Summary NAD+ is an important co-factor regulating metabolic homeostasis and a rate-limiting substrate for sirtuin deacylase. We show that NAD+ levels are reduced in aged mice and C. elegans and that decreasing NAD+ levels results in a further reduction in worm lifespan. Conversely, genetic or pharmacological restoration of NAD+ prevents age-associated metabolic decline and promotes longevity in worms. These effects are dependent upon the protein deacetylase sir-2.1 and involve the induction of mitonuclear protein imbalance as well as activation of stress signaling via the mitochondrial unfolded protein response (UPRmt) and the nuclear translocation and activation of FOXO transcription factor DAF-16. Our data suggest that augmenting mitochondrial stress signaling through the modulation of NAD+ levels may be a target to improve mitochondrial function and prevent or treat age-associated decline.

Transplantation studies in mice and rats have shown that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts1–3, but two critical issues related to their electrophysiological behavior in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear if these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea pig model to show hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia (VT). To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically-encoded calcium sensor, GCaMP34, 5. By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host-graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

C. elegans SIR-2.1, a member of the Sir-2 family of NAD(+)-dependent protein deacetylases, has been shown to regulate nematode aging via the insulin/IGF pathway transcription factor daf-16. Treatment of C. elegans with the small molecule resveratrol, however, extends life span in a manner fully dependent upon sir-2.1, but independent of daf-16. Microarray analysis of worms treated with resveratrol demonstrates the transcriptional induction of a family of genes encoding prion-like glutamine/asparagine-rich proteins involved in endoplasmic reticulum (ER) stress response to unfolded proteins. RNA interference of abu-11, a member of this ER stress gene family, abolishes resveratrol-mediated life span extension, and overexpression of abu-11 extends the life span of transgenic animals. Furthermore, SIR-2.1 normally represses transcription of abu-11 and other ER stress gene family members, indicating that resveratrol extends life span by inhibiting sir-2.1-mediated repression of ER stress genes. Our findings demonstrate that abu-11 and other members of its ER stress gene family are positive determinants of C. elegans life span.

The longevity of Caenorhabditis elegans is promoted by extra copies of the sir-2.1 gene in a manner dependent on the forkhead transcription factor DAF-16. We identify two C. elegans 14-3-3 proteins as SIR-2.1 binding partners and show that 14-3-3 genes are required for the life-span extension conferred by extra copies of sir-2.1. 14-3-3 proteins are also required for SIR-2.1-induced transcriptional activation of DAF-16 and stress resistance. Following heat stress, SIR-2.1 can bind DAF-16 in a 14-3-3-dependent manner. By contrast, low insulin-like signaling does not promote SIR-2.1/DAF-16 interaction, and sir-2.1 and the 14-3-3 genes are not required for the regulation of life span by the insulin-like signaling pathway. We propose the existence of a stress-dependent pathway in which SIR-2.1 and 14-3-3 act in parallel to the insulin-like pathway to activate DAF-16 and extend life span.

Recent studies of subjects infected with human immunodeficiency virus (HIV-1) have produced conflicting results about the extent of reconstitution possible in the CD4+ lymphocyte repertoire after highly active antiretroviral therapy (HAART). The effect of HAART on the incidence of opportunistic infections will probably depend on reconstitution of antigen-specific CD4+ lymphocyte responses to important pathogens, including cytomegalovirus (CMV), the leading cause of blindness in AIDS. Several studies have demonstrated an important role for CD4+ lymphocytes in controlling CMV replication in vitro and in clinical studies. It is now possible to quantitate antigen-specific CD4+ lymphocyte responses by flow cytometry. Using this method, we studied CMV-specific CD4+ lymphocyte responses in individuals infected with HIV-1 with and without a history of active CMV-associated end organ disease (EOD), and in those with quiescent CMV EOD after ganciclovir therapy and HAART. The presence of active CMV-associated EOD strongly correlated with loss of CMV-specific lymphocyte responses (P = 0.0004). In contrast, patients with no history of CMV-associated EOD and most patients with quiescent EOD after HAART demonstrated strong CMV-specific CD4+ lymphocyte responses. These data indicate that the loss of CMV-specific CD4+ lymphocyte responses in individuals infected with HIV-1 who have active CMV EOD may be restored after ganciclovir therapy and HAART, which provides evidence for functional immune reconstitution to an important pathogen.

Biochemical studies with model DNA heteroduplexes have implicated RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X in Escherichia coli methyl-directed mismatch correction. However, strains deficient in the four exonucleases display only a modest increase in mutation rate, raising questions concerning involvement of these activities in mismatch repair in vivo. The quadruple mutant deficient in the four exonucleases, as well as the triple mutant deficient in RecJ exonuclease, exonuclease VII, and exonuclease I, grow poorly in the presence of the base analogue 2-aminopurine, and exposure to the base analogue results in filament formation, indicative of induction of SOS DNA damage response. The growth defect and filamentation phenotypes associated with 2-aminopurine exposure are effectively suppressed by null mutations in mutH, mutL, mutS, or uvrD͞mutU, which encode activities that act upstream of the four exonucleases in the mechanism for the methyl-directed reaction that has been proposed based on in vitro studies. The quadruple exonuclease mutant is also cold-sensitive, having a severe growth defect at 30°C. This phenotype is suppressed by a uvrD͞mutU defect, and partially suppressed by mutH, mutL, or mutS mutations. These observations confirm involvement of the four exonucleases in methyl-directed mismatch repair in vivo and suggest that the low mutability of exonuclease-deficient strains is a consequence of under recovery of mutants due to a reduction in viability and͞or chromosome loss associated with activation of the mismatch repair system in the absence of RecJ exonuclease, exonuclease VII, exonuclease I, and exonuclease X.

The understanding of human thymic function and evaluation of its contribution to T cell homeostasis are matters of great importance. Here we report the development of a novel assay to quantitate the frequency and diversity of recent thymic emigrants (RTEs) in the peripheral blood of humans. Such cells were defined by the presence of T cell receptor (TCR) rearrangement deletion circles (DCs), episomal byproducts of TCR-β V(D)J rearrangement. DCs were detected in T cells in the thymus, cord blood, and adult peripheral blood. In the peripheral blood of adults aged 22 to 76 years, their frequency was highest in the CD4+CD45RA+ CD62L+ subpopulation of naive T cells. TCR DCs were also observed in other subpopulations of peripheral blood T cells, including those with the CD4+CD45RO−CD62L+ and CD4+CD45RO+CD62L+ phenotypes. RTEs were observed to have more than one Vβ rearrangement, suggesting that replenishment of the repertoire in the adult is at least oligoclonal. These results demonstrate that the normal adult thymus continues to contribute, even in older individuals, a diverse set of new T cells to the peripheral circulation.

Melatonin binding sites were localized and characterized in the vasculature of the rat by using the melatonin analogue 2-['25I]iodomelatonin (1251-melatonin) and quantitative in vitro autoradiography. The expression of these sites was restricted to the caudal artery and to the arteries that form the circle of Willis at the base of the brain. The arterial 125I-melatonin binding was stable, saturable, and reversible.Saturation studies revealed that the binding represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 3.4 X 10-11 M in the anterior cerebral artery and 1.05 x 10-10 M in the caudal artery. The binding capacities (Bm,) in these arteries were 19 and 15 fmol/mg of protein, respectively. The relative order of potency of indoles for inhibition of 'II-melatonin binding at these sites was typical of a melatonin receptor: 2-iodomelatonin > melatonin > Nacetylserotonin >>> 5-hydroxytryptamine. Norepinephrineinduced contraction of the caudal artery in vitro was significantiy prolonged and potentiated by melatonin in a concentration-dependent manner, suggesting that these arterial binding sites are functional melatonin receptors. Neither primary steps in smooth muscle contraction (inositol phospholipid hydrolysis) nor relaxation (adenylate cyclase activation) were affected by melatonin. Melatonin, through its action on the tone of these arteries, may cause circulatory adjustments in these arteries, which are believed to be involved in thermoregulation.Melatonin, a hormone produced by the pineal gland of vertebrates, is thought to serve as a neuroendocrine transducer of photoperiodic information (1,2). Through use of the radioiodinated melatonin analogue 2-[125lJiodomelatonin (1251-melatonin) putative melatonin receptors have been described in specific areas of the brain (3-12) and retina (13) Horizontal or coronal sections of the brain and cross sections of the caudal artery (16 ,um) were cut in a cryostat at -17TC, thaw-mounted onto gelatin-coated slides, and dried in a desiccator at 40C overnight.Binding Assays. The binding conditions used have been described (14). Tissue sections were incubated for 60 min in 50 mM Tris HCl buffer (pH 7.4) containing 4 mM CaCl2 and 1251I-melatonin (specific activity, 1800-2100 Ci/mmol; Amersham; 1 Ci = 37 GBq). Nonspecific binding was determined in the presence of 1 uM melatonin. After incubation, the sections were washed twice for 5 min in 50 mM Tris-HCl buffer followed by 30 sec in distilled water at 0C. There was no loss of high-affinity binding when tissue sections were washed in Tris HCI for up to 30 min at 00C.In the anterior cerebral artery, 125I-melatonin binding reached equilibrium within 60 min at 220C and was stable for at least 120 min. Excess of melatonin (1 ,uM), added at equilibrium, was able to compete with 1251-melatonin for binding to the arterial sites, demonstrating reversibility ofthe binding. The apparent dissociation constant (Kd) was 1.27 x 10-11 M, calculated as k2/kl (k1 = association rate constant = 7.5...