Four agents — acarbose (ACA), 17-α-estradiol (EST), nordihydroguaiaretic acid (NDGA), and methylene blue (MB) — were evaluated for lifespan effects in genetically heterogeneous mice tested at three sites. Acarbose increased male median lifespan by 22% (P < 0.0001), but increased female median lifespan by only 5% (P = 0.01). This sexual dimorphism in ACA lifespan effect could not be explained by differences in effects on weight. Maximum lifespan (90th percentile) increased 11% (P < 0.001) in males and 9% (P = 0.001) in females. EST increased male median lifespan by 12% (P = 0.002), but did not lead to a significant effect on maximum lifespan. The benefits of EST were much stronger at one test site than at the other two and were not explained by effects on body weight. EST did not alter female lifespan. NDGA increased male median lifespan by 8–10% at three different doses, with P-values ranging from 0.04 to 0.005. Females did not show a lifespan benefit from NDGA, even at a dose that produced blood levels similar to those in males, which did show a strong lifespan benefit. MB did not alter median lifespan of males or females, but did produce a small, statistically significant (6%, P = 0.004) increase in female maximum lifespan. These results provide new pharmacological models for exploring processes that regulate the timing of aging and late-life diseases, and in particular for testing hypotheses about sexual dimorphism in aging and health.

Amyloid-␤ peptide (A␤) is the toxic agent in Alzheimer's disease (AD), although the mechanism causing the neurodegeneration is not known. We previously proposed a mechanism in which excessive A␤ binds to regulatory heme, triggering functional heme deficiency (HD), causing the key cytopathologies of AD. We demonstrated that HD triggers the release of oxidants (e.g., H 2O2) from mitochondria due to the loss of complex IV, which contains heme-a. Now we add more evidence that A␤ binding to regulatory heme in vivo is the mechanism by which A␤ causes HD. Heme binds to A␤, thus preventing A␤ aggregation by forming an A␤-heme complex in a cell-free system. We suggest that this complex depletes regulatory heme, which would explain the increase in heme synthesis and iron uptake we observe in human neuroblastoma cells. The A␤-heme complex is shown to be a peroxidase, which catalyzes the oxidation of serotonin and 3,4-dihydroxyphenylalanine by H 2O2. Curcumin, which lowers oxidative damage in the brain in a mouse model for AD, inhibits this peroxidase. The binding of A␤ to heme supports a unifying mechanism by which excessive A␤ induces HD, causes oxidative damage to macromolecules, and depletes specific neurotransmitters. The relevance of the binding of regulatory heme with excessive A␤ for mitochondrial dysfunction and neurotoxicity and other cytopathologies of AD is discussed.curcumin ͉ heme deficiency ͉ mitochondria ͉ regulatory heme ͉ serotonin

BackgroundSmall RNAs complex with proteins to mediate a variety of functions in animals and plants. Some small RNAs, particularly miRNAs, circulate in mammalian blood and may carry out a signaling function by entering target cells and modulating gene expression. The subject of this study is a set of circulating 30–33 nt RNAs that are processed derivatives of the 5′ ends of a small subset of tRNA genes, and closely resemble cellular tRNA derivatives (tRFs, tiRNAs, half-tRNAs, 5′ tRNA halves) previously shown to inhibit translation initiation in response to stress in cultured cells.ResultsIn sequencing small RNAs extracted from mouse serum, we identified abundant 5′ tRNA halves derived from a small subset of tRNAs, implying that they are produced by tRNA type-specific biogenesis and/or release. The 5′ tRNA halves are not in exosomes or microvesicles, but circulate as particles of 100–300 kDa. The size of these particles suggest that the 5′ tRNA halves are a component of a macromolecular complex; this is supported by the loss of 5′ tRNA halves from serum or plasma treated with EDTA, a chelating agent, but their retention in plasma anticoagulated with heparin or citrate. A survey of somatic tissues reveals that 5′ tRNA halves are concentrated within blood cells and hematopoietic tissues, but scant in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of 5′ tRNA halves change markedly with age, either up or down, and these changes can be prevented by calorie restriction.ConclusionsWe demonstrate that 5′ tRNA halves circulate in the blood in a stable form, most likely as part of a nucleoprotein complex, and their serum levels are subject to regulation by age and calorie restriction. They may be produced by blood cells, but their cellular targets are not yet known. The characteristics of these circulating molecules, and their known function in suppression of translation initiation, suggest that they are a novel form of signaling molecule.

Methylene blue (MB) has been used clinically for about a century to treat numerous ailments. We show that MB and other diaminophenothiazines extend the life span of human IMR90 fibroblasts in tissue culture by >20 population doubling (PDLs). MB delays senescence at nM levels in IMR90 by enhancing mitochondrial function. MB increases mitochondrial complex IV by 30%, enhances cellular oxygen consumption by 37-70%, increases heme synthesis, and reverses premature senescence caused by H2O2 or cadmium. MB also induces phase-2 antioxidant enzymes in hepG2 cells. Flavin-dependent enzymes are known to use NAD(P)H to reduce MB to leucomethylene blue (MBH2), whereas cytochrome c reoxidizes MBH2 to MB. Experiments on lysates from rat liver mitochondria suggest the ratio MB/cytochrome c is important for the protective actions of MB. We propose that the cellular senescence delay caused by MB is due to cycling between MB and MBH2 in mitochondria, which may partly explain the increase in specific mitochondrial activities. Cycling of MB between oxidized and reduced forms may block oxidant production by mitochondria. Mitochondrial dysfunction and oxidative stress are thought to be key aberrations that lead to cellular senescence and aging. MB may be useful to delay mitochondrial dysfunction with aging and the decrease in complex IV in Alzheimer disease.

Heme is a common factor linking several metabolic perturbations in Alzheimer's disease (AD), including iron metabolism, mitochondrial complex IV, heme oxygenase, and bilirubin. Therefore, we determined whether heme metabolism was altered in temporal lobes obtained at autopsy from AD patients and age-matched nondemented subjects. AD brain demonstrated 2.5-fold more heme-b (P < 0.01) and 26% less heme-a (P ‫؍‬ 0.16) compared with controls, resulting in a highly significant 2.9-fold decrease in heme-a͞heme-b ratio (P < 0.001). Moreover, the strong Pearson correlation between heme-a and heme-b measured in control individuals (r 2 ‫؍‬ 0.66, P < 0.002, n ‫؍‬ 11) was abolished in AD subjects (r 2 ‫؍‬ 0.076, P ‫؍‬ 0.39, n ‫؍‬ 12). The level of ferrochelatase (which makes heme-b in the mitochondrial matrix) in AD subjects was 4.2 times (P < 0.04) that in nondemented controls, suggesting up-regulated heme synthesis. To look for a possible connection between these observations and established mechanisms in AD pathology, we examined possible interactions between amyloid ␤ (A␤) and heme. A␤(1-40) and A␤(1-42) induced a redshift of 15-20 nm in the spectrum of heme-b and heme-a, suggesting that heme binds A␤, likely to one or more of the histidine residues. Lastly, in a tissue culture model, we found that clioquinol, a metal chelator in clinical trials for AD therapy, decreased intracellular heme. In light of these observations, we have proposed a model of AD pathobiology in which intracellular A␤ complexes with free heme, thereby decreasing its bioavailability (e.g., heme-a) and resulting in functional heme deficiency. The model integrates disparate observations, including A␤, mitochondrial dysfunction, cholesterol, and the proposed efficacy of clioquinol.mitochondria ͉ heme-a ͉ iron ͉ clioquinol ͉ ferrochelatase H eme (ferriprotoporphyrin IX) metabolism appears altered in the brains of Alzheimer's disease (AD) patients. Heme oxygenase (HO) increases in AD (1, 2), and the level of bilirubin (one of the products of heme degradation by HO) is increased in AD patients (3). Mitochondrial complex IV, the only enzyme in cells that contains heme-a (see below) (4), declines in AD (5-8). Heme-a is rate limiting for the assembly of complex IV (9). Furthermore, an inhibitor of muscarinic acetylcholine receptor binding, which increased in AD brain, was suggested to be heme (10, 11). Therefore, we studied heme metabolism in AD brain and nondemented age-matched normal subjects.Heme-b, the product of ferrochelatase (FC) (also known as protoheme), is produced in the mitochondrial matrix (12, 13) and is the precursor for heme-c and heme-a. The structure of heme-c is similar to that of heme-b, but it is covalently attached to a few specific proteins (reviewed in ref. 14). Heme-b and heme-a exist in two major pools in the cell, free and protein-associated. Conversion of heme-b to heme-a requires farnasylation and oxidation (15). Farnesyl-pyrophsphate (FPP) is the precursor for the farnesyl moiety in heme-a, cholesterol, dolichol, farnesylation ...

DNA damage ͉ aging ͉ glycosylase ͉ AP site A purinic and apyrimidinic (AP or abasic) sites are common lesions in DNA and are formed either spontaneously or as intermediates during the course of base excision repair (BER) of oxidized, deaminated, or alkylated bases (1, 2). N-glycosylases of BER recognize and excise the aberrant bases from DNA, resulting in formation of an AP site. The AP site is then incised by an AP endonuclease to generate a 5Ј-terminal base-free deoxyribose phosphate at a single-strand break. The repair of the single-strand break (AP site) is accomplished by a DNA polymerase ␤ (␤-pol) and DNA ligase (ligase I or III͞XRCC1) to restore the normal and correct base (3, 4). The rate-limiting step of BER is the lyase activity associated with the 8-kDa domain of ␤-pol, which removes the AP site from DNA (4). AP sites can be mutagenic (5-7) or can cause cell death (8-10).The methods (11-14) that detect AP sites after DNA isolation, including those with ARP (15, 16), may be inaccurate as a result of several factors: (i) artifactual AP sites resulting from base loss and formation of AP sites in DNA by high temperature at neutral pH (12, 17, 18); (ii) AP sites in DNA are subject to loss by ␤-elimination, followed by ␦-elimination catalyzed by high temperatures (19), primary amines in histones, polyamines (19-21), and thiols (22), causing underestimation of AP levels in DNA. Nakamura et al. (23) studied the endogenous level of AP sites in different tissues of the rat using aldehyde-reactive probe (ARP), isolating the DNA before trapping AP sites. They estimate that there are 50,000-200,000 AP sites͞mammalian cell, that the brain of rat contained the most AP sites, and that most of the AP sites were cleaved 5Ј to the AP sites. The first two results are different from the results reported here.In this paper, we describe a method for biotinylating AP sites in live cells and then quantifying the biotin in the isolated DNA. This method is used to determine the age-dependent changes in BER in human fibroblasts (IMR90), leukocytes isolated from human blood, and nuclei isolated from rat tissues. Materials and Methods Materials.A QIAamp blood kit was purchased from Qiagen (Chatsworth, CA). The DNA-precipitating agent, DAPER [N,NЈ-bis(3,3Ј-(dimethyl-amino)propylamine)-3,4,9,10-perylenetetra-carboxylicdiimide] and ImmunoPure 3,5,3Ј,5Ј-tetramethylbenzidine horseradish peroxidase (HRP) substrate kits were from Pierce. Vectastain ABC kit was from Vector Laboratories. O-Methoxyamine, EGTA, and Histopaque 1077 were from Sigma. HK-UNG thermolabile uracil N-glycosylase (UNG) was from Epicentre Technologies (Madison, WI). Hydrogen peroxide (30%) and Tween 20 were from Fisher. Fatty acid-free BSA was from Calbiochem. Rnase A was from Roche Molecular Biochemicals. Calf thymus DNA was from Worthington. ARP NЈ-aminooxy-methylcarbonyl hydrazino-D-biotin, was from Dujindo Laboratories (Kumamoto, Japan). Methyl methanesulfonate (MMS) and 1-octane sulfonic acid were from Fluka.Preparation of Nuclear Fraction from Rat Liver and Bra...

Small noncoding RNAs circulating in the blood may serve as signaling molecules because of their ability to carry out a variety of cellular functions. We have previously described tRNA- and YRNA-derived small RNAs circulating as components of larger complexes in the blood of humans and mice; the characteristics of these small RNAs imply specific processing, secretion, and physiological regulation. In this study, we have asked if changes in the serum abundance of these tRNA and YRNA fragments are associated with a diagnosis of cancer. We used deep sequencing and informatics analysis to catalog small RNAs in the sera of breast cancer cases and normal controls. 5′ tRNA halves and YRNA fragments are abundant in both groups, but we found that a breast cancer diagnosis is associated with changes in levels of specific subtypes. This prompted us to look at existing sequence datasets of serum small RNAs from 42 breast cancer cases, taken at the time of diagnosis. We find significant changes in the levels of specific 5′ tRNA halves and YRNA fragments associated with clinicopathologic characteristics of the cancer. Although these findings do not establish causality, they suggest that circulating 5′ tRNA halves and YRNA fragments with known cellular functions may participate in breast cancer syndromes and have potential as circulating biomarkers. Larger studies with multiple types of cancer are needed to adequately evaluate their potential use for the development of noninvasive cancer screening.

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