Objective-The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. Methods and Results-Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H 2 O 2 -induced apoptosis. Lung fibroblasts from 6-month-old TERT Ϫ/Ϫ mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. Key Words: aging Ⅲ apoptosis Ⅲ mitochondrial functions Ⅲ mitochondrial DNA Ⅲ reactive oxygen species Ⅲ telomerase reverse transcriptase T o date several theories exist to explain the phenomenon of normal and pathological aging. The free radical theory of aging 1 proposes that reactive oxygen species (ROS) in biological systems attack molecules and thereby cause functional decline of organ systems that eventually leads to death. This damage accumulates over time and may contribute to diseases associated with aging like atherosclerosis, neurodegeneration, or cataracts. 2 Recently, Schriner et al produced transgenic mice that overexpressed human catalase localized to the peroxisome, the nucleus, and the mitochondria. Only mice overexpressing mitochondrially targeted catalase showed a significant increase in life span and a reduction in oxidative damage to DNA and consequently in apoptosis. 3 Thus, these data define the mitochondria as compartment of ROS formation, which contributes to aging processes. Further evidence supporting the importance of mitochondria and formation of ROS in the mitochondria comes from findings that overexpression of mitochondrially localized antioxidant enzymes lengthens lifespan of Drosophila 4,5 and that deletion of manganese superoxide dismutase results in the age-related decline of mitochondrial function, culminating in increased apoptosis. 6 Recent studies using isolated complex I of the respiratory chain clearly demonstrated that superoxide production into the mitochondrial matrix is predominantly dependent on flavine-mononucleotide within complex I. 7,8 The enzyme telomerase counteracts the shortening of the physical ends of chromosomes and, thereby, prevents the onset of replicative senescence and genetic instability. 9 -12 The c...

BackgroundEvolutionary transitions from outcrossing between individuals to selfing are partly responsible for the great diversity of animal and plant reproduction systems. The hypothesis of ‘reproductive assurance’ suggests that transitions to selfing occur because selfers that are able to reproduce on their own ensure the persistence of populations in environments where mates or pollination agents are unavailable. Here we test this hypothesis by performing experimental evolution in Caenorhabditis elegans.ResultsWe show that self-compatible hermaphrodites provide reproductive assurance to a male-female population facing a novel environment where outcrossing is limiting. Invasions of hermaphrodites in male-female populations, and subsequent experimental evolution in the novel environment, led to successful transitions to selfing and adaptation. Adaptation was not due to the loss of males during transitions, as shown by evolution experiments in exclusively hermaphroditic populations and in male-hermaphrodite populations. Instead, adaptation was due to the displacement of females by hermaphrodites. Genotyping of single-nucleotide polymorphisms further indicated that the observed evolution of selfing rates was not due to selection of standing genetic diversity. Finally, numerical modelling and evolution experiments in male-female populations demonstrate that the improvement of male fitness components may diminish the opportunity for reproductive assurance.ConclusionsOur findings support the hypothesis that reproductive assurance can drive the transition from outcrossing to selfing, and further suggest that the success of transitions to selfing hinges on adaptation of obligate outcrossing populations to the environment where outcrossing was once a limiting factor.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0093-1) contains supplementary material, which is available to authorized users.

The ubiquitously expressed aryl hydrocarbon receptor (AhR) induces drug metabolizing enzymes as well as regulators of cell growth, differentiation and apoptosis. Certain AhR ligands promote atherosclerosis, an age-associated vascular disease. Therefore, we investigated the role of AhR in vascular functionality and aging. We report a lower pulse wave velocity in young and old AhR-deficient mice, indicative of enhanced vessel elasticity. Moreover, endothelial nitric oxide synthase (eNOS) showed increased activity in the aortas of these animals, which was reflected in increased NO production. Ex vivo, AhR activation reduced the migratory capacity of primary human endothelial cells. AhR overexpression as well as treatment with a receptor ligand, impaired eNOS activation and reduced S-NO content. All three are signs of endothelial dysfunction. Furthermore, AhR expression in blood cells of healthy human volunteers positively correlated with vessel stiffness. In the aging model Caenorhabditis elegans, AhR-deficiency resulted in increased mean life span, motility, pharynx pumping and heat shock resistance, suggesting healthier aging. Thus, AhR seems to have a negative impact on vascular and organismal aging. Finally, our data from human subjects suggest that AhR expression levels could serve as an additional, new predictor of vessel aging.

Background: Evolutionary transitions from outcrossing between individuals to selfing are partly responsible for the great diversity of animal and plant reproduction systems. The hypothesis of 'reproductive assurance' suggests that transitions to selfing occur because selfers that are able to reproduce on their own ensure the persistence of populations in environments where mates or pollination agents are unavailable. Here we test this hypothesis by performing experimental evolution in Caenorhabditis elegans. Results: We show that self-compatible hermaphrodites provide reproductive assurance to a male-female population facing a novel environment where outcrossing is limiting. Invasions of hermaphrodites in male-female populations, and subsequent experimental evolution in the novel environment, led to successful transitions to selfing and adaptation. Adaptation was not due to the loss of males during transitions, as shown by evolution experiments in exclusively hermaphroditic populations and in male-hermaphrodite populations. Instead, adaptation was due to the displacement of females by hermaphrodites. Genotyping of single-nucleotide polymorphisms further indicated that the observed evolution of selfing rates was not due to selection of standing genetic diversity. Finally, numerical modelling and evolution experiments in male-female populations demonstrate that the improvement of male fitness components may diminish the opportunity for reproductive assurance. Conclusions: Our findings support the hypothesis that reproductive assurance can drive the transition from outcrossing to selfing, and further suggest that the success of transitions to selfing hinges on adaptation of obligate outcrossing populations to the environment where outcrossing was once a limiting factor.

Lysosomes contain a variety of enzymes for the degradation of proteins, nucleic acids, polysaccharides, and lipids. Among them is the family of the proteolytic enzymes, the cathepsins (1). Cathepsins are subdivided into three classes based on the active site amino acid. Cathepsins are widely distributed in normal tissues (2) and involved in different physiological processes (1). Several studies suggest that cathepsins are involved in the signaling pathways leading to cell death (3, 4).Among the cathepsins, cathepsin D (CatD) 2 seems to have a pivotal role as a cell death mediator (5, 6). Fibroblasts from CatD-deficient mice are more resistant to etoposide and adriamycin-induced apoptosis than fibroblasts from their wild-type littermates (7). Furthermore, microinjection of CatD induces caspase-dependent apoptosis and staurosporine-mediated apoptotic cell death by CatD-triggered cytochrome c release (8, 9).Reactive oxygen species (ROS), such as H 2 O 2 , induce different cellular effects depending on the concentration and cell type. Several studies have documented the involvement of oxidative stress in apoptosis induction. Upon production of high levels of ROS from exogenous or endogenous sources, the redox balance is perturbed, and cells are shifted into a state of oxidative stress (10). These excessively high concentrations of ROS directly cause oxidative damage of DNA, lipids, and proteins and may impair cellular functions, which can result in apoptosis induction in severely damaged cells (11,12). CatD has been implicated to be involved in oxidative stress-induced apoptotic pathways. Lysosomes, and specifically CatD, have been identified as modulators of oxidative stress-induced apoptosis in alveolar type II cells (6). Moreover, stabilization of lysosomes by imidazoline protected astrocytes from oxidative stress-mediated damage (13).To protect themselves from oxidative stress, cells possess a set of antioxidative enzymes, which maintain the intracellular reactive oxygen species (ROS) at appropriate levels. Among these antioxidative enzymes are superoxide dismutase and catalase (14). Two other major intracellular redox systems are the thioredoxin and glutathione systems (15, 16). The thioredoxin family includes three proteins, thioredoxin-1 (Trx), thioredoxin-2, and Sp-thioredoxin (17-19). They contain a conserved Cys-Gly-Pro-Cys-active site (cysteine 32 and cysteine 35 within Trx), which is essential for the redox regulatory function of thioredoxins (20). Trx is ubiquitously expressed in mammalian cells, and Trx deficiency leads to a lethal phenotype (20,21

T he vascular endothelium is crucially involved in the fundamental regulation of blood flow matching demand and supply of tissue. After transient ischemia, arterial inflow increases. As a response to increased shear forces during reactive hyperemia, healthy arteries dilate via release of NO or other endothelium-derived vasoactive substances. This endothelium-dependent flow-mediated vasodilation (FMD) is impaired in atherosclerosis.1 As the presence of endothelial dysfunction is closely associated with cardiovascular risk and outcome, the measurement of FMD in the brachial artery has become a standard method for the assessment of endothelial function in patients and to evaluate therapeutic interventions targeting atherosclerosis. To date, this has been impossible in mice, although highly desirable to study mechanisms affecting endothelial function and in particular femoral artery dilation in transgenic mouse models.2 Therefore, we here characterize the physiology of FMD in living mice using a methodology analogous to humans and present important examples of vascular pathologies. Materials and MethodsMaterials and Methods are available in the online-only Data Supplement. ResultsIn healthy humans, the relative increase in brachial artery diameter at 45 to 60 sec after resolution of forearm ischemia is typically in the 5% to 10% range. To be able to detect such small diameter changes expected in mice (10-30 μm change at a baseline diameter of ≈200-300 μm) with a high heart rate, we used a high-resolution, high-frequency digital imaging platform and a 30 to 70 MHz linear array microscan transducer © 2014 American Heart Association, Inc. Objective-Endothelium-dependent, flow-mediated vasodilation after an increase in shear stress at the endothelial lining of conduit arteries during reactive hyperemia after ischemia is a fundamental principle of vascular physiology adapting blood flow to demand of supplied tissue. Flow-mediated vasodilation measurements have been performed in human studies and are of diagnostic and prognostic importance, but have been impossible because of technical limitations in transgenic mice to date, although these represent the most frequently used animal model in cardiovascular research. Approach and Results-Using high-frequency ultrasound, we visualized, quantified, and characterized for the first time endothelium-dependent dilation of the femoral artery after temporal ischemia of the lower part of the hindlimb and demonstrated that the signaling was almost exclusively dependent on stimulation of endothelial nitric oxide synthase, similar to acetylcholine, completely abolished after pharmacological or genetic inhibition of endothelial nitric oxide synthase and endothelial denudation, substantially impaired in mice of increasing age and cholesterol-fed ApoE knock outs and increased by the dietary polyphenol (−)-epicatechin. Intra-and interindividual variability were similar to the human methodology. Conclusions-The

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

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