Regulation of SIRT6 protein levels by nutrient availability.

Sirtuins have been shown to regulate life-span in response to nutritional availability. We show here that levels of the mammalian sirtuin, SIRT6, increased upon nutrient deprivation in cultured cells, in mice after fasting, and in rats fed a calorie-restricted diet. The increase in SIRT6 levels is due to stabilization of SIRT6 protein, and not via an increase in SIRT6 transcription. In addition, p53 positively regulates SIRT6 protein levels under standard growth conditions but has no role in the nutrient-dependent regulation of SIRT6. These observations imply that at least two sirtuins are involved in regulation of life-span by nutrient availability.

Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment.

Strategies that increase homing to the bone marrow and engraftment efficacy of ex vivo expended CD34(+) cells are expected to enhance their clinical utility. Here we report that nicotinamide (NAM), a form of vitamin B-3, delayed differentiation and increased engraftment efficacy of cord blood-derived human CD34(+) cells cultured with cytokines. In the presence of NAM, the fraction of CD34(+)CD38(-) cells increased and the fraction of differentiated cells (CD14(+), CD11b(+), and CD11c(+)) decreased. CD34(+) cells cultured with NAM displayed increased migration toward stromal cell derived factor-1 and homed to the bone marrow with higher efficacy, thus contributing to their increased engraftment efficacy, which was maintained in competitive transplants with noncultured competitor cells. NAM is a known potent inhibitor of several classes of ribosylase enzymes that require NAD for their activity, as well as sirtuin (SIRT1), class III NAD(+)-dependent-histone-deacetylase. We demonstrated that EX-527, a specific inhibitor of SIRT1 catalytic activity, inhibited differentiation of CD34(+) cells similar to NAM, while specific inhibitors of NAD-ribosylase enzymes did not inhibit differentiation, suggesting that the NAM effect is SIRT1-specific. Our findings suggest a critical function of SIRT1 in the regulation of hematopoietic stem cell activity and imply the clinical utility of NAM for ex vivo expansion of functional CD34(+) cells.

DeubiKuitylation: a novel DUB enzymatic activity for the DNA repair protein, Ku70.

The Ku70 protein was shown to be involved in multiple cellular pathways including DNA repair, telomere maintenance, V(D)J recombination and Bax mediated apoptosis. Yet, despite this wide spectrum of pathways, until recently the enzymatic activity of Ku70 was elusive. Recent findings demonstrate that Ku70 is associated with the proapoptotic protein Bax and possesses a deubiquitin enzyme (DUB) activity on it. These data suggest a dual role for Ku70 in apoptotic regulation; on one hand the association with Ku70 sequestered Bax away from the mitochondria and plays an antiapoptotic function. On the other hand, this association mediates and promotes Bax deubiquitylation which will block its labeling for proteasomal degradation and in this manner Ku70 will have a proapoptotic role. The exciting finding of Ku70's DUB activity opens numerous avenues for future research. Here we suggest candidate substrate proteins and indicate how the DUB activity of Ku70 on these, might affect the known Ku70's related pathways.

Differential staining of Western blots of human secreted glycoproteins from serum, milk, saliva, and seminal fluid using lectins displaying diverse sugar specificities.

Human milk, serum, saliva, and seminal fluid glycoproteins (gps) nourish and protect newborn and adult tissues. Their saccharides, which resemble cell membrane components, may block pathogen adhesion and infection. In the present study, they were examined by a battery of lectins from plants, animals, and bacteria, using hemagglutination inhibition and Western blot analyses. The lectins included galactophilic ones from Aplysia gonad, Erythrina corallodendron, Maclura pomifera (MPL), peanut, and Pseudomonas aeruginosa (PA-IL); fucose-binding lectins from Pseudomonas aeruginosa (PA-IIL), Ralstonia solanacearum (RSL), and Ulex europaeus (UEA-I), and mannose/glucose-binding Con A. The results demonstrated the chosen lectin efficiency for differential analysis of human secreted gps as compared to CBB staining. They unveiled the diversity of these body fluid gp glycans (those of the milk and seminal fluid being highest): the milk gps interacted most strongly with PA-IIL, followed by RSL; the saliva gps with RSL, followed by PA-IIL and MPL; the serum gps with Con A and MPL, followed by PA-IIL and RSL, and the seminal plasma gps with RSL and MPL, followed by UEA-I and PA-IIL. The potential usage of these lectins as probes for scientific, industrial, and medical purposes, and for quality control of the desired gps is clearly indicated.