Regulation of SIRT 1 mediated NAD dependent deacetylation: a novel role for the multifunctional enzyme CD38

The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.     

Influence of different levels of ammonium concentrations on cell growth,RNA and protein production byCandida albicans

Candida albicans starved cells were incubated in minimal synthetic liquid media containing different concentrations of ammonium sulphate (0.00, 0.02, 0.05, 0.10, 0.03, 0.50 g/L). Culture growth was monitored by measuring daily the optical density and by evaluating RNA and protein cellular content after 48 and 96 hours from the inoculum. The environmental availability of ammonium ion influenced the biomass production, that was maximum when its concentration was 0.10 and 0.30 g/L. In addition, an effect on cell duplication time was observed, this was particularly evident when the (NH₄)₂SO₄ concentration was 0.10 g/L. The protein content increased in relation to the increase of ammonium ion availability, with a peak in correspondence to 0.30 g/L and a drop when the greatest concentrations were employed. RNA production was inversely proportional in respect to protein production. The optimal range of ammonium sulphate concentration forC. albicans growth was 0.10–0.30 g/L; over these concentrations there was an inhibitory effect. The rate of the protein and RNA syntheses seems to indicate the growth phase and the nitrogen nutritional conditions of the cultures, respectively.     

Development of 99mTc(CO)₃-dendrimer-FITC for cancer imaging

Study of fluorophore and technetium labeling of poly(amido)-amine (PAMAM) generation 4 (G4) dendrimer and its evaluation as potential molecular imaging agent in both normal and melanoma-bearing mice, are described. Dendrimers were first conjugated with FITC (fluorescein isothiocyanate). Dendrimer-FITC was then incubated with the intermediate [(99m)Tc(CO)(3)(H(2)O)(3)](+) and purified by gel filtration. Biodistribution and scintigraphy images were performed administrating (99m)Tc(CO)(3)-dendrimer-FITC to normal mice (NM) or melanoma-bearing mice (MBM). Cryostat tissue sections from MBM mice were analyzed by confocal microscopy. Radiolabeling yield of dendrimer was approx. 90%. The (99m)Tc(CO)(3)-dendrimer-FITC complex was stable for at least 24h. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with high tumor uptake that allowed tumor imaging. Confocal microscopy analysis showed cytoplasmic distribution of (99m)Tc(CO)(3)-dendrimer-FITC.     

Recombinant expression, characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

HIV Risk Perception and Behavior among Sex Workers in Three Major Urban Centers of Mozambique

HIV risk perceptions and behaviors of 236 commercial sex workers from three major Mozambican urban centers were studied using the International Rapid Assessment, Response and Evaluation (I-RARE) methodology. All were offered HIV testing and, in Maputo, syphilis testing was offered as well. Sixty-three of the 236 opted for HIV testing, with 30 (48%) testing positive for HIV. In Maputo, all 30 receiving HIV tests also had syphilis testing, with 6 (20%) found to be positive. Results include interview excerpts and qualitative results using I-RARE methodology and AnSWR-assisted analyses of the interviews and focus group sessions.     

Four different forms of interferon-induced 2',5'-oligo(A) synthetase identified by immunoblotting in human cells

Antibodies against synthetic peptides derived from the cDNA sequence of interferon-induced 2',5'-oligo(A) synthetase, and which immunoprecipitate the native enzyme activity, were found to detect multiple enzyme forms in denaturing electrophoretic immunoblots. In some human cell lines, four different interferon-induced proteins of 40, 46, 67, and 100 kDa were found to react with the same peptide antibodies. Each isolated form was shown to have 2',5'-oligo(A) synthetase activity, but the dependence on double-stranded RNA was markedly different for activation of the individual enzymes. The four enzyme forms also differ in their intracellular localization, on microsomes (100 kDa), in nuclei (67, 46, 40 kDa), and on membrane structures (67 kDa). Plasma membranes from interferon-treated Daudi lymphoblastoid cells are highly enriched in the 67-kDa 2',5'-oligo(A) synthetase form. The 2',5'-oligo(A) synthetase activity induced by interferons in human cells appears, therefore, as a complex multienzyme system.