Cloning and characterization of two mouse genes with homology to the yeast Sir2 gene

The yeast Sir2 gene encodes a protein (Sir2p) that plays an essential role in silencing regulation at mating-type loci, rDNA, and telomeres. Recent studies have also shown that the protein participates in cell cycle regulation, DNA double-strand break repair, meiotic checkpoint control, and histone deacetylation. Overexpression of wildtype Sir2p in yeast resulted in an extended life span but mutant Sir2p shortened the life span, suggesting its function in aging processes. Sir2p is evolutionarily conserved from prokaryotes to higher eukaryotes. However, its function(s) in mammals remains unknown. To investigate Sir2p function(s) in mice, we cloned and characterized two mouse Sir2-like genes. Our results revealed that the two mouse Sir2-like proteins (mSIR2L2 and mSIR2L3) are most similar to the human Sir2-like proteins SIR2L2 and SIR2L3, respectively. Sir2 core domains are highly conserved in the two proteins and yeast Sir2p; however, the intracellular localizations of both mSIR2L2 and mSIR2L3 differ from that of yeast Sir2p and from one another. The two mouse genes have completely different genomic structures but were mapped on the same chromosome. It seems that the two mouse proteins, though they have Sir2 conserved domains, may function differently than yeast Sir2p.     

Differences between fruit-bearing and non-bearing apple spurs in activity of an enzyme system decomposing phloridzin

The activity of an enzyme system decomposing phloridzin was investigated in fruitbearing and nonbearing spurs of apple trees, Landsberger Reinette cv., throughout vegetation. Acetone powder obtained from xylem sap of apple spurs was incubated with phloridzinsubstratum in citric buffer at pH 5.5 for 12, 18 and 24 h at 30 °C. A paper and thin-layer chromatography as well aa a spectrophotometric assay were employed for tentative identification of enzymic degradation products. Phloretic acid (PA), co-factor of IAA-oxidase, as well as phloretin (Pin), and phloroglucinol (PI) were found after the digestion of phloridzin. The chromatographed enzyme reaction products were measured densitometrically. The activity of the enzyme system was estimated by its efficiency in PA production and phloridzin disappearance. Obtained values, expressed in percentages, showed that the enzyme activity in fruitbearing spurs was much higher than in nonbearing ones; 30 and 10% of released PA in July, respectively. Because fruitbearing spurs of the apple tree are possibly additionally supplied with auxin translocated from developing seeds, an adaptive character of the enzyme system producing PA, a known auxin repressor, is suggested.     

Pleiotropic effects of the yeast Sal1 and Aac2 carriers on mitochondrial function via an activity distinct from adenine nucleotide transport

In Saccharomyces cerevisiae, SAL1 encodes a Ca2+ -binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Delta exacerbates the respiratory deficiency and mtDNA instability of ggc1Delta, shy1Delta and mtg1Delta mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+ -binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96 V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria.     

Budding yeast silencing complexes and regulation of Sir2 activity by protein-protein interactions

Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo.     

Anti-aging effects of hesperidin on Saccharomyces cerevisiae via inhibition of reactive oxygen species and UTH1 gene expression

This study used a replicative lifespan assay of K6001 yeast to screen anti-aging food factors in commercial flavonoids. Hesperidin derived from the Citrus genus extended the lifespan of yeast at doses of 5 and 10 μM as compared with the control group (p<0.01, p<0.01). Reactive oxygen species (ROS), real-time PCR (RT-PCR), and lifespan assays of uth1 and skn7 mutants with the K6001 background were used to study the anti-aging mechanisms in yeast. The results indicate that hesperidin significantly inhibits the ROS of yeast, and UTH1 gene expression, and that SKN7 gene are involved in hesperidin-mediated lifespan extension. Further, increases in the Sir2 homolog, SIRT1 activity, and SOD gene expression were confirmed at doses of 5 (p<0.01) and 10 μM (p<0.05). This suggests that Sir2, UTH1 genes, and ROS inhibition after administration of hesperidin have important roles in the anti-aging effects of yeast. However, the aglycon hesperetin did not exhibit anti-aging effects in yeast.     

Involvement of Drosophila Sir2-like genes in the regulation of life span

In Drosophila melanogaster, the Sir2 gene and four Sir2-like genes have been found to be homologous to yeast SIR2 genes. To examine whether the fly Sir2, CG5216, and two Sir2-like genes, CG5085 and CG6284, affect life span, we suppressed their expression using RNAi. Decreased expression of the Sir2 and Sir2-like genes in all cells caused lethality during development. Suppression of the Sir2 in neurons and ubiquitous silencing of the Sir2-like genes shortened life spans. The effects were severer at 28 degrees C than at 25 degrees C. These results suggest that Sir2-like genes as well as Sir2 are involved in the regulation of life span in Drosophila.     

Kinetics of inhibition of polyphenol oxidase mediated browning in apple juice by beta-cyclodextrin and L-ascorbate-2-triphosphate

Polyphenol Oxidase (PPO) mediated browning in raw fruits and vegetables is a major cause of quality deterioration in fruits and vegetables and derived food products. Here the rate of browning reaction in apple juice treated individually and in combination (1:1) of beta-Cyclodextrin (beta-CD) and L-Ascorbate-2-triphosphate (L-AATP) is described. It was observed that the rate of quinone formation can be minimized using a combination of beta-CD and L-AATP as compared to individual treatment with these agents. Kinetic experiments revealed that both compounds are non-competitive inhibitors of PPO.     

Functional conservation between the human, nematode, and yeast CK2 cell cycle genes.

Protein kinase CK2 (formerly casein kinase II) is a highly conserved serine/threonine protein kinase ubiquitous in eukaryotic organisms. Previously, we have shown that CK2 is required for cell cycle progression and essential for the viability of the yeast Saccharomyces cerevisiae. We now report that either the human or the nematode Caenorhabditis elegans CK2alpha catalytic subunit can substitute for the yeast catalytic subunits. Additionally, expression of the human CK2 regulatory subunit (CK2beta) can suppress the temperature sensitivity of either of the two yeast CK2 mutant catalytic subunits. Taken together, these observations reinforce the view that the CK2 cell cycle progression genes have been highly conserved during evolution from yeast to humans, not only in structure but also in function.     

Timescale effects on the environmental control of carbon and water fluxes of an apple orchard

Model parameterization and validation of earth–atmosphere interactions are generally performed using a single timescale (e.g., nearly instantaneous, daily, and annual), although both delayed responses and hysteretic effects have been widely recognized. The lack of consideration of these effects hampers our capability to represent them in empirical‐ or process‐based models. Here we explore, using an apple orchard ecosystem in the North of Italy as a simplified case study, how the considered timescale impacts the relative importance of the single environmental variables in explaining observed net ecosystem exchange (NEE) and evapotranspiration (ET). Using 6 years of eddy covariance and meteorological information as input data, we found a decay of the relative importance of the modeling capability of photosynthetically active radiation in explaining both NEE and ET moving from half‐hourly to seasonal timescale and an increase in the relative importance of air temperature (T) and VPD. Satellite NDVI, used as proxy of leaf development, added little improvement to overall modeling capability. Increasing the timescale, the number of variables needed for parameterization decreased (from 5 to 1), while the proportion of variance explained by the model increased (r² from 0.56–0.78 to 0.85–0.90 for NEE and ET respectively). The wavelet coherence and the phase analyses showed that the two variables that increased their relative importance when the scale increased (T, VPD) were not in phase at the correlation peak of both ET and NEE. This phase shift in the time domain corresponds to a hysteretic response in the meteorological variables domain. This work confirms that the model parameterization should be performed using parameters calculated at the appropriate scale. It suggests that in managed ecosystems, where the interannual variability is minimized by the agronomic practices, the use of timescales large enough to include hysteretic and delayed responses reduces the number of required input variables and improves their explanatory capacity.     

Enhancement of Telomere-Plasmid Segregation by the X-Telomere Associated Sequence in Saccharomyces Cerevisiae Involves Sir2, Sir3, Sir4 and Abf1

We have previously shown that circular replicating plasmids that carry yeast telomere repeat sequence (TG(1-3)) tracts segregate efficiently relative to analogous plasmids lacking the TG(1-3) tract and this efficient segregation is dependent upon RAP1. While a long TG(1-3) tract is sufficient to improve plasmid segregation, the segregation efficiency of telomere plasmids (TEL-plasmids) is enhanced when the X-Telomere Associated Sequence (X-TAS) is also included on the plasmids. We now demonstrate that the enhancement of TEL-plasmid segregation by the X-TAS depends on SIR2, SIR3, SIR4 and ABF1 in trans and requires the Abflp-binding site within the X-TAS. Mutation of the Abflp-binding site within the X-TAS results in TEL-plasmids that are no longer affected by mutations in SIR2, SIR3 or SIR4, despite the fact that other Abflp-binding sites are present on the plasmid. Mutation of the ARS consensus sequence within the X-TAS converts the X-TAS from an enhancer element to a negative element that interferes with TEL-plasmid segregation in a SIR-dependent manner. Thus, telomere associated sequences interact with TG(1-3) tracts on the plasmid, suggesting that the TASs have an active role in modulating telomere function.     

Growth phase-dependent roles of Sir2 in oxidative stress resistance and chronological lifespan in yeast

Silent Information Regulator 2 (Sir2), a conserved NAD⁺-dependent histone deacetylase, has been implicated as one of the key factors in regulating stress response and longevity. Here, we report that the role of Sir2 in oxidative stress resistance and chronological lifespan is dependent on growth phase in yeast. In exponential phase, sir2Δ cells were more resistant to H₂O₂ stress and had a longer chronological lifespan than wild type. By contrast, in post-diauxic phase, sir2Δ cells were less resistant to H₂O₂ stress and had a shorter chronological lifespan than wild type cells. Similarly, the expression of antioxidant genes, which are essential to cope with oxidative stress, was regulated by Sir2 in a growth phasedependent manner. Collectively, our findings highlight the importance of the metabolic state of the cell in determining whether Sir2 can protect against or accelerate cellular aging of yeast.