself asexually (via the ramet) may be immortal while individual ramets are mortal [12]. Therefore, a distinction between the longevity of genets and ramets is important.

The evolutionary framework The three most popular theories explaining patterns in the evolution of longevity are the antagonistic pleiotropic theory of Williams [12], the mutation accumulation theory of Haldane [13] and Medawar [14] and the disposable soma theory of Kirkwood [15]. In the mutation theory of Haldane and Medawar, late onset genetic diseases contributing to senescence and mortality will encounter only weak negative selection since reproduction is over at the time of their onset. This theory has been supported experimentally [16]. In the disposable soma theory, there is a trade-off between investment in the maintenance of somatic and reproductive tissues; consequently ageing sets in when somatic tissues become disposable after reproduction has ceased, and when the organism is no longer likely to survive. Several good reviews of these theories exist [4, 17, 18] and they will not be dealt with in detail in this paper. The antagonistic pleiotropy theory will, however, be given more attention.

According to the antagonistic pleiotropy theory, senescence occurs because certain genes that are beneficial in early life may have a negative effect during life after reproduction; natural selection is therefore unable to remove such genes from the population owing to their combined beneficial and antagonistic effects. Consequently, this theory uses the concept of life history trade-offs such that early fecundity is obtained at a cost to later-life reproductive success. Important deductions from this theory which are relevant to an examination of the diversity of longevity patterns seen in nature and discussed in this paper are: a) low adult extrinsic mortality rates should be coupled with low rates of senescence, and b) organisms with determinate growth should show greater senescence than those with indeterminate growth. Additional predictions are available in Williams [12] who also claimed that senescence would only be found in those organisms where there is a distinction between germline and soma. The relevance of this claim for senescence and longevity patterns [19] will be discussed later in this paper. Perhaps the best direct test of this evolutionary theory of

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senescence was conducted in Drosophila, where flies that were subjected to different adult mortality rates evolved shorter lifespans when subjected to higher adult mortality [20].

At proximate, mechanistic levels, many theories and processes have been invoked to explain ageing and thereby longevity in organisms. The following are the principal processes and associated theories affecting ageing at various levels: a) Molecular (gene regulation, codon restriction, error catastrophe, epigenetic); b) Cellular (telomere reduction, free radical theory, wear-andtear, programmed cell death or apoptosis, autophagy); and c) Systemic (neuroendocrine, immunological, rate-of-living). These processes have been ably reviewed by several authors [17, 21–33], and will not be dealt with in any detail here. Only selected examples of some of these theories with relevance to particular cases of longevity in plants and animals will be discussed.

The Methuselahs of the plant and animal kingdoms Humans crave an understanding of outliers. What causes progeria? What makes an Usain Bolt? Why were tyrannosaurs so large? So too with longevity. Why do adult mayflies live for only a few minutes while larvae of cicadas live for 17 years?

The oldest living plants are either trees or clones of non-woody species.

The gymnosperm Pinus longaeva is recorded to live for about 4700 years [34, 35], the cedar Thuja plicata regularly lives for between 800–1000 years [36] and clones of the shrub Lomatia tasmanica (Proteaceae) have been dated to 43,600 years with individual ramets living for about 300 years [37].

Rockfish are known to live for 157 years [8], while lifespans of whales and tortoises can exceed a couple of centuries [38, 39].

Longevities of clonal and non-clonal organisms While trees can reach great ages, this extreme longevity applies to their meristematic lineages and not to individual cells or modules that may not be more than a few decades old. Indeed much of the tree consists of dead cells in the form of heartwood, cork and bark. Still, long-lived trees or plants with tree-like forms can also have long-lived cells; e.g. the ray parenchyma cells of the giant saguaro or tree-cactus Carnegiea gigantea and the cortex cells of the barrel cactus Ferrocactus wislizenii [40, 41] can live for 100 years.

Human neurons can also reach great ages, even more than a century, but humans do not live to be as old as trees.

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Recently Aarssen [42] has sketched an interesting scenario to provide a solution to “the problem of the small� via an analysis of plant reproductive economy. This scenario has useful implications for an understanding of how plants might be strategizing longevity. In this view, plants are competing for limiting resources such as space and light, and woody plants are selected to grow into tall trees as a result of this competition [43]. Trees do not usually reproduce clonally. By virtue of their indeterminate growth and meristematic tissue, trees reproduce for extended periods of time by meiosis in their germlines; this results in ovules or pollen that can form zygotes which may be vectored to new germination sites. Small and usually short-lived plants adopt another strategy. Besides sexual reproduction, these plants also reproduce clonally and thus ensure the longevity of the original genet.

According to Aarssen [42], clonality can provide a survival advantage to small plants since it can move the genet from areas of competition or nutrient deprivation to more suitable sites. Correspondingly, a trade-off between sexual and clonal reproduction can influence clonal senescence since greater asexual reproduction via ramets will ensure genet survival and retard genet senescence [44]. Interestingly, during plant evolution, while all early vascular plants were clonal, clonality was lost in some clades only after the appearance of erect and arborescent forms, e.g. Lepidodendron [45].

Similarly, while all extant pteridophytes are relatively small and clonal, their now extinct large and non-clonal relatives gave rise to the mostly large and non-clonal gymnosperms [45]. As mentioned earlier, trees generally lack clonality, and their reproduction is not limited by small body size. Trees have relatively long-lived meristematic lines while small non-woody plants employ clonality to achieve reproductive economy. Thus according to Aarssen [42], some plants may sacrifice the size of a ramet in order to produce ramets in larger numbers via clonality with reductions in the longevity of each individual ramet, while others trade off clonality to achieve tallness (as happens in trees) in which case they can escape local competition and send their gametes and zygotes to far distances through wind or biotic pollination and seed dispersal. This reproductive process also occurs for longer periods of time in tall trees compared to smaller clonal plants.

Separation between germline and soma Williams [12] asserted that senescence could only occur in organisms where there is separation between germline and soma. The unity of germline and soma was therefore considered to be a barrier to the evolution of senescence in unicellular organisms such as bacteria in which germline and

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soma were considered indistinguishable. Recently, however, senescence has been shown to occur in bacteria which undergo either asymmetrical [46] or symmetrical division [47]. In these cases, the older cell retains the old pole and only the newly synthesized components enter the daughter cell [47].

Consequently, even in unicellular organisms there may be separation between germline and soma [19] which is also perhaps indicated by the presence of two types of nuclei, the germline micronucleus and the somatic macronucleus, in amoebae such as Paramoecium [48]. Similarly, the budding of smaller yeast daughter cells from older and larger mother cells which show signs of ageing [49] may also constitute a germline–soma distinction [19].

Other authors have also questioned whether the separation between germline and soma is necessary for the evolution of senescence [50, 51], pointing out additionally the consequences of this point of view for differences between senescence in ramets and genets [52]. The amount of clonal reproduction in an organism can also affect the evolution of senescence since clonal reproduction tends to retard but does not preclude senescence [53, 54].

Therefore, even “immortal� Hydra [55] may show senescence [56], as does the asexually reproducing marine oligochaete Paranais litoralis [50].

Similary zooid senesence in the marine bryozoan Electra pilosa has also been observed [57]. Yet, the evolutionary theory of senescence does not offer clear predictions for the presence of colony-level senescence in clonal organisms [50], and further exploration of this interesting subject area is warranted.

The physiological integration between ramets may also affect the evolution of senescence at the level of the genet [52]. If an individual plant is structurally and functionally a single-rooted integrated physiological unit (IPU) and does not produce asexual ramets to which it is attached, then this IPU will undergo senescence since it will continue to grow and may even reach a size level that is beyond its physiological optimum; at this point the genet will begin to senesce. This may be what occurs in the iteroparous sea beet Beta vulgaris ssp. maritime, which never produces ramets, exists as a single IPU, and in which ageing effects have been observed [58]. According to this view, if the genet is physically connected to ramets, and retains this connectivity, senescence in the genets will set in. However, if the ramets produced by the genets are physically separated from the genet by fragmentation, then while the individual fragments or ramets may undergo senescence, there will be limited evolution of senescence in the genet as long as new ramets are produced by clonal growth. Thus ramets will senesce but the genet may escape senescence. Therefore, with respect to plants, the critical distinction is not between germline and soma or even between clonal and non-clonal plants but between plants in which the genet is a single-rooted

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IPU and one in which it is physically connected to its clones [52]. From this perspective, senescence will evolve in clonal plants that are physiologically integrated rather than in those that fragment. The unity of the germline and soma therefore appears to be a necessary but not sufficient condition to prevent the evolution of senescence in plants and clonal animals [52].

Ageing in trees It therefore appears that despite indeterminate growth and great longevity in plants, especially in trees, they do exhibit senescence. What are the correlates of this senescence and how may ageing be explained in trees?

Plants can only live as long as they continue to grow. This is because plants are autotrophs and continually require to produce new photosynthetic units (leaves) as well as nutrient-gathering units (roots) in order to grow. New leaves often require new branches in the growth process. Thus plant growth and plant size are likely to have strong relationships with plant age.

In trees, several factors may govern reduced growth with age [59]. a) Respiration/Photosynthesis Ratios: As trees grow, there is an increase in non-productive relative to productive tissue [60, 61]. Increasing respiration relative to photosynthesis results in slower growth. b) Hydraulic Limitation: Increasing tree height results in increasing length and corresponding decreasing conductance of water-conducting xylem tissue. This has led to the hydraulic limitation hypothesis of tree growth and tree height [62, 63], which although applicable to many trees, may not be universal [64]. c) Nutrient Limitation: This hypothesis refers to self-limitation of tree growth by locking up nutrients in tree biomass, and thus making nutrients unavailable for further growth [65]. Individual trees may only be released from such limitation if there is continuous nutrient flux into the system or by nutrient resorption from senescing tissues such as leaves. d) Genetically Programmed Senescence: According to this hypothesis, there is programmed senescence leading to decreased growth potential of meristems. Whereas this type of senescence brought about by DNA methylation and changes in gene expression has been shown in transitions from juvenile to adult stages in plants [66, 67], it has not been clearly demonstrated in trees after they have acquired adult reproductive status. Hence the evidence for this process is still equivocal [59].

The effect of size must also be uncoupled from that of age while examining senescence in trees [68]. Experiments in which meristems from the canopy of old tall trees were grafted onto young rootstocks in angiosperms and gymnosperms showed that there was no reduction in growth

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potential of these meristems, while the meristems left in their original locations exhibited reduced vigour [68]. Therefore, factors extrinsic to the meristem such as its location can influence its growth and vigour, and thereby its senescence, rather than factors intrinsic to the meristem. To demonstrate this, young shoots grafted onto the tall crowns of Japanese cedar trees exhibited the limitations of photosynthesis and stomatal conductance inherent to their new locations, and their performance was as poor at that of the ageing crowns [69]. Even in non-clonal plants with a single terminal meristem (e.g. palms), there is a decline in reproductive output with size and height which is probably due to problems in vascular transport with increasing height [70].

Some palms may, however, have resolved the problem of a reproductive slow down by becoming clonal [71].

It appears, therefore, that senescence can occur in single-rooted genets of trees as a result of size-mediated effects. Other studies have also shown a decline in growth rates with lifespan in trees [9]. The maximum height of trees (ca. 130 m) is probably dictated by hydraulic [72] as well as biomechanical constraints [73, 74], although tallness itself is a trait subject to escalation owing to competition between plants for light [43]. Hydraulic and biomechanical constraints are also present in long-lived desert cacti [75]. The longest-lived cacti are those that can reach heights of 12–15 m and have treelike forms (e.g. Carnegiea gigantea); such cacti regularly live for more than 100 years [40]. Most trees reach heights of only about 30–50 m; consequently their growth potentials, and thereby their longevity, would be influenced by the numerous constraints outlined above. Furthermore, since plants are immobile, their longevity is also subject to their ability to escape degradation of their supporting dead and living tissues by attacks from pests and pathogens, particularly fungi [2, 36, 76, 77]. It is therefore not surprising that most of the Methuselahs of the plant world occur in taxa which are strongly defended by terpenoid resins (e.g. conifers) and phenolic compounds [60].

Indeed these resins are so resistant to degradation that intact DNA can even be recovered from ancient organisms that have been trapped in amber [78]. It would be valuable therefore to correlate the maximum longevity of trees with their pest- and pathogen-resisting abilities; e.g. the great longevity of some white cedar Thuja occidentalis populations is probably also due to their decay-resistant properties [36]. The Methuselahs of the plant tree world also appear to inhabit extreme environments such as deserts or arid mountains. Is longevity then an inevitable outcome of adaptations for survival in such harsh environments? It might also be worth investigating bonsai plants and shortstatured plants of the Mediterranean which are probably very old and in which height is not a correlate of their longevity [79].

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Adaptive iteration, vascular modularity and somatic genetic mosaicism In evolutionary time, some plants have overcome various limitations to growth, and thereby to longevity, by the process of adaptive reiteration [80].

This is a type of epicormic branching which occurs from dormant buds on the trunk or branches of woody trees, not in response to injury or trauma such as defoliation or damage to the apical bud, but as an adaptive response to changing light, nutrient, water and stomatal conductance levels [59].

Adaptive reiteration is believed to free plants from light limitation by initiating new shoots in more illuminated parts of the crown, and to solve hydraulic limitations by initiating these new shoots where hydraulic conductance and water-use efficiency is higher, as well as by redirecting nutrients from senescing tissues by the formation of strong and vigorous nutrient sinks [59]. In ancient 450-year old Pinus menziesii trees, nonepicormic branches in the middle portion of the crown have 148 growth rings on average while epicormic branches in the lower crown are younger with only 94 growth rings [81]. Thus individual trees consist of a mosaic of modules of different ages, and adaptive reiteration can be a very important response leading to the extension of longevity in such trees. The phenomenon of epicormic branching occurs in angiosperms and gymnosperms in which it increases with tree height [82].

The vascular modularity of plants [83] also confers a survival advantage since it allows the localization of trauma or damage to the hydraulic system and consequently avoids systemic failure [2]. Sectoral hydraulics may have contributed to the longevity of many tree species [36]. Similarly, the ability to resprout after damage such as fire [84] is another mechanism that endows greater survival ability on plants [2]. Furthermore, since plants are somatic genetic mosaics, intraindividual selection can purge mutational load from somatic mutations and allow only the more adapted meristematic modules to survive [2, 85, 86]. Genetic heterogeneity within organisms can be beneficial for survival, and thereby contribute to longevity, especially in modular organisms [87].

Biomechanical and physical constraints on longevity Biomechanical constraints on longevity especially in taxa with indeterminate growth could also be important. While trees have great longevity coupled with woodiness, genes for making the vascular cambium of woody plants are also present in non-woody Arabidopsis [88]. This may

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explain why woodiness and the tree habit could evolve in many plant lineages [89], resulting in the appearance of long-lived taxa across the plant kingdom. The large size of trees is probably facilitated by the cellulosic properties of wood just as the longevity of corals is possibly a result of a calcium carbonate skeleton which can protect it from biomechanical stresses [36]. Indeed, colonies of proteinaceous deep-sea corals may live for four thousand years [90]. Similarly, a “redwood� of the coral reef is the giant barrel sponge Xestospongia muta which in Carribean reefs may live for up to 2300 years [91]. Yet, even in these environments, physical constraints can limit growth and thereby longevity; e.g. the constraint of flow-induced energy intakes limited the maximum size of intertidal sea anemones [92], and turbidity–light gradients in the oceans affected the photosynthetic abilities of zooxanthellae and correspondingly coral growth [93]. Importantly, however, partial colony mortality, colony fission and fusion may also confuse any straightforward relationship between size and age in reef corals [94] and associated colonial organisms. Examination of relationships between size and age in such colonial organisms is, therefore, fraught with difficulties.

Protected environments, effects of ROS, and telomerase activity Long-lived animals and plants can provide important insights into the mechanisms and environments influencing the evolution of the ageing process [95]. An ocean mollusc, the Arctic quahog (Arctica islandica), can live for 400 years [96, 97], and a species of deep-sea oyster for over 500 years [98]. The imperceptible ageing of the ocean quahog is apparently due to its great antioxidant capacities [99]. Among mammals, bats have the longest lifespan for their body size [100] and in some long-lived bat species this is correlated with resistance to protein oxidation coupled with enhanced protein homeostasis abilities [101]. The eusocial naked mole rat Heterocephalus glaber lives in captivity for more than 28 years, almost 9 times longer than mice of similar sizes [102]. Surprisingly, these mole rats produce similar amounts of reactive oxygen species (ROS) compared to shorter-lived rodent species and have similar repertoires of antioxidants as these species; however, some biochemical parameters such as glucose tolerance, glycated haemoglobin, and antioxidant activity remain unchanged with age in H. glaber while those of laboratory mice and rats decline [102]. The mechanisms for such biochemical prowess remain unknown. Naked mole rats also live in underground burrow systems and may be exposed to less adult predation pressure resulting in greater longevities. Similarly, volant

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organisms such as birds, bats, and flying squirrels which can fly away from predators, as well as animals such as tortoises and bivalves which have thick anti-predator body armour are believed to have lower adult extrinsic mortality and have therefore evolved longer lifespans [12, 103, 104, 105].

Enhanced longevity and lower senescence coupled with lower extrinsic adult mortality seem to have evolved even in tiny Daphnia, since water fleas from low-mortality risk pond habitats exhibited lower amounts of senescence than those from high-risk areas [106]. It is similarly predicted that the unusually long lifespan of rodents such as the African porcupine Hystrix brachyura (greater than 27 years) is probably facilitated by its quills which are efficient anti-predator armaments [105]. From this same perspective, a comparison of chemically protected versus non-protected fish, reptiles and amphibians, showed that, after correcting for body size, the former had longer maximum lifespans than the latter species [107]. Similarly, clown anemonefish Amphiprion percula protected by anemones have lifespans six times greater than the longevity expected for their size [108]. Protection against extrinsic mortality may also induce plasticity in ageing as demonstrated by intraspecific differences in the parasitic nematode Strongyloides ratti. Those nematodes inhabiting the intestinal mucosa of rats can live for 400 days and reproduce by mitotic parthenogenesis, as opposed to the free-living soildwelling generations that live for only 5 days [109]. Similarly workers of the weaver ant Oecophylla smaragdina that conduct risky foraging outside the nest (major workers) have higher rates of ageing than those that perform activities within the nest (minor workers) and that rarely venture outside the protected confines of their arboreal leaf nests [110].

Deep-sea environments also seem to select for long-lived organisms, e.g. scorpaenid fish [111]. Deep-sea environments have lower oxygen concentrations; consequently deep-sea organisms probably have less exposure to environmentally-generated ROS compared to surface or shallow marine organisms. Therefore, while ROS-scavenging antioxidants are needed to a greater extent in shallower water, the selection pressure for these compounds is lower in deep-sea creatures. It is therefore extremely interesting that bioluminescence has evolved to a considerable extent in deepsea creatures. Coelenterazine, the type of luciferin present in many marine bioluminescent groups, is a powerful antioxidant with efficient ROSscavenging capabilities [112]. It has been suggested that when ROS scavenging was no longer so critical in the deep-sea, ROS detoxification was diverted into a communication tool by deep-sea dwellers in the dark depths of the ocean resulting in deep-sea bioluminescence [112]. Furthermore, coelenterazine is found in all tissues and not just in the bioluminescent organs [112]. While all reasons for the greater longevity of deep-sea fishes are not

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yet known, and their low metabolic rates may also be involved in this phenomenon [113], there are many exciting possibilities for research in this area [111].

In the rainbow trout (Oncorhynchus mykiss) which does not age perceptibly, high telomerase activity has been found in all analysed organs [114], as was also found for long-lived lobsters [115], both organisms with indeterminate growth. Newer studies using non-conventional model organisms are being conducted to test the proximal mechanistic theories of senescence that were mentioned before. For example, the free radical theory of ageing was tested using long-lived and short-lived colubrid snakes and it was found that longer-lived species had lower free radical production than shorter-lived ones [116]. Within the same garter snake species Thamnophis elegans, longlived ecotypes in areas of low extrinsic mortality had more efficient mitochondria and more efficient antioxidant capacities compared to shorterlived ecotypes living in areas with higher extrinsic mortality [117]. The evidence for these proximal theories has also been recently reviewed for birds [118] and will not be dealt with here in any detail.

Dormancy and diapause Dormancy and diapause can be important bet-hedging strategies [119, 120] prolonging life in a variety of organisms ranging from desert annual plants [121], through sponges [122], many arthropods [123], and vertebrates including fish and amphibians [124]. In plants, many types of seed dormancy mechanisms exist and there is also a general positive relationship between ratios of embryo to seed volumes and seed dormancy [125]. Here too, plants exhibit exceptional longevity. A 1288-year old seed of the sacred lotus Nelumbo nucifera was germinated from an ancient lake in China, and plants were successfully grown from lotus seeds that were at least 332 years old [126]. While the preservation of such seeds in anoxic highly reducing clay lake sediments must have also contributed to their longevity, such ancient seeds were also found to be functionally and enzymatically robust, especially in L-isoaspartyl methyltransferase activity [126].

Dormancy can be an evolutionary stable strategy when there is intense competition for space in the above-ground environment and when survival in the soil seed bank is high [127]. In such cases, sexually produced offspring can inherit the limited above-ground space after parental mortality. Density dependence and severe competition for space has been shown theoretically to result in greater adult longevity [128]; this may also explain greater juvenile (seed) longevity. Periods of dormancy with very slow growth or prolonged

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development have been seen, for example, in the periodical cicadas which have larval development periods of 13 or 17 years, which are the longest larval development periods for any insect [129]. During this period, cicada nymphs feed on root xylem tissues in the relatively protected rhizosphere environment. It is possible that this prolonged juvenile development has been selected for since it increases fecundity with little simultaneous increased risk of mortality [130]. In this vein, it is also useful to consider the evolutionary and reproductive consequences of vegetative dormancy in plants which occurs when an herbaceous perennial does not sprout aboveground but lives underground as rootstock for one or more growing seasons [131]. The relationship between this type of vegetative dormancy, senescence and lifespan is recently being investigated and presents a new exciting area of research [132].

Sociality and ageing Sociality can also have an effect on ageing in animals, perhaps in a way analogous to that between attached and non-attached ramets and genets in plants and in clonal as well as colonial animals. In insects, the evolution of eusociality was associated with a 100-fold increase in lifespan [133]. In birds, however, no relationship between sociality and lifespan was detected after correcting for body size [134, 135]. The findings for eusocial insects are consistent with evolutionary theories of ageing, since their colonies are usually situated in extremely sheltered places where the mortality of queens or kings (i.e. the sole reproductives of the colony) from extrinsic causes is likely to be much lower than in non-social taxa [133]. In accordance with theory, queens of ant species that found monogynous colonies live longer than those of polygynous colonies, and this is believed to be related to the mortality risk of colony founding via polygyny compared to monogyny [133]. Similarly, termite kings and queens have greater longevity compared to reproductives of other solitary insect species, living as they do within the protected environment of their mounds [136].

In social organisms, ageing may be delayed and longevity prolonged even during post-reproductive periods if by this means care-giving and intergenerational transfers to offspring or grand-offspring can enhance the survival and in turn the reproduction of the recipients of the transfer [137, 138]. Queens of some ant species can live for 30 years [139]. Hive bees that are prevented from becoming foragers can live for many times longer than forager bees since as nurse bees restricted to the hive they are capable of transferring more investment to the next generation than as foragers [140].

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Furthermore, since the risk of mortality for hive bees is much lower than that for foragers, investment in the forager soma should be lower than that for hive bees; foragers may therefore be more expendable and have shorter lifespans in accordance with the disposable soma theory of ageing [140, 141].

In social organisms, therefore, the reproductive success, ageing and longevity of individuals rely on the structure and ecology of the social group for their explanation. Social insects, therefore, can contribute greatly to the study of ageing [142].

In the branching coral Acropora palmate, regeneration rates are higher in the younger distal parts of the colony while older basal parts show lower regeneration and therefore greater senescence [143]. Physiological integration between parts of the colony may be responsible for this phenomenon wherein older parts transfer nutrients and metabolites to the younger growing tips which serve as active growth sinks [144]. Thus proximal senescence which occurs at the base of the colonial organism may be offset by the increased growth and reproductive potential of the distal tips and this is achieved by transfer of nutrients from older to young modules [145]. Furthermore, tradeoffs in stem cell allocations between requirements for tissue repair and for reproduction are important factors influencing survival and longevity in these clonal and colonial animals [146]. Thus integration of reproduction as well as tissue repair over the entire colony could be responsible for the long-lived nature of the colony, i.e. of the genet, while individual ramets (modules) may senesce [147–149]. This pattern of increased senescence at the proximal part of the colony compared to the distal part has also been observed in a variety of other organisms, e.g. hydroids [150], ascidians [151] and bryozoans [57], and appears to be a common strategy for such growth forms.

While sexual selection and sexual dimorphism have been invoked to explain higher mortality rates in males compared to females in both animals (see review of this controversial subject for animals [152]) and plants [153], an examination of this topic is beyond the scope of the present paper.

Longevities of interacting species Evolutionary theories of ageing concern themselves with the longevities of individuals which influence the longevity characteristics of species. When species are involved in mutualistic or parasitic interactions, which may even be symbiotic in nature, there is likely to be co-evolution of the longevities of the interactants. For example, most plants depend on the activity of mycorrhizae for successful existence, and since many plants require mycorrhizal inoculum for successful establishment, the mycorrhizal spores

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must also have considerable longevity to match with the germinating time of their host seeds [154]. Similarly, the longevity of parasites must match that of their hosts, especially during the juvenile development stage, as occurs during the parasitism of fig wasps developing within fig inflorescences [155].

Some parasites may even be able to increase the lifespans of their vectors if by doing so they can increase their own transmission [156]. Therefore, an examination of arms races in the longevities of interacting partners [157] should emerge as a fascinating avenue for investigation. Similarly, mutualistic ants influence life-history traits including the longevity of their aphid partners from whom they derive nutrition and to whom they provide protection from predators and fungi [158]. Thus the impact of partner behaviour and/or physiology on the longevities of interactants is an area worthy of serious investigation.

Regeneration in plants and animals Understanding regeneration abilities across plants and animals will also aid in an understanding of longevity [159]. This is because regeneration abilities vary greatly in these kingdoms, whether at tissue, organ or whole organism levels [159]. Somatic embryogenesis in plants in which a single somatic cell can give rise to an embryogenic-like ontogeny is quite unique [160]. A comprehensive comparison of regeneration abilities relative to longevity in clonal versus non-clonal plants and animals would provide interesting pointers to relationships with longevity. While stem cells also age [161], how stem cells in plants and in animals are able to erase epigenetic marks and to reset their ages on being elicited to develop into whole organisms are very exciting research areas.

Conclusions While all the above evolutionary theories and proximate mechanism can explain general patterns of ageing, there is variation in lifespans even when genetically identical organisms are reared in constant environments [162].

Investigations of the molecular, cellular and systemic sources of such intrinsic variability will therefore provide important insights into the ageing process. A multidisciplinary and multifactorial approach to the problem of longevity is undoubtedly necessary since factors contributing to longevity are not mutually exclusive [79].

What implications do all these perspectives have for human ageing? The intergenerational transfer theory may tell us that over evolutionary time those

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human populations that invest more in their offspring and grand-offspring may be able to postpone senescence, while other theories predict that any factors that reduce extrinsic mortality in adult age and contribute to greater fecundity may also result in the evolution of longer lifespans. From the perspective of development, research that can reveal how plants undergo somatic embryogenesis from a single cell will have important implications for our understanding of how cell, tissues and organisms can forget elapsed time and reset their development clocks.

In conclusion, it is important to remember that evolution of reduced ageing or enhanced longevity is a population process, while ageing is an individual phenomenon. While an individual experiences the effect of natural selection in the past, its offspring will experience the natural selection of the future. Thus past, present and future dwell within an individual, and provide a measure of its immortality.

Acknowledgements I thank Vidyanand Nanjundiah for critical comments on the manuscript. I am grateful to Abdullah Olgun for inviting me to contribute to this volume and for accepting this idiosyncratic but hopefully informative review.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

The Field of Biological Aging: Past, Present and Future, 2011: 83-86 ISBN: 978-81-7895-513-1 Editor: Abdullah Olgun

5. Long living plants as longevity models and sources of anti-aging medicines Abdullah Olgun Erzincan Mil. Hospital, Biochemistry Lab.24000 Erzincan Turkey

Abstract. Many organisms have no chance to reach old ages in wild environments due to predation and accidents. Therefore natural selection likely effects early life traits as fitness and reproduction, but fails to prevent aging and age related diseases. As described in antagonistic pleiotropy theory, aging could be a side effect of the molecular mechanisms ensuring early life fitness and reproduction. Very long living trees like Pinus longaeva that lives up to 5000 years, live in an almost protected environment and are not subject to predation. Natural selection seems to have succeeded in finding a way to prevent late life’s deleterious effects in at least these trees. The discovery of the mechanisms of their longevity can have immense benefits in our efforts to increase healthspan and lifespan in humans.

Introduction Aging can be defined as time dependent progressive degenerative changes that decrease the chance of survival of the organism and ultimately lead to death. Aging is one of the most complex and challenging problems in

111 Correspondence/Reprint request: Dr. Abdullah Olgun, Erzincan Mil. Hospital, Biochemistry Lab.24000 Erzincan Turkey. E-mail: aolgun@yahoo.com

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biology to study. Therefore, many models from simple organisms to mammals as well as “in silico” models are widely used to discover its mechanisms. As a result of these studies, we hope to postpone aging and prevent age related diseases. Aging theories can be divided into two main groups as stochastic (accumulation of damage) and genetic programming (longevity but not aging genes) [1; 2].

The evolutionary theory of aging [1] states that there is no natural selection for advanced ages due to early death by predation and accidents in natural environment. Therefore natural selection likely affected only early life traits like fittness and reproduction. But it failed to prevent late life phenotypes like aging and even contributed or caused them by antagonistic pleiotropy. Additionally some features which are good early in life can even be harmful later. The testosteron-prostate cancer, estrogen-breast cancer and tumor supression via cellular senescence-ageing relationships can be given as examples to such effects.

But in contrast to animals, some plants, especially trees, live in a protected environment and do not seem to be subject to predation. This should have allowed natural selection to prevent late life’s deleterious effects and aging in at least some plants. Therefore the discovery of the mechanisms of plant longevity can provide us tools to fight human aging and age related diseases.

Aging in plants As observed in animals, the life span of plants differs among species. It can change from days to thousands of years [2]. For example, while Lemna minor (duckweed, water lentil) lives only ~21 days, Pinus longaeva lives up to 5000 years without the signs of aging [3;4] and an 9550 years old spruce was discovered as the oldest living tree. The studies on plant aging have been mainly using leaf senescence as a model. Another area is seed longevity. The studies on seed longevity promise great opportunities to decipher the mechanisms of longevity in plants.

“Leaf senescence” and “senescence syndrome” The term “leaf senescence” is used for both “senescence –time dependent functional decay due to the accumulation of damage- of leaf” which is consistent with animal senescence literature and for “senescence syndrome” which states the yellowing of leaves in autumn. Leaf senescence can trigger senescence syndrome [2]. Therefore senescence syndrome can not be accepted as a model for aging.

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Senescence syndrome Senescence syndrome is the final controlled stage of leaf development. It is very important for the relocation and recycling of nutrients from leaves to other requesting organs like seeds of the plant. It is highly regulated at the genetic level [5]. It requires the activation of > 2,000 genes in Arabidopsis [6]. It is an age and environment dependent process of programmed cell death [7].

Leaf senescence The normal metabolism and especially some reactions of photosynthesis can generate damage (i.e. oxidative) in leaves. Therefore plants, as in animals, have developed many preventive and repair mechanisms to deal with the damage [2].

Seed longevity The seed longevity record is ~2000 years dated by radiocarbon dating and is hold by date (Phoenix dactylifera L.) seeds found in an Herodian fortress overlooking the Dead Sea during 1963–1965 excavations of Masada [8]. Plants have many elaborate systems (protection, detoxification, repair) to assure seed longevity [9].

The rate of seed aging mainly determined by environmental and genetic factors and also by physical, chemical, and molecular factors. Environmental factors are especially temperature and seed moisture content. There are studies in rice and Arabidopsis showing the control of seed longevity by genetic factors. The testa (seed coat) plays a very important role in seed longevity by maintaining the weakest metabolic activity and protection (physical, phytochemical, enzymatic etc.) from many biotic and abiotic environmental stresses. Seed longevity is influenced by protective chemical compounds like flavonoids, vitamin E and GABA. Protective proteins like late embryogenesis abundant (LEA) proteins, heat shock proteins (HSPs) and seed storage proteins also play an important role. Detoxification of free radicals is assured by many antioxidant enzymes such as such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GSHPx), and glutathione reductase (GSSGR). Seeds have mechanisms to remove other toxic compounds like cyanide. Efficient repair and turnover of macromolecules like DNA and protein is very crucial for seed longevity [9].

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Future prospects The presence of very long living tree species like Pinus longaeva [3] and 9550 years old spruce [4], and the discovery of ~2000 years old germinating date seed suggest that natural selection has possibly found a way to prevent aging in these organisms. This provides us very great opportunities in aging studies. The wide range health benefits of pycnogenol [10], a bark extract of Pinus maritimus that is also a long living tree, gives the hope to discover other phytochemicals ensuring plant longevity from especially oldest living plants and seeds that have the potential to be the weapons in our war against aging and age associated diseases. We can easily test the extracts and phytochemicals from longest living plants and seeds on model organisms like yeast and C. elegans in a very short time.

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Ljubuncic, P. and Reznick, A. Z. 2009. Gerontology 55, 2, 205.

Guarente, L., Ruvkun, G. and Amasino, R. 1998. Proc Natl Acad Sci U S A 95, 19, 11034.

3. Lanner, R. M. and Connor, K. F. 2001. Exp Gerontol 36, 4-6, 675.

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5. Lim, P. O., Kim, H. J. and Nam, H. G. 2007. Annu Rev Plant Biol 58, 115.

6. Gepstein, S. 2004. Genome Biol 5, 3, 212.

7. Yoshida, S. 2003. Curr Opin Plant Biol 6, 1, 79.

8. Sallon, S., Solowey, E., Cohen, Y., Korchinsky, R., Egli, M., Woodhatch, I., Simchoni, O. and Kislev, M. 2008. Science 320, 5882, 1464.

9. Rajjou, L. and Debeaujon, I. 2008. C R Biol 331, 10, 796.

10. Rohdewald, P. 2002. Int J Clin Pharmacol Ther 40, 4, 158.

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The Field of Biological Aging: Past, Present and Future, 2011: 87-102 ISBN: 978-81-7895-513-1 Editor: Abdullah Olgun

6. Plant seed: A relevant model to study aging processes Erwann Arc, Laurent Ogé, Philippe Grappin and Loïc Rajjou Institut Jean-Pierre Bourgin (IJPB), Unité Mixte de Recherche 1318 Institut National de la Recherche Agronomique-AgroParisTech – Team: Physiology of Seed Germination. Route de St-Cyr, F-78026 Versailles Cedex, France

Summary. Seeds are the major form of dispersal of plants in the environment. Seeds of many plant species are exceptionally adapted to harsh environmental conditions provided they are in a state of desiccation. Spectacular cases of seed longevity have been reported. It’s one of the singular case of pluricellular, differentiate eukaryotic organ able to survive several years in anhydrobiosis.

Plant scientific community explore these fascinating aspects of seed aging thanks to the immense possibilities now offered to create/modify plants at a much faster rate and in a more accurate way than through classical and molecular genetic approaches and genomic tools. These investigations allowed unveiling seed specificities against aging processes.

Introduction Aging in photosynthetic plants differs in some major ways from the process in animals. Plant biologists clearly differentiate aging from

111111 Correspondence/Reprint request: Dr. Loïc Rajjou, Institut Jean-Pierre Bourgin (IJPB) Unité Mixte de Recherche 1318 Institut National de la Recherche Agronomique-AgroParisTech – Team: Physiology of Seed Germination. Route de St-Cyr, F-78026 Versailles Cedex, France. E-mail: loic.rajjou@agroparistech.fr

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senescence [1, 2]. Senescence in higher plants is a type of programmed cell death included in a genetically determined developmental process [3]. The term “senescence” is consequently applicable to a process that leads to the death of a cell, an organ, or a whole plant and occurs at the final stage of its development [4]. Plant cells display either mitotic senescence or post-mitotic senescence [5]. Loss of cell division ability in the meristematic cells is called mitotic senescence or replicative senescence. On the other hand, post-mitotic senescence occurs in mature organs such as leaves, flowers and fruits. Unlike senescence, aging affects all living systems and can be defined in a population as an increase of the probability of death throughout time. It corresponds to the progressive alteration of normal biological functions and structural cell components resulting from a gradual accumulation of molecular damages that increases the susceptibility to environmental stresses.

These changes will consequently have a direct impact on the functional integrity of organs, biological systems, and ultimately on the organism as a whole [6]. As in other kingdoms, survival of plant species mainly relies on the sexual reproduction, which gives birth to new individuals [7, 8]. Thus, the seed, resulting from this process, represents the main unit of dispersal and spread of flowering plants (Angiosperms). It contains an embryo (the future plant resulting from the fusion of the gametes), storage tissues (necessary for the establishment of the seedling) and a protective outer coat. According to their desiccation-tolerance properties, three main seed groups are differentiated: (i) viviparous-seeds, (ii) recalcitrant-seeds and (iii) orthodoxseeds. Several plant families of predominantly large-seeded tropical species are not tolerant to desiccation (viviparous-seed) or weakly tolerant to desiccation (recalcitrant-seed) and germinate prior to, or coincident with late seed maturation on the maternal plant [9]. Viviparous and recalcitrant plant conservation by seed storage is not possible. Indeed, these seeds rapidly lose viability if they are dried. On the other hand, in most plant species of temperate climates, so called “orthodox seeds” acquire tolerance to desiccation during their maturation on the mother plant and are dispersed in the soil in a quiescent dehydrated state [10]. This reduce metabolic activity allows seed survival in stressful environment sometimes during several years until the proper conditions for germination (transition from seed to seedling) will be encountered. If mature dry seeds are not collected for agronomical or industrial purposes, they will be stored buried in the soil after dispersal. The orthodox seed is consequently exposed to aging processes but is not concerned by senescence. As it gets older, the seed progressively loses its germination vigor and viability. In order to overcome adverse environments and optimize plant propagation “orthodox seeds” have consequently developed several resistance mechanisms responsible for an increased

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longevity. Seed longevity can be defined as the time during which the seed will keep its aptitude to germinate. It mainly depends on the plant species, the genetic background, the storage environment and the quality of the seed lot.

Spectacular cases of seed longevity have been reported. Indeed, radiocarbon dating allowed the determination of the age of date (Phoenix dactylifera L.) seeds at about 2000 years [11], sacred lotus (Nelumbo nucifera) seeds at 1300 years [12] or canna (Canna compacta) seeds at 600 years [13], all were found in the soil still alive and able to give progeny. These spectacular records of survival among eukaryotic living organisms make the seed an appropriate model to study the mechanisms allowing to cope with the progression of aging deteriorations. The comprehension of the survival mechanisms occurring during seed life and of theirs consequences in term of germination vigor is of paramount importance for seed industry and crop yield, as well as to manage seed conservation for the preservation of the plant genetic resources and biodiversity. Moreover seed physiology also provides experimental facilities to investigate aging. Seed aging can be artificially accelerated through a process called controlled deterioration treatment (CDT) and reverse by a pre-germination treatment that is called priming [14, 15].

Therefore, the physiological versatility of the seed has been extensively used as a powerful tool in functional genomics and genetic approaches that will be described, in order to document seed aging and their many controlling mechanisms of survival.

I. Seed life Following maturation on the mother plant, the orthodox seeds are dispersed in the environment in a low hydrated state (5-10% moisture content) resulting from a developmentally regulated period of dehydration [16]. As a consequence of this desiccation, the cytoplasm condenses and intracellular components become more crowded leading to an increase probability of undesirable interactions [17]. It can indeed result in an alteration of the proteome or membrane fusion [17]. Following their maturation, seeds of many plant species can also display a period of dormancy when they fail to germinate under otherwise favorable conditions [18]. The exact requirements for dormancy breaking and subsequent germination depend on the species and thus contribute to the adequacy of the plant to its environment by delaying germination until the meeting of appropriate conditions for seedling development [19, 20]. As a result of this blockage, the time between the end of maturation and germination can be drastically increase and in the case of physiological dormancy so do the longevity.

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One of the main problems from the seed point of view is that it can’t move by itself. Different evolutionary adaptations have enabled the optimization of seed dispersion through different means of transportation like wind or animals but finally most seeds will end buried in the soil. As a consequence, the seeds will have to cope with diverse biotic and abiotic stresses, proper to their direct environment, which can affect their viability and eventually participate in the progressive natural deteriorations induced by aging. But, it should be stressed that sudden loss of viability related to an instant damage as a mechanical stress, insect or animal attacks or sudden burst of temperature cannot be considered as aging alterations [21].

Among the numerous environmental factors known to affect seed longevity, the temperature and the moisture content appear to be the most important and this seems to be true both in controlled storage conditions and in the soil [22, 23]. Indeed, the longevity of a seed lot can be estimated quite accurately providing the temperature and the moisture content of the seed are known [24]. Moreover, it has been suggested that each 1% decrease in seed moisture content or 5-6°C reduction in temperature doubles the life span of the seed [25]. The rate of most biochemical reactions including theses implicated in the respiration process is strongly correlated to these two parameters. The lower the seed temperature and moisture content is, the slower the metabolism will be. The exact interplay between these two parameters is complicated and remains to be detailed. In the nature, the environment surrounding the seed can buffer variations in temperature and humidity related to the climate. The preservation of low moisture content is essential for seed survival in case of exposition to sub-zero temperature.

Otherwise, uncontrolled freezing might cause fatal damage to the structure [26].

Different experiments carried in controlled conditions have shown that most of the extrinsic factors are of negligible importance in cold dry storage of orthodox seeds [21]. But, in the field, numerous constraints such as the pH of the soil, the salinity, the oxygen availability, the light, the presence of toxic compounds and microorganisms can promote seed aging [16]. Seed structure also has an important influence on the aging rate (ratio surface area/volume, hardness and permeability of the seed coat, presence of mucilage…).

Moreover, the large heterogeneity and inequality of seeds to retain their germination vigor and viability among plant species and even among seeds within a lot highlight the importance of the genetic component in the determinism of seed longevity [27]. Seed deterioration is inexorable and the best that can be done is to control its rate [28].

Under aerobic conditions, a slow progressive alteration of cell constituents (lipids, sugar, proteins, nucleic acids…) has been reported [29].

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Many of these damages have been associated to the deleterious effect of reactive oxygen species (ROS) due to their high reactivity towards biomolecules [28, 30-37]. Indeed, most of the modifications observed during aging including lipid peroxidation, nucleic acid alteration, enzyme inactivation and protein degradation can be the result of oxidative events induced by ROS [38, 39]. In plants, it has been described that developmental processes and/or environmental stresses can induce endogenous ROS production [40-42] though the mechanisms involved in their generation in orthodox seeds remain elusive [38]. Besides their metabolically quiescent state, dry seeds can endure several non-enzymatic oxidative reactions such as lipid peroxidation [28, 39, 43, 44], protein carbonylation [45, 46] or Amadori and Maillard reactions [47-51] leading to a progressive accumulation of ROS associated damages during storage. This results in a weakness that can be fatal to the embryo during imbibition.

If the seed encounters suitable conditions for germination during its life, it may, if still viable, fulfill its purpose and release the young seedling. But as a consequence of the aging process, the seed germination vigor can be severely affected. In other word, the capacity of a seed lot to germinate rapidly, uniformly and in a wide range of environmental conditions can be impaired or destroyed. As the seed germination process mainly relies on stored mRNA and proteins [52], damages at the DNA level can result in an aborted development of the seedling.

II. Morphology and physical structure of seeds During its life a seed can be exposed to numerous adverse conditions reducing the embryo lifespan. To ensure the reproductive success of the species, plants have consequently developed mechanisms to enable long-term seed survival in soil. Some of these rely on the integuments and the properties of the glassy state of the cytoplasm.

The seeds of flowering plants consist of three genetically distinct components: (i) the embryo resulting from the fertilization of the egg cell in the embryo sac by one of the male pollen tube nuclei, (ii) the usually triploid endosperm (about 70% of angiosperm species) formed by the fusion of the two polar nuclei with the second spermatic nucleus, and (iii) the seed coat (or testa), representing the maternal tissues of the ovule, formed by the layers of the inner integument and the inner epidermis of the outer integument [53].

Due to its physico-chemical properties, the integument is of great importance for the preservation of the embryo for which it acts as a shield. Thanks to its hardness resulting from the strong drying and compression of the cell layers constituting the testa at the end of seed maturation, it represents a physical

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and chemical barrier that enable the seed to withstand environmental attacks which could otherwise alter its structure [54, 55].

In most seeds, one of the very important features of the testa is its brown color conferred by the presence of phenolic compounds [56]. It results from the flavonoids oxidation occurring during the later stages of seed maturation. This high concentration of flavonoids has often been associated with resistance to fungi [57-62] due to their antifungal and antinutritionnal properties [58, 63] allowing the seeds to maintain a healthy testa, byword for longevity. Other studies emphasize the importance of the impermeability of seed coat in particular to avoid solute leakage from the seed towards the medium, which has the effect of increasing the probability of pathogen infection [64], but also to regulate the absorption of water that can be deleterious to the embryo during imbibition [65]. For instance, in Vicia faba, tanin concentration has been shown to be correlated with the rate of water absorption [66]. Thus, in legumes, unpigmented seeds deteriorate faster and are more susceptible to imbibition damage upon germination [67, 68].

As discussed above, oxidative stress and reactive oxygen species (ROS) play an important role in seed aging processes during storage. It’s consequently interesting to note that the polyphenols of the seed coat also act as antioxidants and protect the embryo against oxidative stress [69, 70].

Moreover, flavonoids act as a UV screen to protect the inner tissues from ROS production by this mean [71-75]. Thus, the physico-chemical properties of the seed coat associated with the low moisture content (slowing down enzymatic activities) tends to limit the production of ROS in orthodox seeds.

The particular structure of the cytoplasm also participates in the protection against oxidative alterations. The seed completes its development, for most species growing in temperate climates, with a desiccation phase during which the cytoplasm switches from the liquid state to a glassy state [76-79]. The removal of water induces a super saturation of cytoplasmic components leading to an increase in cohesive forces between molecules and a decrease of molecular mobility in the cytoplasm [80, 81]. This glassy state corresponds to a relatively stable phase, near a solid except that it has properties of physical disorder of liquids. Even if the composition of the glass has been well studied there are still many uncertainties [79, 82-87]. It’s a complex structure in which different compounds interacts including sugars and proteins [82, 88, 89]. The main role of the glassy state in the protection of the seed embryo is the preservation of structural integrity of macromolecules [90-92]. To illustrate that, it has been demonstrated that the secondary structure of proteins in dry seeds of wheat is very stable and can be conserved during storage even after total loss of seed viability [93]. The

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cytoplasm viscosity has also an impact on the diffusion of the ROS within the cell, resulting in an action range restricted to the targets closest to the production site [94]. A very strong correlation has been demonstrated between the intercellular molecular mobility, the moisture content and seed life span [80, 81, 87]. To conclude, the glassy state is very important to prevent seed deterioration [49, 84, 87].

III. Chemical and biochemical feature of seed Since orthodox seeds are dispersed in natural environment in a dehydrated state, waiting for suitable germination conditions, the occurrence of oxidative injuries is reduced. However, the low water content in the embryo is also limiting for certain biochemical activities that are required to limit age related cellular damages and enable seed survival during long-term storage. It has been hypothesized that enzymatic detoxification and repair mechanisms are decisive to limit molecular damage at the end of seed development and to repair and renew macromolecules and cellular structures until seed stored in soil encounter hydrated conditions that could induce germination and growth [30].

A large panel of antioxidant components accumulates in dry seeds during the late maturation step on the mother plant and contributes to control seed storability. As discussed previously, flavonoids localized in the testa play an important role in seed protection during storage. Flavonols are also abundant in the embryo where they can apply their protective action by scavenging ROS and protecting membranes. Recent papers reported that germination changed the distribution profile of isoflavone [95, 96]. For instance, in soybean seeds, the total flavonoid aglycones content increased by five times during germination, whereas acetyl-conjugated forms remained at a low and constant level [96]. Flavonoid aglycones should be hydrophobic enough to incorporate into cell membranes and lipoprotein lipids and interfere with lipid peroxidation [97]. It is very likely that flavonoid protective role at the cell level is not due exclusively to their antioxidant and prooxidant properties.

Indeed, inductive or signaling effects may occur at lower concentrations than these required for effective radical scavenging. Further research will be needed to reveal in more detail the true signaling pathway of flavonoids in plants.

The α-Tocopherol is the member of the vitamin E group (α-, β-, γ-, and δ-tocopherolsand tocotrienols) with the most biologically significant properties. Members of vitamin E group are associated with antioxidant function [98]. These lipophilic molecules are very abundant in seeds in particular in oleaginous seeds (Arabidopsis, sunflower, rape, olives, palm,

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etc...). The primary function of vitamin E in plants is to limit non-enzymatic lipid oxidation during seed storage, germination and early seedling development. When α-tocopherol is attacked by lipid peroxy radicals, it becomes, via one-electron oxidation, the α-tocopheryl radical. The ascorbate–glutathione cycle recycles tocopheroxyl radicals to tocopherols. As a consequence of two-electron oxidation, α-tocopheryl radical becomes α-tocopherylquinone [99]. It was observed that the ratio α-tocopherylquinone/ α-tocopherol increased in general with increasing oxidative stress. Also, recent works tend to demonstate that tocopherols influence cellular signaling in seed and may modulate gene expression in the nucleus by affecting lipid peroxidation, and therefore the levels of oxylipins such as jasmonic acid [100, 101].

IV. Genetic approaches to identify genes involved in seed survival: QTL The advent of molecular markers and QTL (Quantitative Trait Loci) mapping has opened a new insight for improving agronomic species especially for multigenic and quantitative traits such as yield [102, 103], resistance or tolerance to biotic and abiotic stresses [104, 105] or nutritional quality [106, 107]. A large number of studies have been done on Rice, which is one of the most important crops worldwide, as it feeds over half of the world's population. Rice seed vigor and longevity traits were strongly concerned. It has been described a rapid deterioration of this crop under humid tropical regions [108] and a poor germination of aged seeds under anaerobic conditions [109]. Therefore, in a direct seeding system, breeders have been working since the last decade on the selection of age-tolerant rice varieties to ensure consistent quality in fields. QTL analyses conducted in parallel on rice [110] and Arabidopsis [111, 112] showed that longevity is controlled by several genetic factors. The resistance to stress conditions during seed storage is one aspect of longevity. Therefore it would be coherent to observe a co-localization between QTL for longevity in Arabidopsis and QTL associated with environmental stress tolerance such as germination in saline conditions [112], in drought conditions [113] or under low temperature [114, 115]. The presence of genes implicated in ROS detoxification in these regions like catalase, superoxide dismutase, and a gene homolog to glutathione S-transferase is in accordance with the assumption that seed deterioration is mediated by the production of ROS. Interestingly, a colocalization between QTL of longevity and QTL controlling oligosaccharide contents (sucrose, raffinose and stachyose) has been pointed out [111]. This

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result could be expected keeping in mind that these compounds are known to play a role in membrane and macromolecule protection upon desiccation and storage [116, 117] and also due to their ability to form glassy state in the cytoplasm [78]. Vigor and longevity are traits of a complex nature and it is therefore interesting to proceed by quantitative genetic methods but it is now necessary to continue this work with fine mapping and study of candidate genes by analysis of mutants to understand genetic mechanisms of these traits.

V. Application of high-throughput genomic analysis in order to identify candidate genes involved in seed longevity High-throughput genomic analyses of expression profiles were realized by transcriptomic [118, 119], proteomic [45, 52, 120, 121]and metabolomic [122] to unveil complex molecular mechanisms involved in seed germination. About 12 000 mRNA species were quantitatively detected in Arabidopsis dry mature seed [118]. These transcripts correspond either to the residues of the maturation program or those that are necessary for starting of the germination program. It has been demonstrated that the germinating Arabidopsis seed has a distinctive transcriptome pattern, divergent from those found in other plant organs such as roots, leaves, hypocotyls, siliquae and flowers [123]. A proteomic investigation of the joint liability of transcription and translation in seed germination concluded that only protein translation is indispensable for radicle emergence suggesting that germination-specific proteins are translated from stored mRNAs [52]. However, if de novo mRNAs synthesis in not required for germination, transcription appear necessary to enhance seed vigor. In addition to storage proteins, three major group of gene products (mRNA and proteins), corresponding to metabolism, protein synthesis, folding and stability and stress responses are predominant in dry seeds and synthesized during early stage of seed germination.

Interestingly, proteins associated with stress responses cover a large range of plant defense mechanisms such as detoxification (i.e: reactive oxygen species, reactive nitrogen species, cyanide‌), cell repair, chaperones or pathogenesis related proteins.

Seed storage proteins, localized in protein storage vacuoles (PSVs) constituting protein bodies, are degraded by hydrolytic enzymes to generate amino acids required for seedling growth. These enzymes are synthesized de novo in germinating seeds and transported to the PSV through the lytic vacuole (LV) pathway [124]. Previous studies demonstrated that vacuolar sorting receptor (VSR) proteins are associated with seed germination because

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VSR knockout Arabidopsis seeds are not able of germinate [125, 126]. The role of these storage proteins is not limited as a source of nitrogen but also as major reactive radical scavengers that prevent cellular damages [45, 46, 121].

Recently, lower germination rates of Arabidopsis mutants upon application of oxidative and/or osmotic stress strongly suggested the involvement of specific genes in stress response [127-129]. A close relationship seems to exist between the abundance of characteristic proteins and seed longevity [46, 130].

VI. Biotechnology tools for candidate gene identification and validation In order to confirm the involvement of candidate genes identified, several mutants have been analyzed. Control deterioration treatment (CDT) allows to artificially accelerate seed aging [14] and has been demonstrated to mimic molecular and biochemical events occurring during natural aging [46, 112].

Therefore, it constitutes a powerful tool to characterize the impact of mutations on seed longevity. However, up to date, due to the heaviness of a large-scale genetic screen based on CDT, no large mutant collection has been directly screen for defect in seed storability. Indeed, most of the seed longevity phenotypes have been characterized in already identified mutants mostly in the Arabidopsis plant model species or by reverse genetic approaches.

The reduce longevity of transparent testa (tt) and banyuls (ban) mutants [131] altered in the flavonoid synthesis pathway and consequently in the composition of the testa is in accordance with the protective role attributed to these compounds. It has also been shown that mutants with defects in the integument (ats, aberrant testa shape ; ap2, apetala2 ; gl2, glabra2) display an impaired viability phenotype during storage, underlining the importance of the tegument integrity [112, 131, 132].

Deficient mutants for antioxidant biosynthesis such as vitamin E (vte1 and vte2) also exhibit aging phenotype in CDT treatments [133]. The orthodox seed is well equipped to control the prooxidant/antioxidant balance in order to prevent damages of cellular content. Nevertheless seed sensitivity phenotypes to aging treatments for other mutants affected in free radical detoxification such as vitamin C deficient (vtc1-1) mutant, catalase double mutant (cat1cat3), or glutathione deficient mutant (cad2-1) are not clear [112]. Although these mutants still accumulate residual amount of the antioxidant or of the detoxifying enzyme targeted by the mutation, it is thought that specific ROS scavengers, such as vitamin E, peroxiredoxin or superoxide dismutase act predominantly in seed [134].

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Recently, genetic modulation of the Protein L-isoAspartyl Methyltransferase 1 (PIMT1) using T-DNA insertion mutant and transgenic lines [135] allowed to demonstrate that protein repair of age related protein damage contributes importantly to seed longevity. PIMT enzyme activity reduces the level of deamidated and isomerized aspartyl and asparaginyl residues that represents a significant part of the spontaneous damage to proteins [136]. Alteration of protein conformation by isoaspartyl (isoAsp) accumulation has been described as a root case of seed aging. Although oxidative stress is known to enhance these protein alterations, isoAsp formation is not chemically an oxidation. This suggests that not only oxidative process contribute to aging. How this metabolism of protein modification control seed survival remains to be elucidated. The identification of altered proteins that are repair by PIMT will be promising to discover important actors of seed longevity.

Mutants affected in seed development such as leafy cotyledon1 and 2 (lec1, lec2) and fusca3 (fus3) or impaired in the plant hormone abscisic acid (ABA) synthesis and signaling such as aba1, abi3 and green seed (grs, enhancer of abi3-1) [14, 137-139] are also afflicted in seed longevity. It has been documented that abi3 and lec1 seeds are impaired in the accumulation of seed storage protein accumulation that could have protective property for seed cellular compounds. Nevertheless resistance mechanisms that are controlled by these loci remain to be understood. A recent screen for suppressor of abi3-5 (sua) aging sensitivity phenotype permitted to select 4 interesting mutants and their molecular characterization will be helpful to describe subtle controlling processes of seed storability [130]. Interestingly seed-specific overexpression of the heat stress transcription factor HSF in tobacco (Nicotiana tabacum) both enhances the accumulation of the heat shock protein HSP101 and small HSPs and improves resistance to controlled deterioration [140]. These findings point out the importance of the control of the expression of stress protein in seed longevity.

Conclusion Plant seeds belong to the most impressive examples of organism longevity in eukaryotes and provide a suitable model to study resistance mechanisms against aging. This system used along with the diverse techniques available on plant material such as functional genomics, insertion mutant lines and quantitative trait locus (QTL), constitutes an extremely powerful tool to study aging. Therefore, the seed constitute a pertinent model to dissect much further the complex mechanism associated with the longevity.

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Transworld Research Network 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

The Field of Biological Aging: Past, Present and Future, 2011: 103-122 ISBN: 978-81-7895-513-1 Editor: Abdullah Olgun

7. Gerontology in Russia: Past, present and future 1

Vladimir N. Anisimov1 and Olga N. Mikhailova2

Department of Carcinogenesis and Oncogerontology, N.N. Petrov Research Institute of Oncology, St. Petersburg, Russia; 2St.Petersburg Institute of Bioregulation and Gerontology, St. Petersburg, Russia

Abstract. The chapter presents major stages of gerontology development in Russia. The issues of training in gerontology and geriatrics, institutional infrastructure within Gerontological Society of the Russian Academy of Sciences have been considered therein.

Special attention is given to the prospects of gerontology in Russia.

Major stages of gerontology development in Russia The analysis of gerontological science in Russia has been given in recent papers which also outlined prospects for its development in the coming years (1-8). There are few key issues in the history of Russian gerontology. First of all, this is a book by I. Metschnikoff «Etudes sur la nature humaine: Essai philosophie optimiste» (1903) (9), where he introduced the term «gerontology» and put the cornerstone of the scientific discipline in biology and physiology of aging. In the 20-ies of the ХХ century the works of N.A. Belov, A.A. Bogdanov, S.A. Voronov, M.S. Milman, I.I. Schmalhausen not only evoked interest towards the investigation in the processes of aging per se, 11111 Correspondence/Reprint request: Prof. V.N. Ansisimov, N.N. Petrov Research Institute of Oncology, Pesochny-2 St.Petersburg, 197758, Russia. E-mail: aging@mail.ru

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but also raised the question on the possible increase in the life span of animals and humans. The 30-40-ies are characterized by the origin of the first national gerontological schools in this country – in Kiev and Kharkov (A.A. Bogomolets, A.V. Nagorny, I.N. Bulankin) and in Leningrad (Z.G. Frenkel, E. S. Bauer, V.G. Baranov). In 1938 in Kiev there took place the first scientific conference on aging. In 1957 in Leningrad on the initiative of Z.G. Frenkel there was organized the very first in this country City Scientific Society of Gerontologists and Geriatricians (3). The same year in Moscow there was organized the section of gerontology within Moscow Society of Nature Testers (MSNT) (10). In 1958 there was established Research Institute of Gerontology of the USSR Academy of Medical Sciences in Kiev with Academic Councils in Gerontology of the USSR Academy of Medical Sciences and Academy of Sciences on its basis (11). In 1963 in Kiev there took place the first All-Union Conference (Congress) on Gerontology and Geriatrics where the All-Union Research Medical Society оf Gerontologists was set up. It functioned successfully till the end of the 80-ies. The Academic Council in Gerontology and Geriatrics of the USSR Academy of Medical Sciences alongside with section «Biological and Social Bases of Gerontology» of the Joint Research Council in Human Physiology of the USSR Academy of Sciences and Academy of Medical Sciences coordinated research work in all union republics. In 1970 Kiev Medical Institute for Postgraduate Education opened the first in the country Chair of Gerontology and Geriatrics. In this period, the network of geriatric rooms at the health institutions was set up. A long-term All-Union comprehensive research programme in gerontology and geriatrics was elaborated in the period from 1981 to 1990. This period is characterized by active development of gerontology in the Ukraine (D.F. Chebotarev, V.V. Frolkis, V.N. Nikitin), and other regions of the country – in Leningrad (I.I. Likhnitskaya, N.S. Kosinskaya, M.D. Alexandrova, V.M. Dilman), Moscow (I.A. Arshavsky, N.M. Emanuel, B.F. Vanyushin, I.V. Davydovsky, L.V. Komarov), Tbilissi (N.N. Kipshidze), Kishinev (V.Kh. Anestediadi), Minsk (T.L. Dubina). Of great importance appeared to be workshops «Basic problems of aging» organized by N.M. Emanuel (1970-1984). The Group (Laboratory) of Mechanisms of Aging was organized by V.M. Dilman in 1973 at the Institute of Experimental Medicine in Leningrad. Four All-Union Congresses were held in 1972, 1976, 1982 and 1988. In 1986 there was organized the first in Russia Chair of Geriatrics at the Leningrad Institute for Postgraduate Studies, in 1990 in Kiev the first issue of the All-Union (further Ukrainian) Journal “Problems of Aging and Longevity” saw the light. Major stages of Russian gerontology development up to middle 80-ies of the last century are described in the monograph of Yu.K. Duplenko (12).

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Desintegration of the USSR resulted in the collapse of all former AllUnion structures and actual closure of systematic studies in gerontology and geriatrics on the territory of the Russian Federation. Practically anew we started collecting professionals and establishing research and practical institutions of this profile. The first regional gerontological centre was set up in Nizhny Novgorod in 1989, then followed city geriatric centre in St. Petersburg in 1994. In 1992 there was founded the Institute of Bioregulation and Gerontology in St. Petersburg. In 1992 on the initiative of Dr. L.D. Itkina there was organized Moscow (later – inter-regional) association “Gerontology and Geriatrics”, which consolidated physicians, practicing in the field of geriatrics.

The convocation of the All-Union founding conference «Medical and social aspects in gerontology and geriatrics» organized by the St. Petersburg scientific gerontological society in March 1994 in St. Petersburg became a crucial moment in the modern history of Russian gerontology. It set up Gerontological Society which in November 1995 was granted the status of the Institution at the Russian Academy of Sciences (RAMS) (Resolution of the Presidium of the Russ. Acad. of Sci. № 241 of 28 November 1995).

Gerontological Society united leading scientists in gerontology and geriatrics around the country irrespective of their agency belonging. In 1994 there was set up a Chair of Gerontology and Geriatrics at the Russian Medical Academy for Postgraduate Studies in Moscow. In 1995 by the resolution of the RF Health Ministry there was adopted a new medical speciality «physician-geriatrist». The same year the first issue of the Journal «Clinical Gerontology» (Moscow) and the manual for physicians «Practical geriatrics» (Samara) saw the light. In 1996 Gerontological Society joined European Regional Branch of International Association of Gerontology (IAG). The same year at the premises of the Samara Regional Hospital for War Veterans there was opened a research institute «International Centre for the Problems of the Aged». In 2007 the Institute was reorganized into geriatric centre.

Regular issuing of the information bulletin «Herald of the Gerontological Society of RAS» (www.gersociety.ru) started since 1996. In 1997 in Moscow there was set up Russian Research Institute of Gerontology of the RF Ministry of Health, and in the Russian Academy of Medical Sciences there took place first elections on the speciality “Gerontology and Geriatrics” (V.S. Gasilin). In August 1997 at the 16th IAG World Congress in Adelaide (Australia) Gerontological Society was accepted into the IAG and its representatives entered IAG Council. The textbook «Gerontology and Geriatrics» (13), the first issue of the journal «Advances in Gerontology» (St. Petersburg) and the 1st issue of the journal «Psychology of Maturity and Ageing» (Moscow), Abstract Bulletin «Gerontology and Geriatrics»

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appeared in 1997. In 1998 Scientific council on gerontology and geriatrics of the Russian Academy of Medical Sciences and RF Ministry of Health, International centres for older people on the basis of regional hospitals for war veterans in Ul'yanovsk and Yaroslavl were set up and started their work.

The first Russian Congress of Gerontologists and Geriatrists was held in 1999 in Samara. Resolution of the Russian Ministry of Health № 297 of 28 July 1999 «On the improvement of medical assistance to old and senile citizens in the Russian Federation» played a significant role in the development of national geriatric service. This document provided for organization of geriatric centres throughout the country, departments of medical and social assistance to the elderly within out-patient clinics and other important measures, including those on professional training. In 2000 Saint Petersburg hosted the 2nd European Congress on Biogerontology with 300 participants from 33 countries. Scientific Journal “Bulletin of Experimental Biology and Medicine” has a permanent section «Biogerontology». Since 2001 annual almanac «Gerontology and Geriatrics» has been issued by the Russian Research Institute of Gerontology.

In June 2002 in Moscow there was held the 6th European Congress of Clinical Gerontology, and in October 2003, also in Moscow, – the 2nd Russian Congress of Gerontology and Geriatrics. The 6th European Congress of IAGG held on 5-7 July 2007 in St. Petersburg was an event of utmost importance for European and Russian gerontology. It gathered over 1500 participants from 70 countries of the world.

It is worth noting that for the first time ever the Russian institution has been designated a Collaborating Centre of the International Association of Gerontology and Geriatrics. This institution is St. Petersburg Institute of Bioregulation and Gerontology of the North-Western Branch of the RAMS. This is a sign of international recognition of the research achievements’ of Russian gerontology.

Chronology of important events in the development of Russian gerontology is given in Table 1.

Table 1. Chronology of Russian gerontology development (1957-2009).

Year 1957

Event Set up of the Leningrad scientific society of gerontologists and geriatricians Organization of the gerontological section in the Moscow Society of Nature Testers (MSNT) 1958 Establishment of the Research Institute of Gerontology of the USSR Academy of Sciences (Kiev) 1963 1st All-Union Conference (Congress) of Gerontologists and Geriatricians; Set up of the All-Union Research Medical Society of Gerontologists and Geriatricians 1970-84 Workshops «Fundamental problems of aging» (Moscow)

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Table 1. Continued 1973 1986 1991 1989 1992

1994

1995

1996

1997

1998

1999

2000 2001

The Group (Llaboratory) of Mechanisms of Aging at the Institute of Experimental Medicine, Leningrad The Chair of Geriatrics at the Leningrad State Medical Institute for Postgraduate Studies The 1st issue of the journal “Problems of aging and longevity” (Kiev) Nizhny Novgorod Regional Geriatric Centre Foundation of the Moscow (later Inter-regional) association “Gerontology and Geriatrics”; Foundation of the St. Petersburg Institute of Bioregulation and Gerontology (since 1999 – member of the North-Western Branch of the Russian Academy of Medical Sciences) All-Union founding conference «Medical and social aspects in gerontology and geriatrics» (St. Petersburg); Organization of the Gerontological Society of the Russian Academy of Sciences; The City Geriatric Medical and Social Centre (St.Petersburg); The Chair of Gerontology and Geriatrics at the Russian Medical Academy for Postgraduate Studies (Moscow) Adoption of the medical specialty “physician-geriatrist”; The 1st issue of the journal “Clinical Gerontology” (Moscow); Institution of the annual award of the Gerontological Society of the Russian Academy of Sciences for young scientists from Russia for the best work in gerontology Samara Research Institute “International Centre for the Problems of the Aged”; The 1st issue of the “Herald of Gerontological Society of the Russian Academy of Sciences” (St. Petersburg); The 1st issue of the journal “Older Generation” (Samara) Set up of the Gerontological Society of the RAS joined International Association of Gerontology; Foundation of the Russian Research Institute of Gerontology of the Russian Federation Ministry of Health (Moscow) The 1st issue of the journal “Advances in Gerontology” (St. Petersburg); The 1st issue of the journal “Psychology of Maturity and Aging” (Moscow).

The 1st issue of the abstract bulletin “Gerontology and Geriatrics” (Moscow); First elections in “Gerontology and Geriatrics” at the Russian Academy of Medical Sciences Organization of the Scientific council on gerontology and geriatrics of the Russian Academy of Medical Sciences and Russian Federation Ministry of Health; Organization of International centers on the problems of the aged in Ul’yanovsk and Yaroslavl’ 1st Russian Congress of Gerontologists and Geriatrists (Samara); Issue of the textbook “Gerontology and Geriatrics” (Samara); Organization of section “Gerontology and Geriatrics” at the Academic Council of the Russian Federation Ministry of Health 2nd European Congress on Biogerontology (St. Petersburg) Organization of the Institute of Social Gerontology at the Moscow State Social University; 1st Congress of gerontologists and geriatrists of Siberia and Far East (Novosibirsk); European School of Oncology “Cancer in the elderly: achievements and prospects” (Moscow); Institution of a new scientific specialty 14.00.53 - “Gerontology and Geriatrics”; Organization of two dissertation councils for upholding doctorate and candidate thesis in gerontology and geriatrics (Moscow, St.Petersburg)

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Table 1. Continued 2002

2003 2004

2005 2007

2008

2009

6th European Congress on Clinical Gerontology (Moscow) Decision of the Board of the RF Ministry of Health of 25 June 2002 “On the state of the art and development of investigation in the field of gerontology in the Russian Federation” 2nd Russian Congress of Gerontologists and Geriatricians (Moscow) Dissertation Council on Gerontology and Geriatrics at the Research Centre of Clinical and Experimental Medicine of the RAMS Siberian Branch in Novosibirsk; The Highest Attestation Committee of the RF Ministry of Education for the first time awarded an academic title of “Professor” in “Gerontology and Geriatrics”.

2nd International School on Gerontology and Geriatrics (St. Petersburg) th The 6 European Congress of the International Association of Gerontology and Geriatrics (St. Petersburg); The 3rd International School on Gerontology and Geriatrics (St. Petersburg) Programme “Prevention of age-related pathology and accelerated aging, decrease of premature mortality from biological causes and an extension of healthy period of the life for thew population of Russia”; UN Workshop on Formulation and Implementation of Policy on Aging for the countries of the former USSR (St.Petersburg) The first edition of a complex interdisciplinary programme of basic research “Science against aging”

Professional training in gerontology and geriatrics in Russia A uniform system of education in geriatrics has been set up in Russia with respect to International experience. Therefore, there have been set up Chairs of gerontology and elaborated regulations (7). Teaching of gerontology in Russia has been included into curricula since 1993, and speciality "physician-geriatrist" was adopted by the resolution of the RF Ministry of Health and RF Ministry of Education № 33 of 1995. However, back in 1977 there was issued a Resolution of the USSR Ministry of Health "On the organization of Chairs of geriatrics in medical institutes and institutes for postgraduate studies to train physicians-gerontologists». The system of personnel training in gerontology in the USSR takes its beginning from the onset of postgraduate course. As it was noted above, the first in the country Chair of Gerontology and Geriatrics was organized in 1970 on the basis of the Research Institute of Gerontology of the USSR Academy of Medical Sciences in Kiev for the needs of the Kiev Institute for Postgraduate Studies of Physicians, and in 1986 there was set up the first in Russia Chair of Geriatrics in Leningrad State Institute for Postgraduate Studies, where the Course on Geriatrics has been launched since 1980 at the Chair of Therapy.

The significance of created system for personnel training at all levels (physicians, medical assistants, nurses) in the field of geriatrics was stated in

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the Resolution of the RF Ministry of Health № 297 of 28.07.99 «On the improvement of medical assistance to old and senile citizens in the Russian Federation».

In 1994, the Chair of Gerontology started its work at the faculty for postgraduate studies of the Moscow Medical Dentistry Institute. Later on, in 1995 it was re-subordinated to the Russian Medical Academy for Postgraduate Studies. There were elaborated qualification requirements to physician-geriatrist in 1995 with its second edition in 1998; statute of physician-geriatrist in 1996, with its second edition in 1998; qualification tests for speciality physician-geriatrist in 1996 with its second edition in 1998; uniform programme for postgraduate training of physicians in gerontology and geriatrics (1997). On the basis of these documents there was developed the document «Standard training of physician-geriatrist» at the I.M. Sechenov Medical Academy in Moscow.

According to the requirements developed on the basis of generalized world experience, geriatric education in Russia should be multilevel and multifocal (13). Undergraduate teaching in fundamental, social and medical gerontology and gerontotechnologies should be conducted during the whole course of studies at therapeutic and dentistry faculties of medical higher schools with the help of uniform end-to-end programme of teaching at all Chairs. Postgraduate training of doctors should include general advancement in geriatrics, social gerontology and prevention of premature aging (for physicians of general practice, district out-patient doctors and therapists working with old people) during 144 academic hours; primary specialization in «geriatrics» (for the staff of geriatric centres, hospitals and departments of medical and social care) for at least 144 hours; attestation cycles for certified specialists – 72-144 hours; topical advancement according to separate sections of gerontology for health care organizers and «narrow» specialists.

Paramedical personnel needs training in medical, social and psychological rehabilitation, as well as gerontotechnologies.

Along with the Chair of Gerontology at the Russian Medical Academy for Postgraduate Studies other institutions carry out postgraduate training in gerontology and geriatrics. Thus, the Chair of Gerontology and Geriatrics of the Russian State Medical University is open at the premises of the Russian Research Institute of Gerontology; I.M. Sechenov Moscow Medical Academy has the Chair of Geriatrics and Hematology. The Chair of Gerontology and Geriatrics of the St.Petersburg Academy for Postgraduate Studies conducts training in clinical gerontology and medical-social expertise, as well as rehabilitation of old and senile patients. Advancement cycles are held for geriatrists and therapists with subsequent examination for the certificate of specialist – geriatrist, as well as for paramedical personnel

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with further exam and award with the certificate of geriatric nurse. Chair researchers carry out substantial scientific and practical work on age-related pathology of cardio-vascular, gastro-intestinal and genitourinary systems (14). In St. Petersburg, the license for educational activity in the area of professional (postgraduate and additional) training in gerontology and geriatrics was granted also to the St. Petersburg Institute of Bioregulation and Gerontology of the North-Western Branch of the Russian Academy of Medical Sciences. The Chairs or courses in gerontology and geriatrics are open in medical higher schools in Ekaterinburg, Yoshkar-Ola, Nizhny Novgorod, Novosibirsk, Rostov-on-Don, Yaroslavl and other Russian cities.

As for undergraduate training, there should be first of all noted a tremendous work carried out by the Chair of Geriatrics of Samara State Medical University. Moreover, the courses in gerontology and/or geriatrics are conducted more than in 10 medical higher schools. For example, St. Petersburg State Medical University named after I.P. Pavlov initiated elective cycle on gerontology and geriatrics for students of the 6th course.

The cycle includes lectures on demography and geography of aging, concepts and mechanisms of aging, geroprotectors, interrelation of cancer and aging, major aspects of geriatrics, its diagnostic peculiarities, characteristic features of nutrition in old and senile age. Practical studies are focused on organization and medical and social assistance to the aged, participation in their therapy. The students and masters of the St. Petersburg State University (the Chair of genetics and selection of biology and soil faculty and medical faculty) enjoy lectures in gerontology.

Of great importance is the task of training paramedical personnel. Since 1997 there has been introduced into practice the State educational standard for paramedical personnel (speciality «nurse management»), where issues of gerontology and geriatrics are given within the following disciplines: «The stages of human life and medical services to various groups of population» (the 1st year of education) and «Geriatrics» (the 3rd year of education.)

70 hours are allocated in the curricula to the issues of gerontology and geriatrics. Similar training of nurses has started in several regions. Thus, training programmes in geriatrics have been elaborated with the help of leading specialists of St. Petersburg City Geriatric Centre and the Chair of Family Medicine of the Medical Academy for Postgraduate Studies and the training process has been carried out in St. Petersburg medical college №2.

Experience and methodological assistance of professionals from the USA, England and Finland is widely utilized in the training process.

Alongside training of medical workers in gerontology and geriatrics there has been undertaken personnel training in social sphere starting from 1992 (15). It has been conducted in compliance with the state educational

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standards of higher professional education on specialties 350500 “Social work” and 031300 “Social pedagogy”. «Social work» standard has a special discipline «Social gerontology» therein. Demographic aspects are included into the course “Social politics”, the issues of pensioners' rights protection – into the course “Legal coverage of social work”, gender issues – into the course «Genderology and Feminology». According to the state educational standard, professional social work embraces population social protection, work with different social, age, gender and ethnic groups, individuals in need of social assistance and protection. Basic curricula include humanitarian, socio-economic and natural sciences. This multidisciplinary training has an integrative character. Postgraduate courses for social workers in the field of gerontology are open in many national universities.

Personnel training for social and medical gerontology is well organized in Republic Bashkortostan at the Institute for Postgraduate Studies of the Medical University, Bashkir State University, Medical colleges (16). During several years professional training in social gerontology is carried out at the M.V. Lomonosov Pomorsky State University in Arkhangelsk.

International schools in gerontology launched in Russia gave a new impulse to personnel training. Bearing in mind acute interest at the national level towards geriatric oncology and contribution of Russian gerontologists to the development of this issue, European school of oncology «Cancer in the elderly: achievements and prospects» was organized in November 2001 at the Russian Cancer Research Centre named after N.N. Blokhin of the Russian Academy of Medical Sciences (Moscow). In 2002 and 2004 there were held International schools on gerontology and geriatrics organized by the International Institute on Aging –UNO (Malta), St. Petersburg Institute of Public Health at the Medical Academy for Postgraduate Studies and City Geriatric Medical and Social Centre.

Joint Finland - St. Petersburg projects «Personnel training for geriatric services» and «Development of geriatric services in St. Petersburg» may serve a vivid example of International collaboration in professional training.

Within the framework of these projects doctors, nurses and social workers take postgraduate course in gerontology in Finland (Turku and Tampere).

Textbooks in gerontology issued in Russia serve a good practical basis for personnel training (17-33). A valuable tutorial for students and doctors is «Glossary on social gerontology» (34). International editions and manuals are being translated into Russian. There are manuals summarizing experience of colleagues outside Russia, in particular, the experience accumulated in the United States and Great Britain (23,24,25).

Distant Internet-based course seem to be a prospective undertaking.

Thus, Center «Compassion» within the project «Personnel training for the

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care of the aged» supported by Foundation «EuroAsia» made a web site (www.openweb.ru/gerocomp), which includes 6 sections containing information regarding medical and social care. One of these section «Training» contains materials describing social work useful for distant training of social workers of Siberian region (34). Regional Public Foundation for Aged People Assistance «Dobroye Delo» has a web site www.dobroedelo.ru, containing materials ment for the personnel of nonprofit organizations working in the area of social and medical assistance to the elderly and old people, as well as for students of sociology faculties and those who are interested in the issues of social gerontology. On the basis of this site there has been made a Virtual Resource Centre of gerontological non-profit organizations providing distant training (www.dobroedelo.ru/vrc).

Of utmost importance for the development of national gerontology is training of researchers. Recognition of the research speciality «Gerontology and Geriatrics» would be an essential step towards the development of the international system of training.

On the initiative of the Gerontological society a scientific specialty No 14.00.53 «Gerontology and Geriatrics - medical and biological sciences» has been introduced into the official list of specialties of the Russian Federation Ministry of Industry and Science in 2001, 2 dissertation councils were set up: at the St. Petersburg Institute of Bioregulation and Gerontology of the NorthWestern Branch of the Russian Academy of Medical Sciences and Russian Research Institute of Gerontology of the Russian Federation Ministry of Health; first hundred and eighty thesis were defended on the new specialty.

The dynamics of dissertations upheld in gerontology and geriatrics in 20012006 is presented in Fig. 1. The team of St. Petersburg professionals prepared and published in 2002 a guidebook for education and training of gerontologists and geriatricians on specialty 14.00.53 (Rules and Regulations) (36).

This specialty (14.00.53) was for the first time introduced into the list of specialties of researchers in this country by the Decree of the Minister of Industry, Science and Technology of the Russian Federation of January 31 2001. The Decree of the Highest Attestation Committee of the Russian Ministry of Education of June 2001 established the Dissertation Councils for Doctorate and Candidate. thesis in gerontology and geriatrics at the Russian Research Institute of Gerontology of the Russian Ministry of Health (Moscow) and St. Petersburg Institute of Bioregulation and Gerontology of the North-Western Branch of the Russian Academy of Medical Sciences. The first dissertation was upheld in December 2001 at the St. Petersburg Institute of Bioregulation and Gerontology. Professor G. Gutman emphasized that this event could be instrumental for the development of gerontology worldwide at the Assembly of IAG Council in Valencia in April 2002. In June 2004 the

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Figure 1. Dynamics of the number of dissertations upheld on specialty 14.00.53 — gerontology and geriatrics, 2001–2008.

third Dissertation Council in gerontology and geriatrics was opened at the Research Centre on Clinical and Experimental Medicine of the Siberian Branch of the Russian Academy of Medical Sciences in Novosibirsk. It is worth noting that numerous researchers from Ukraine, Belarus, Kazakhstan and Uzbekistan upheld their theses at the Dissertation Councils in Russia.

The award and mutual recognition of scientific degrees in different countries will foster education and training of researchers and finally, progress of gerontological studies.

The work of young researchers in the laboratories outside Russia in the framework of joint projects constitutes an important aspect in education and training. Young scientists awarding with grants and prizes of national gerontological societies plays its role as well. For instance, Gerontological Society of the Russian Academy of Sciences instituted in 1995 and awards annually since then the best work among young scientists and recommends young professionals for training at the International schools and courses in gerontology. Presidium of the Board of the Ukrainian Scientific Medical Society of Gerontologists and Geriatricians instituted acad. V.V. Frolkis award for researchers, tutors, postgraduate students from national and international institutions aged under 35 years in order to attracts young scientists to basic studies in gerontology. Academic Council of the Research Institute «International Centre for the Problems of the Aged» (Samara) instituted an annual prize named after T.I. Eroshevski for the best research work among Russian scientists in the field of medical gerontology. The prize is granted for the best monograph, thesis, cycle of papers or other publications. In accord with recommendation of the IAGG President

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G. Gutman many national gerontological societies set up sections for young professionals and made up a programme for exchange of young specialists.

Postgraduate training of geriatricians is carried out at 10 Chairs of Gerontology in Medical Academy for Postgraduate Studies and faculties for advanced studies. Students get education at the Chair of Geriatrics in Samara Medical University, Chair of Haematology and Geriatrics of the I.M. Sechenov Moscow Medical Academy, course of gerontology in Perm Medical Academy and some other medical higher schools. In October 2001 for the first time ever in Russia St. Petersburg Institute of Bioregulation and Gerontology got a license of the Russian Ministry of Education and Science authorizing professional postgraduate education on speciality 14.00.53 «Gerontology and Geriatrics», professional training and advanced qualification of administrative staff on the Institute profile.

Thus, during recent decade there has been created a research and institutional infrastructure in Russia for the development of gerontology and geriatrics: research institutions (Russian Research Institute of Gerontology of the Russian Health Ministry, Moscow; St. Petersburg Institute of Bioregulation and Gerontology of the North-Western Branch of the Russian Academy of Medical Sciences), number of big gerontological (geriatric) centres. There have been issued special scientific journals and guidebooks providing information basis for research and training in new scientific speciality «gerontology and geriatrics” and new medical speciality «geriatrician».

Organizational activity of the gerontological society of the Russian academy of sciences (RGS) on development of research in Russia The primary objectives of the Gerontological Society consist in promoting the development of gerontology and related fields of physiology and biology; integrating research results with practice; establishing and maintaining contacts with scientific gerontological institutions of the CIS and other countries and with international non-governmental scientific organizations; organizing and convening meetings to exchange and discuss research and practical issues; assisting Society members in improving their professional skills and research activities; providing research and methodological assistance in teaching gerontology and geriatrics at higher schools and those for paramedical personnel; membership in international scientific associations and participating in the international meetings; fostering and distributing knowledge and recent scientific achievements in the field of Society's activity.

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RGS was founded in 1994 and included 7 regional branches at that moment. The participants of the Founding conference numbered 100 people.

Today it embraces 45 branches with about 1500 members from over 50 regions: Moscow, Saint Petersburg, Altay (Barnaul), Arkhangelsk, Astrakhan, Bashkir (Ufa), Buryat (Ulan-Ude), Chelyabinsk, Chita, Sverdlovsk (Ekaterinburg), Far East (Khabarovsk), Irkutsk, Ivanovo, Kabardino-Balkarian (Nalchik), Karelian, Krasnoyarsk, Kursk, Magadan, Mari El Republic (Ioshkar-Ola), Mordovian (Saransk), Minsk, Nizhny Novgorod, Novokuznetsk, N5ovosibirsk, Obninsk, Perm, Primosky (Vladivostok), Pushchino, Rostov, Ryazan, Samara, Saratov, Sochy, Stavropol, Syktyvkar, Tomsk, Tula, Tver, Tyumen, Ulyanovsk, Volgograd, Voronezh, Yaroslavl, Yakutsk.

Honorary members of the RGS are Professors V.V. Bezrukov (Kiev, Ukraine), L.A. Bokeria (Moscow), Butenko (Kiev, Ukraine), F.I. Komarov (Moscow), B.A. Lapin (Sochi-Adler), W.B. Ershler (Norfolk, USA), C. Franceschi (Bologna, Italy), M. Passeri (Parma, Italy), Y. Touitou (Paris, France).

Great attention in the Society is given to young researchers. In 1995 there was instituted the Award for young Russian scientists which is annually granted for the best research work in the field of gerontology and geriatrics.

Since then about 20 young researchers became its laureates. In 1999 within the framework of the National congress «Man and Medicine» (Moscow) there was open «the School of gerontologists». Upon the recommendation of the Gerontological Society a few young researchers and practitioners participated in the International courses and schools in gerontology and geriatrics (Romania, 1997; Italy, 1999, Malta, 2001-2007, Turkey, 2003, 2009). International schools in gerontology and geriatrics organized by the International Institute on Aging – UN (Malta) and Gerontological Society of the Russian Academy of Sciences were held in 2002, 2004 and 2007 in St. Petersburg at the City Geriatric Medical and Social Centre.

Over 120 scientific conferences and symposia, including 20 international ones have been organized since the date of Gerontological Society foundation in 1994. During recent years (1994 - 2007) there were held over 250 scientific conferences on different aspects of gerontology and geriatrics.

Among them it's worth mentioning such significant events as the 1st Russian Congress of Gerontologists and Geriatricians, Samara, 1999; the 1st Congress of Gerontologists and Geriatricians of Siberia and Far East, Novosibirsk, 2000; the 2nd European Congress on Biogerontology, 2000, St. Petersburg; the 6th European Congress of Clinical Gerontology, 2002, Moscow; the 6th European Congress of the International Association of Gerontology and Geriatrics, 2007, St. Petersburg; annual International conferences «Elderly Patient. Quality of life» (1996-2002), Moscow; International symposium

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«Gerontological aspects of peptide regulation of functions», 1996; annual International workshops on the problems of the aged «Samara lectures» (1996-2002), Samara; International conference «Contemporary approaches to geriatric estimation of a patient», 2000, St. Petersburg; International conference «Free radical processes and anti-oxidants in the development and functions of the nervous system: from fetus to aging», 2000, St. Petersburg; European school on oncology «Cancer in the Aged: Advances and Prospects», 2001, Moscow, etc.

Prospects of Russian gerontology development Despite growing interest to research in gerontology in Russia during recent 15 years, creation of infrastructure (establishment of profile research institutions, issue of new specialized journals, introduction of a new scientific speciality «Gerontology and Geriatrics», etc.) and a number of obvious scientific achievements of Russian gerontologists, it should be noted that there is definite lack of governmental support, financial, in particular, especially in regard to basic research. It dooms national gerontology to backlog in development and inhibits solution of urgent problems the country faces. Demographic situation in Russia (decreased birth rate, increased proportion of old people in the structure of population, especially in big cities, such as Moscow, St. Petersburg, Ekaterinburg and other, unprecedented decrease of expected life span, decreased number of people of the working age and their premature aging) and unfavorable demographic prognosis for the coming decades (36,37), put forward not only the issue of health in Russia, but its economic and political safety.

Has young and immature Russian gerontology a chance to «find feet»?

Isn't it doomed to stay an outsider, hardly making itself known in the world science on aging?

Table 2 contains a detailed list of priorities in up-to-date fundamental gerontology with reference to leading Russian institutions engaged in the studies at a high professional level providing publication of the results obtained in the reputable peer-reviewed national and international journals.

The contribution of Russian science into major priorities of the world biogerontology is manifested by few groups of researchers conducting up-todate studies. The research made by them in respect to above directions produce considerable and sometimes decisive impact on the solution of particular scientific tasks, which is confirmed by the level of publications, and their lecturing as invited speakers at the top International forums on gerontology, where they organize symposia and topical sessions and often are awarded with international grants.

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Table 2. Priorities in current biogerontology: Participation of Russia. â„– 1.

2.

Research Directions Population genetics of aging Genetics of aging and longevity in humans, Studying of centenarians

3.

Genetics of aging and longevity in animals

4.

5.

Progeria Use of transgenic and mutant animals in aging research

6.

DNA damage, DNA repair and aging

7.

Cell aging, telomere, telomerase Apoptosis and aging

8.

9.

Free radical theory of aging

10.

Aging of the brain and nervous system

11.

Aging of the neuroendocrine system

12.

Pineal gland and aging

13.

Peptide regulation of aging Geroprotectors

Leading Institutions in Russia N.I. Vavilov Institute of General Genetics, RAS (Moscow); Institute of Therapy, RAMS (Novosibirsk) St.Petersburg Institute of Bioregulation and Gerontology, RAMS; Institute of Therapy, RAMS (Novosibirsk); Research Institute of Gerontology of the RF Health Ministry (Moscow); D.O. Ott Research Institute of Obstetrics and Gynecology, RAMS (St.Petersburg); St.Petersburg City Geriatric Medical and Social Centre St. Petersburg State University; Institute of Genetics and Cytology, RAS (Novosbirsk); Institutes of Biology, RAS (Syktyvkar) Institute of Cytology, RAS (St.Petersburg).

N.N. Petrov Research Institute of Oncology (St. Petersburg); M.V. Lomonosov Moscow State University; Institute of Genetics and Cytology, RAS (Novosibirsk).

Institute of Theoretic and Experimental Biophysics, RAS (Puschino); Institute of Cytology, RAS (St.Petersburg); Central Research Roentgenology and Radiology Institute (St.Petersburg).

Instutute of Molecular Biology, RAS (Moscow) Institute of Cytology, RAS (St.Petersburg) A.N. Belozersky Institute of Physical and Chemical Biology (Moscow); M.V. Lomonosov Moscow State University; N.N. Petrov Research Institute of Oncology (St. Petersburg); Institute of Biology, RAS (Syktyvkar).

M.V. Lomonosov Moscow State University; N.M.Emanuel Institute of Biochemical Physics, RAS (Moscow); D.O. Ott Research Institute of Obstetrics and Gynecology, RAMS (St.Petersburg).

Institute of the Brain, RAMS (Moscow); Institute of the Human Brain, RAS (St.Petersburg); Ural Medical Academy (Ekaterinburg) I.P. Pavlov Institute of Physiology, RAS (St. Petersburg); I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, RAS (St.Petersburg); N.N. Petrov Research Institute of Oncology (St. Petersburg); St. Petersburg Institute of Bioregulation and Gerontology, RAMS; Research Institute of Medical Primatology, RAMS (Sochi-Adler); Tyumen Medical Academy St.Petersburg Institute of Bioregulation and Gerontology, RAMS; N.N. Petrov Research Institute of Oncology (St.Petersburg); A.N. Belozersky Institute of Physical and Chemical Biology, Moscow State University.

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Table 2. Continued 14.

Aging and cancer

15.

Theories of aging

16.

Mathematic models of aging

17.

Demography of aging

18.

Biomarkers of aging

N.N. Petrov Research Institute of Oncology (St.Petersburg); N.N.Blokhin Russian Cancer Research Center, RAMS (Moscow) M.V. Lomonosov Moscow State University; N.M.Emanuel Institute of Biochemical Physics, RAS (Moscow); The Institute of Chemical Physics, RAS (Moscow); Research Institute of Experimental Medicine of the RAMS (St.Petersburg) The Institute of Control Sciences, RAS (Moscow) The Institute of Numerical Mathematics, RAS (Moscow); St. Petersburg State University; Ul’yanovsk State University St.Petersburg Economy and Mathematics Institute, RAS; St.Petersburg Institute of Bioregulation and Gerontology, RAMS Perm Medical Academy; St. Petersburg Institute of Bioregulation and Gerontology, RAMS; The Institute of Systemic Analysis, RAS (Moscow)

Figure 2. Dynamics of publications of Russian authors in gerontology and geriatrics, 1994–2006.

The bulk of investigation carried out by Russian researchers according to certain directions is quite substantial, but unfortunately their publications are rather rare in leading international journals. Many of them, do not meet the requirements of such journals due to weak methodological basis, thus they cannot contribute to the development of the issue they dwell on. At the same time some of them could’ve undoubtedly shown up-to-date professional level should their research be supported and their laboratories up-graded.

Fig. 2 depicts dynamics of publications of Russian authors in gerontology

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and geriatrics in the period from 1994 to 2006 (39). Over 13% of them are published in International peer-reviewed journals.

In general, it may be stated that gerontology has not yet entered the range of sciences, supported by the government on a constant basis.

Historically, research schools in Russian gerontology got shape within the framework of other disciplines. However, Gerontological Society of the Russian Academy of Sciences set up in 1994 gave an impulse to the development of gerontology in Russia (37), and today we can talk about the existence, rather formation of research schools, where gerontology studies occupy a substantial proportion. The criteria for referring this or that group to the rank of a research school in gerontology could be as follows: 1. Presence of a leader whose personal contribution to the development of particular aspects in gerontology is most valuable, and who has publications of international standard and outlines the area of investigation conducted by his team.

2. Presence of the followers, who develop certain areas of the research undertaken by the team.

3. Wide-scale participation in the national and international research forums devoted to the problem under study.

4. Research collaboration with other groups of scientists.

The most notable role in the national basic gerontology play schools which got shape on the basis of leading research institutions of this country, entering the system of the Russian Academy of Sciences (RAS) (N.M. Emanuel Institute of Biochemical Physics of the RAS, the Institute of Numerical Mathematics of the RAS, V.A. Engelhardt Institute of Molecular Biology of the RAS, N.I. Vavilov Institute of General Genetics of the RAS, the Institute of Economic Forecasting of the RAS, the Institute of Control Sciences of the RAS, N.N. Semenov Institute of Chemical Physics of the RAS, - all in Moscow; the Institute of Theoretical and Experimental Biophysics, Puschino; the Institute of the Problems of Chemical Physics of the RAS, Chernogolovka; I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the RAS, the Institute of the Human Brain of the RAS, I.P. Pavlov Institute of Physiology of the RAS, the Institute of Nuclear Physics of the RAS, - all in St. Petersburg; the Institute of Genetics and Cytology of the Siberian Branch of the RAS, Novosibirsk, Institutes of Biology and Physiology of the Komi Research Centre of the Ural Branch of the RAS, Syktyvkar; the Institutes under the auspices the Russian Academy of Medical Sciences (RAMS) (St. Petersburg Institute of Bioregulation and Gerontology of the North-Western Branch of the RAMS, Research Institute of Experimental Medicine of the RAMS, D.O. Ott Institute of Obstetrics and

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Gynecology of the RAMS,- all in St. Petersburg; Research Institute of the Brain of the RAMS, Moscow; the Institute of Therapy of the Siberian Branch of the RAMS, Novosibirsk); the Institutes of the Ministry of Health and Social Development of the Russian Federation (Russian Research Institute of Gerontology of the RF Ministry of Health and Social Development, Moscow, N.N. Petrov Research Institute of Oncology of the RF Ministry of Health and Social Development, St. Petersburg) and leading higher schools of the country (M.V. Lomonosov Moscow State University, St. Petersburg State University, Ulyanovsk State University, I.M. Sechenov Moscow Medical Academy, Russian State Medical University, St. Petersburg State Medical University named after I.P. Pavlov, St. Petersburg Medical Academy named after I.I. Metschnikoff, Samara State Medical University, Ural Medical Academy (Ekaterinburg), Tjumen Medical Academy and other).

It should be emphasized that formation of steady research schools in gerontology is a marker of progress in this discipline and its growing topicality, which in its turn, reflects global changes in demographic situation and society demands in general. We believe that the development of gerontology would be more effective under governmental support.

Enrollment of gerontology into the classifier of research directions sponsored by the Russian Foundation for Basic Research could play its positive role. Of utmost importance seems the “Programme for prevention of age-related pathology and accelerated aging, reduction of premature mortality due to biological reasons and extension of healthy period of life for the population of Russia” developed on the basis of the latest achievements of Russian researchers on the initiative of the St.Petersburg Institute of Bioregulation and Gerontology (40). On the initiative of the non-government organization Russian Foundation for the support of scientific research “Science for Life Extension”, a complex interdisciplinary programme for fundamental research “Science against aging” was prepared. Both programmes were presented at the 19th IAGG World Congress of Gerontology and Geriatrics, Paris, 2009 (41).

In spring 1907 in his introduction to the first Russian edition to «Etudes of Optimism» E. Metschnikoff wrote that science in Russia survived long and hard crisis. Not only the demand for science was absent, it was kept down. Unfortunately, we must admit that the situation in today’s Russia little differs from that at the beginning of the century. As mentioned by another Russian citizen Nobel Prize Laureate A.I. Solzhenitsyn in 1999 at the celebration of the 275th anniversary of the Russian Academy of Science: “Never ever during three centuries of its existence in Russia science has been neglected as such and left in poverty”. And still Metschnikoff entitled his book optimistically. Impetuous development of gerontology in this country gives us every reason to feel optimistic about future.

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40. Anisimov, V.N., Baranov, V.S., Khavinson, V.Kh. et al. 2008, Program “Prevention of age-related pathology and accelerated aging, decrease of premature mortality from biological causes and an extension of working period of the population life”, Firma KOSTA, St.Petersburg.

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The Field of Biological Aging: Past, Present and Future, 2011: 123-135 ISBN: 978-81-7895-513-1 Editor: Abdullah Olgun

8. A short look at aging, anti-aging, geriatry and death in Turkish history before the 19th century Nil Sarı Cerrahpaşa Medical School of Istanbul University, Basic Medical Sciences Division, Medical History and Ethics Department, Kocamustafapaşa, 34098 İstanbul, Turkey

Introduction The belief in life after death does not lessen the desire to live and to help others to live a healthy and long life. Both the legends of Gilgamesh, the king and Lokman, the patron saint of Islamic medicine, reflect the idea of a plant of immortality. According to legend in regions like Amasya, Bergama and Adana, Lokman not only treated illnesses, he also learned the cure for death.

However, the book in which this information was written was dropped into the water by Gabriel, who said: “You shall not find the cure for death.” The water into which the book had been dropped was used to water barley fields; as a result, in these regions barley water is believed to be good for the health.

An early 17th century miniature picture of Lokman dressed in Ottoman costume is found in an album kept at the Topkapı Palace Library. Alexander also could not find the way to immortality no matter how hard he tried to.

However, Khidr was said to drink the water of life. A miniature picture of 111111 Correspondence/Reprint request: Dr. Nil Sarı, Cerrahpaşa Medical School of Istanbul University, Basic Medical Sciences Division, Medical History and Ethics Department, Kocamustafapaşa, 34098 İstanbul, Turkey E-mail: hnilsari@gmail.com

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Khidr drinking water from the water of life is depicted in Zübdetü’t-Tevarih.

In Kazvini’s Acaibü’l Mahlukat (The Wonders of Creation), a late 16th century miniature of the Safavid period illustrates Khidr and Elijah, who were said to have drunk the water of life, depicted before the fountain of youth.

Iskendername by Ahmedi relates the adventures of İskender zü’l-Karneyn in order to find the fountain of youth and the changes Khidr went through after finding the fountain of youth. Khidr and Alexander were favored subjects in Turkish literature. The belief that water is the essence of life basically relies on the verse 30 of the Enbiya Surah of the Koran writing, "We created all living things from water".

Medical literature describing health, disease and treatment before the 19th century rely on philosophy and empirical findings. Knowledge about the health and medical treatment of the aged is studied in medical manuscripts basically under the title “regimen of old age”, while the subject is touched under various other topics, when needed. In Turkish, the aged is called “koca” or “pîr”. The aged are advised to be treated as a special group of people who ought to be intervened cautiously and medical treatment to be applied with precaution.

The philosophical theory of growing old The humoral theory: Elements (unsur) and their qualities Evaluation of biological mechanisms and effects of aging given in medical manuscripts rely on the humoral theory, the basic philosophy that backs up the ideas about preventive health measures and treatment in general.

According to this philosophy, the universe is made up of four main elements: fire, air, water and earth. These are the basic elements that make up all inanimate and animate objects. The four elements are not material but express characteristics of mass and energy. These elements have hot, cold, moist/humid or dry qualities, described as their "state" (keyfiyet). These qualities are inherent and described as "nature" (tabîat). Not only human beings but all animate and inanimate entities are under the influence of one or a combination of the above-mentioned four qualities. The essential substance of any object is characterised by these qualities, each being hot, cold, moist or dry. Each object has a hotter, colder, drier or moister nature in comparison to others. Organs also have these characteristics. For example, the heart has the tendency to be hot and humid; the liver hot and dry; the brain humid and cold; the spleen dry and cold. These natural qualities are also present to a greater or lesser extent in every food or medicament. For example, a food or medicament might be hot in the first, second, third or fourth degrees, and

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hence the heating effect of that food or medicament varies according to the degree it possesses that characteristic. The combination of qualities in a food or medicament determines the balance in the body of the person who consumes it.

Humours (khilt) and their qualities The human body contains four fluids or humours which are blood, phlegm, yellow bile and black bile. We cannot define the four humours in the literal sense of the words used to express them. Humours are the fluids first generated by the process of digestion. Blood has a hot and moist nature (sanguine); phlegm is cold and moist (phlegmatic); yellow bile is hot and dry (choleric); black bile (atrabilious humor) is cold and dry (melancholic). A good and healthy humour or combination of humours can be digested and assimilated into the tissues. These are restorative, beneficial humours.

Humours with the appropriate qualities taken in moderate amounts nourish the body. Each of the four humours also has sub-categories.

The ways in which the opposing qualities of the four elements combine and interact are called "temperaments". If the opposing characteristics are present in proper amounts there will be a balance. We can talk about the proper ratio of characteristics for each individual person rather than an ideal combination identical for all. But if one or more of the hot, cold, moist or dry qualities are dominant the balance is disturbed. When this occurs the person's temperament is distorted, and their equilibrium is upset. Balance between the humours indicates health. When the quality or amount of the humours deviate from the normal, the humoral balance is disturbed and this causes disease.

Abnormal humours that cannot be digested or transformed into a proper form that have caused the disease need to be expelled from the body by means of diet and / or medicaments. For example, the atrabilious humor, the sediment or precipitation of blood, has a cold and dry nature. Morbid atrabilious humor is a dense - oxidised - by-product of unhealthy digestion.

Temperament of the aged According to the humoral theory, the “vital faculty� accomplishes various functions of life. The innate heat (hararet-i gariziyye) which is the vital spirit manifests itself as breath. It is the role of the innate heat to neutralize pathogenic agents and toxic products of poisons.

Food consumed by the aged is not digested efficiently. Therefore, humidity (rutubet) originating from blood left immature (ham) turns into phlegm. Phlegm increasing too much becoms a corrupter humidity (mĂźfsid

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rutubet). While pure blood, the source (gıda) of vitality (can) weakens and reduces, the innate heat which is the capital (sermaye) of vitality and life (ömür) also lessens, because it nourishes from blood. Undue reduction of the innate heat means loss of vitality and aging. The stomach gets cold, nerves grow lean and feeble. The body’s energy comes from the vital faculty, but vitality is weakened, therefore, the body also weakens. During aging, nature is increasingly directed to the black bile (sevda), in other words, the atrabilious humor, which is cold and dry in nature.

Agents essential to vitality- means of anti -aging In the ancient Turks, the shaman’s (kam) games, equipment and medications were turned to in an attempt to prevent illnesses. The ancient Turks applied a number of different methods to protect against illnesses of the öt öz, that is the flesh and the spirit. Kam the shaman, otacı the herbalist, afsuncu the charmer did all they could to postpone death. During the Islamic period, physicians (hekim / tabib) argued about agents required for a healthy long life. Besides agents that effect human health from the beginning to the end of life, special means for saving the health of each organ were also discussed seperately in medical manuscripts.

The first essential agent is inhalation of “fresh air” which is described as clear (duru) air without dust, smoke or vapor and far away from garbage, filth or mud. This means well oxygenated blood as it issues from the lungs. Air of high altitude strengthens the nature¸ reddens the color of the face, sharpens the appetite and it is anti-aging. Places of low altitude slows the body (süst endam-gets it languid), weakens the inner heat and grows the face pale. The weather should neither be too cold to make one tremble, or too hot to make one perspire. The hotness of the weather in summer is harmful, for it dries and burns the humidity of the body. Walking in the sun and washing with hot water are harmful. Hot drugs and medicaments should be consumed cautiously. Impatience, greed, anger, covetousness / avarice, abstaining from diet / regimen gets one’s temperement too hot, hence are enemies of health.

The breath (nefes) ought to be treated with fine (latif) aromas / fragrances (koku); and vinegar with rose juice (gülsuyu); vinegar with red bole (kilermeni); rose juice with sandalwood (sandal); and clove (karanfil) soaked in rose water / juleb (gülab) and vinegar.

Food and drink are the second necessary means of a healthy and long life. Food reach organs (aza) as blood which turns into flesh (et) and vitality (can). Consuming easily digestible food that makes blood pure and strengthens the inner heat is beneficial. One must beware eating before digestion is completed and should not eat if not hungry; but when there is

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appetite one ought not miss it. Eşref bin Muhammed writes: “When one’s stomach is empty one eats by themselves, but when one is full whatever they eat eats them; although one is not aware of it before they digest the old food, if they eat again, this is the enemy of health.” One shouldn’t eat too much, but in small amounts. If not proper for the temperament, too hot, too cold, too wet and dry natured food are harmful for health. Eating too much sour (ekşi) food accelerates aging (tiz kocaldır), dries the body, causes neural disorders.

Continuously eating sweets cause hotness (ısıtma). Good fresh water is water flowing through clean ground- earth or rock.

The following are proper food for the aged: bread of pure (arı) wheat; black cumin (çörek out), fennel seed (raziyane), anise seed (anason), cumin seed (kimyon), ajowan / (nanahan) added to wheat dough; male lamb in its second year (erkek şişek eti); kid less then a year old (bir yaşını geçmemiş oğlak eti); fleshy calf not yet weaned (semiz buzağı eti); hen fed with digestible (latif ) food; partridge (keklik); hazel grouse / francolin / grey patriot (aç çil) that has not been fed; fresh water fish (tatlı su balığı); white honey (ak bal); blanched almond with red raisons (kızıl kuru üzümle ağardılmış badem); walnut with dried fine fig (kuru iyi incirle ceviz / koz içi); juicy, sweet and large berried grape (iri taneli sulu tatlı üzüm); ripe fig (iyi olmuş incir); delicious and pleasantly tart pomegranate (latif , mayhoş nar); sweet and juicy apple (tatlı sulu elma); delicious and juicy pear (latif sulu armut). All should be eaten in season.

Exercise is another necessary agent for vitality. Physical activities such as walking and playing should be carried out after the food has been digested.

Exercising should be started slowly and one ought to prepare for it and massage the limbs. There are separate exercises for each limb. The body and nerves of those who do not do the necessary exercise are weakened (süst) and his/her mind gets dull (aklı künd eyler), obstruction (südde) occur in the passages / channels of the body and phlegm increases. Emotions, such as cheerfulness, happiness, anger and rage are named as spiritual activities (ruhani hareket). Emotions such as wrath and revenge / vengeance cause perspiration and blushing as a result of vitality (can) having moved outward.

Sleep is also essential for vitality. While asleep, the body and the mind rest and are refreshened, vitaliy (can) gets stronger. During the night one should sleep eight to nine hours. He who sleeps at night shouldn’t sleep during the day. Sleeping late makes one dozy, drying out the moisture and nerves of the mind.

Besides the essential means of vitality, the body should be freed of superfluous humours. However, evacuation ought to be applied carefully. For example, emetics should not be given to the dry and cold natured or those short of blood. Therefore, the aged (gayet kocalıkta) should not be caused to

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vomit. Intercourse is also a way of releasing superfluous humours. It refreshes, gets rid of bad thoughts, reduces anger, awakens the inner heat, and prevents the excess of atrabilous humour and phlegm. If one does not have intercourse when young, the blood would boil and black bile would increase; but one should be cautious when aged (kocalığa eriştiğinde).

In addition to efforts for maintaining a healthy and long life, the search for eternal life led to the elixir of life (âb-ı hayatı ) formulations composed of plants, metals, and precious stones. Elixir of life prescriptions in books on alchemy are usually not written plainly, but in a symbolic style, sometimes too complex to be understood clearly. An early anti-aging prescription named “powder for youthfulness” (gençlik tozu) is registered in an Uighur Turkish medical text. It is composed of long pepper (darifülfül), black pepper (karabiber), cinnamomi (tarçın), cardamom (kakule), ginger root (kuru zencefil), ajowan fruit (nanahan) and sesame (susam) pounded, then kneaded with honey. Other substances named kurbi, bitbul and kurnu are included in the composition, but these could not be defined. Some prescriptions in manuscripts on pharmacy and medicine were named so as to reflect their miraculous effects, such as “The Philosopher’s Paste”, also named as the “The Substance of Life” (Hayat Maddesi) and “The Hand of God” (Yedullah’ın Eli).

Health problems and treatment of the aged It could be suggested that growing old is a type of illness as well. In old age, the natural forces (kuvvetler) decrease. When one reaches sixty years of age, weakness (kuvvetsizlik) certainly starts and movement of bones and joints slow down. The aged get tired quickly and are in distress caused by physical and mental loss. Administration of drugs would not be as beneficial as needed.

Preventive health measures such as diet, sleep, exercise, bathing (food should be eaten after bathing) and massage applied in a proper way are advised for the old. Bathing with warm water is recommended as it moistens the body. Moderate exercise and slow activities such as walking (diseased parts of the body should not be strained) would help digestion and evacuation of superfluous material from the body. Too much exercise (riyazet) is harmful. Walking is enough exercise for digesting the food consumed. Mild, gentle massage of the body with almond, iris (süsen) or jasminum (yasemin) oil is advised to be applied in the morning. Aromatic oils specially with a hot quality are recommended. Painful parts of the body should not be massaged.

The aged ought smell pleasant aromas / fragrances (kokular), but beware smelling cold aromatics / fragrances such as nymphaea (nilüfer), camphor

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(kafur) and vinegar (sirke). Extremely cold or hot weather, vapors / fumes, unpleasant odors, smoke and dust are harmful for the aged.

The aged need to go to sleep in due time on a soft bed. They need sleep and rest more than the young. The aged who wants to do something, but could not do it gets worried and anxious of it. However, the aged should be made feel happy and relieved, while sorrow and fear should be avoided.

Dietetic treatment for the aged: Food as medicine Food changes the elements and the major functions of the organisms.

Therefore, selection of food in accordance / in harmony with the constitution of the patient is important for health. Priority is given to the diet of the aged.

Old people are advised to consume food more frequently then the young. One meal is advised to be eaten at two times. Only little amounts of easily digested light food should be consumed at night. Hunger is also very harmful.

As the temperament of the basic organs of the aged is drier and colder in nature / quality than it should be, their temperament should be balanced by moist and hot food and medicine. The aged must beware of cold and dry natured food. Drugs that dry the body are contraindicated for the old. The aged should be careful of pickels (turşu), dry meet (kuru et), dry cheese (kuru peynir), salty and hot natured herbs and constipation. The cavities of organs of the aged contain the moistness of phlegm. Treatment is to be applied accordingly. The main principle is to avoid food that would cause too much phlegm and black bile, the humors contraindicated for the old. If too much phlegm is found, first it ought to be dissolved, than moistening food should be consumed. The aged need hot and moist food. Food that increases blood and ends phlegm are the following: -

juice of chickpea (nohut) and broth/bouillon of sheep stew (koyun yahnisi), The inside of bread with aniseed (anason), black cumin (çörek otu) and cumin (kimyon), vermicelli with milk (sütlü erişte), Compote (hoşaf) of soaked figs, aniseed and honey.

The aged should evade having heavy meals difficult to digest that cause black bile, except those who have moist in their stomach. Milk, especially milk of goat and donkey has a moistening nature/constituent. Milk consumed with honey and salt could be digested more easily. Milk is advised for the old who have no digestion problem. Although rice should not be eaten, as it causes phlegm, flatulence (yel) and constipation, rice with milk could be consumed with honey, if bile does not get sour and give trouble.

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The aged suffering indigestion should not eat meet, but consume soup prepared with meat boiled in water. Indigestible (rich) food, such as fish pickled in brine (tuzlu balık), dry meet, eggplant (patlıcan), cucumber (salatalık), melon (kavun), pickles, spiced foods should be consumed attentively.

The aged should beware having dry and cold natured sour (ekşi) food that dry the body, causing growing older, specially of vinegar that causes one to grow old. (However, honey and vinegar /oxymel (sirkencubin) is used as medicine.) Those who could not resist having sour food may eat a little unripe grape (koruk), sumac (sumak) or pomegranate (nar) in summer, only if their primary temperament is blood and have a stomach and liver that is hot in nature. If not used as medicine, pungent food biting to the taste, like salt and pepper (biber) ought to be consumed in little amounts. The following are proper food for the aged: -

Pistachia terebinthus (terebentin sakızı), mastic (damla sakızı), black cumin (çörek otu), fennel seed (raziyane), anise seed (anason), cumin seed (kimyon), wild saffron seed mixed with dried fig, delicious and pleasantly tart pomegranate (latif , mayhoş nar), sweet and juicy apple (tatlı sulu elma), delicious and juicy pear (latif sulu armut). ripe fig (iyi olmuş incir), sweet figs, juicy, sweet and large berried grape (iri taneli sulu tatlı üzüm); blanched almond (badem) with red grapes; blanched almond with red raisons (kızıl kuru üzümle ağardılmış badem), walnut (ceviz içi) with quinces (ayva) and figs; walnut with dried fine fig (kuru iyi incirle ceviz / koz içi), sweet basil (reyhan) syrup, rose paste (gülengebin), balm leaf (bazrencbuye- kovan otu yaprağı) juice, white honey (ak bal); delicious (latif) halva, soup prepared with arm beet or cabbage, four drachma (dirhem) Cuscuta europaea (eftimun), a few fig (incir), and some safflower seed (asfur tohumu) pounded and taken orally, arm beet (pancar), celery (kereviz), leek (pırasa) with olive oil, bread of pure (arı) wheat;

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inside of bread soaked in honey syrup, ajowan (nanahan) added to wheat dough; wheat bread soaked in lamb (lamb in its second year) stew (şişek yahnisi) broth, soft- boiled egg, meet of hen (tavuk); hen fed with digestible (latif ) food, fern radix cooked in chicken soup, meet of lamb (kuzu); male lamb in its second year (erkek şişek eti), kid less then a year old (bir yaşını geçmemiş oğlak eti), fleshy calf not yet weaned (semiz buzağı eti); partridge (keklik), hazel grouse / francolin / grey patriot (aç çil) that has not been fed, fresh water fish (tatlı su balığı), Drink composed of barley (arpa) flour, peppermint (nane) and common fennel (rezene); and ginger (zencefil) are said to heat the body, but do not cause dryness, and help digestion of moist food.

All should be eaten in season.

Treatment of obstructions The body was imagined as a complex of actual, as well as potential tubes which vary in size. This is supposed to be a continuous system which includes the alimentary-canal, air passages, blood vessels, the cavernous tissues, the serous cavities and the intracellular channels. The metabolic, secretory and excretory products of ingested food materials may be traced through this system. So long as these channels are unobstructed throughout, and these various substances can flow freely through them, the body was said to be in a state of health. Therefore, a basic cause of disease was the obstruction of the canal / channel system.

Resolvents The obstruction in a tissue prevents the flow of tissue fluids and is the forerunner of a disease. When an obstruction is due to the humors being simply over-abundant, their injurious effect can be removed by evacuating them by purgation; but thick or coarse humor requires attenuation by a resolvent. A resolvent is an agent which dissolves the accumulated matter which is the cause of obstruction. In the action of resolution hot medicaments are to be used to dissolve the over-abundant humors. Therapeutic constituent of some drugs act as an agent that promotes resolution of humors by

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attenuating the dense particles of the matter, causing them to flow and be removed. For instance an inflammatory mass or tumor can be dissolved in this way. This is a description of the lysis process.

Obstructions in the aged are advised to be cleared by peppermint (nane), hot pepper (karabiber) and onion; syrup of barley (arpa) or oat (yulaf) or honey. For the treatment of urinary tract obstructions, dissolvents/ resolvents such as celery (kereviz) radix and seeds are to be added to honey syrup. If a stone obstructs the way, stronger dissolvents are needed. In pulmonary obstruction, for clearing the pulmonary tract / passages, hyssopus (zufaotu), fern / bracken (eğrelti otu), Cinnamomum cassia (çin tarçını) are added to honey syrup; or two drachma gum of the East Indian mastic tree (iklü’l-butm) pounded with sugar and taken orally are advised.

Evacuation of the digestive system The catabolic process of effete substances are removed by deposition in tissues or by discharge from the body. As phlegm is heaped in the stomach and intestines of the aged, it is necessary to clean the bowels (telyin) from time to time. Phlegm should be evacuated through the intestines, though purgatories are not advised for the aged. Purgatives should not be prescribed for very old people (gayet kocalıkta). One with little blood, that is one whose temperament is dry and cold should not be given emetics. Blood letting is harmful for the old, too. Diuretics are used when needed. The aged should be treated moderately (itidal).

Some aged people with a dry nature have constipation. The aged should beware of constipation. To treat constipation one must have vegetables cooked in olive oil, so that the nature will become laxly (yumuşak) and hot.

Olive oil and almond oil have laxative effect on the nature. These also have to be consumed according to the temperament. The following prescriptions are advised for laxing the nature of the aged and the treatment of constipation: -

five drachma of polypodium (besfayiç) cooked with one pinch (tutam) of cabbage (kelem), half spoon almond oil (badem yağı) added to boiled kernel of safflower seed (asfur tohumu), two drachma gum mastic (mastaki), pounded with two drachma sugar, sweet plum (erik) boiled in water, fresh fig (incir) soaked in water and one or two consumed before meals, dried fig cooked with water mixed with honey, - dish of vermicelli (erişte) and spinach (ıspanak).

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Submittion to the inevitable fate: Death The ancient Turks believed in a life force known as kut, which had been granted by the Creator. This was needed throughout life and a long life was believed to depend on this force. People could bless one another with this life energy; they would pray that the life force kut would be protected and that they would be forgiven by the God of the Sky (Gök Tengri). When the kut, which was perceived as the source of life, was expended, or when it was taken back, it was believed that there was no longer any hope for a person. In many sources it is stressed that the ancient Turks not only believed in preventive medicine and medical treatment, but also in the inevitability of death. They did not believe that the living beings on earth could attain immortality. They believed that it was not possible to live forever and that the length of one’s life was not under the control of the human will; it can be determined from the oldest inscriptions that the ancient Turks thought that every person had a certain amount of time to live. For example, in the Kül Tegin inscription it is written “When the time of Kül tegin came he died” (Kül Tiğin özinçe kerğek boldı) and “Time is granted by God; humans are created to die” (öd tenri yasar, kişi oglı kop ölüğli törümiş). In the inscriptions, we can find striking expressions like “the God of Time” (öd tengri) gives an order; people were born to die”. Today, genetic scientists have stated that death may be partly programmed by the shortening of the telomeres. Like an hour-glass, the lifespan of every person could be recorded on the genes.

Today, when someone dies, we say “their time was up”. In the Diwan of Kaşgarlı Mahmud, the cruelty of time (ödhlek) is explained as follows: “Time hurries to reduce the power of man; man have been thinned. Even those who flee attain death”. In Kutadgu Bilig as well, in the last will of Ay-Toldı, the concept of time, which limits the life of mankind, is examined: “Where is the one who has fled death, saving themselves Postponing fate and overstaying their time?

When one is born and given a name The traveler has mounted the horse of time O, my glowing sun, this death has been prepared For you as well, the time is merely waiting.”

The Moslem Turks did not believe in surrendering to fate during an illness. Quite the contrary, medical sciences were around before religious sciences; we find in almost all the introductions to medical manuscripts the idea that a person who has poor health cannot have complete faith. In many

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medical books there are various reports that Prophet Muhammad replied to those who demanded reliance on Allah instead of searching for a cure by saying “Allah has provided a remedy for every illness”. In these reports it is stated that Prophet Muhammad insisted that ill people should summon a doctor. In many medical manuscripts dating from the Seljuks and the classical Ottoman period, as a continuation of traditional Islamic medical practice there is the idea that health can be found in nature and that the secrets of medical science are hidden in nature; thus the physician is our guide through nature. For these reasons, there is a medication for every illness and if the medicine is suited to the illness and the disposition of the patient, and if Allah permits, then the patient will regain health, as it is believed that Allah created the good and bad effects of medication. This idea can be found in all the manuscripts of Ottoman physicians.

“Sooner or later every living being will taste death”, however, it is clearly stated in the verses of the Qur’an that no one knows when, where or how they will die. The practicing physicians of the Seljuk and Ottoman eras, who were a part and a continuation of Islamic medical practices, expressed the same belief.

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3. Erdemir Ayşegül Demirhan: Şifalı Bitkiler Doğal İlaçlarla Geleneksel Tedaviler.

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5. Eşref bin Muhammet: Hazâinü’s Saâdet. Edt. Bedi N. Şehsuvaroğlu, Türk Tarih Kurumu Yay. XI seri-Sa. 9, 1961. Topkapı Sarayı Ktp. III Ahmed Ktp., Hazine 557.

6. Falname, early 17’th century, Topkapı Palace Museum Library, No. H. 1703, f.

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7. Gruner O. Cameron: The Canon of Medicine of Avicenna Incorporating A Translation of the First Book, New York 1970.

8. Gürkan Mahmut : Orta Asyadaki Eski Türk Tıbbının, Başlangıçtan 14. Yüzyıla Kadarki Döneme Ait Bilinen Türkçe Tıp Metinlerinde, Tıp Tarihi Açısından Değerlendirilmesi. İÜ Cerrahpaşa Tıp Fakültesi Tıp Tarihi ve Etik Anabilim Dalı, unpublished PhD thesis, İst. 2010. (See: Türkische Turfan-Texte VII - T III M 66, p. 76 / 4.3.1.)

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11. İbn-i Sînâ: El-Kânûn fi’t Tıbb, Birinci Kitap. Trans. Esin Kahya, Atatürk Kültür, Dil ve Tarih Yüksek Kurumu Atatürk Kültür Merkezi, sayı 103, Külliyatlar Dizisi sayı 5, Ank. 1995.

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14. Muhammed Mü’min et-Tenkabunî: Gunyetü’l-Muhassılîn (1699), trans. Ahmed Sânî: Gunyetü’l-Muhassılîn fi Tercemet-i Tuhfeti’l Mü’minîn (1733), Cerrahpaşa Tıp Tarihi Müzesi, no. 730. Editors: Sarı Nil, İzgöer Zeki Ahmet, Tuğ Ramazan, Okutan Mehmet Yahya: Klasik Dönem İlaç Hazırlama Yöntemleri ve Terkipleri.

Novartis Publ. İst. 2003.

15. Sarı N.: “Türkçe Tıp Yazmalarında Hamam Konusuna Verilen Önem.” Yeni Symposium. Ocak/Nisan 1984, Sa. 1-2, p. 90-96.

16. Sarı N.: “Osmanlı Tıbbında Beden ve Nefis Terbiyesi”, Yeni Sempozyum, Temmuz/Ekim 1984, Sa. 3-4, p. 76-95.

17. Sarı N., Zülfikar B.: “Kuşlarla Tedavi”. Tarih ve Medeniyet, Ocak 1996, Sa. 23., p. 64-68.

18. Sarı Nil: Osmanlı Tıbbında Besinlerle Tedavi ve Sağlıklı Yaşam (Food as Medicine). Türk Mutfağı (Editors: Arif Bilgin, Özge Samancı), Kültür ve Turizm Bakanlığı Sanat Eserleri Dizisi:476, Ankara 2008, p.137-151.

19. Sarı Nil: Türklerde Ölüm İnancı- Tıp Etiğinin İnanç Boyutuna Tarihten Bir Bakış (Concepts About Death in Turkish History- A Historical Perspective of Religious Ethics in Relation With the Dying Patient). 2. Tıp Etiği ve Tıp Hukuku Kongresi Bildiri Kitabı, 21. Yüzyıl Başında Yaşama Destek Tedavileri Etik ve Hukuksal Yönler, Nobel Publ. No. 1459, Ank. 2009, p. 4-17.