Minimization of free radical damage by metal catalysis of multivitamin/multimineral supplements

Multivitamin/multimineral complexes are the most common dietary supplements. Unlike minerals in foods that are incorporated in bioorganic structures, minerals in dietary supplements are typically in an inorganic form. These minerals can catalyze the generation of free radicals, thereby oxidizing antioxidants during digestion. Here we examine the ability of a matrix consisting of an amino acid and non-digestible oligosaccharide (AAOS) to blunt metal-catalyzed oxidations. Monitoring of ascorbate radical generated by copper shows that ascorbate is oxidized more slowly with the AAOS matrix than with copper sulfate. Measurement of the rate of oxidation of ascorbic acid and Trolox® by catalytic metals confirmed the ability of AAOS to slow these oxidations. Similar results were observed with iron-catalyzed formation of hydroxyl radicals. When compared to traditional forms of minerals used in supplements, we conclude that the oxidative loss of antioxidants in solution at physiological pH is much slower when AAOS is present

Expression of thymidylate synthase in human cells is an early G1 event regulated by CDK4 and p16INK4A but not E2F

Thymidylate synthase (TS) is the enzyme that catalyses the last step in de novo thymidylate synthesis. It is of interest clinically because it is an effective target for drugs such as 5-fluorouracil, often used in combination therapy. Despite a number of earlier reports indicating that TS is a cell cycle-dependent enzyme, this remains equivocal. Here, we show that in HCT116 cells synchronised by serum starvation, there is a clear dissociation between the expression of cyclin E (a well-characterised cell-cycle protein) and TS. Although both cyclin E and TS mRNA and protein increased during G1, TS upregulation was delayed. Moreover, TS levels did not decrease following S-phase completion while cyclin E decreased sharply. Similarly, clear differences were seen between cyclin E and TS as asynchronously growing HCT116 cells were growth-inhibited by low-serum treatment. In contrast to previous reports using rodent cells, adenovirus-mediated over-expression of E2F1 and cyclin E in three human cell lines had no effect on TS. Cell-cycle progression was blocked by treatment of cells with pharmacological inhibitors of CDK2 and CDK4 and by ectopic expression of p16INK4A. Whereas CDK2 inhibition had no effect on TS levels, inhibition of CDK4 was associated with decreased TS protein levels. These results provide the first evidence that drugs targeting CDK4 may be useful with anti-TS drugs as combination therapy for cancer

Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies

BACKGROUND: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications. METHODS: Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe3O4 nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy. RESULTS: Surface plasmon resonance analyses revealed a medium affinity (KD\ufffd40\ufffd50 nM) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe3O4 NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb- PEGylated Fe3O4 NPs selectively in GBM vessels. CONCLUSIONS: Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.Peer reviewed: YesNRC publication: Ye

The lipid droplet coat protein perilipin 5 also localizes to muscle mitochondria

Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, 14C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation

Genetic control of quantitative variation in carbonic anhydrase isozymes of mammals. I. The concentration within single erythrocytes of the mouse

The levels of the carbonic anhydrase isozymes (CA and CA II) in single erythrocytes of DBA/2J mice were assayed by measuring the specific immunofluorescence of CA I and CA II with a microspectrofluorometric technique. Measurements of 100 randomly selected cells showed a range (in relative fluorescence units) of 21–52 (mean 31.3±7.5) for CA I and 30–80 (mean 45.6±10.7) for CA II. The CA II/CA I ratio of the means obtained by the single-cell fluorescence assay was similar to the ratios obtained for the two isozymes from hemolysates of DBA/2J mice using an immunodiffusion assay. The influence of cell age on the variation in carbonic anhydrase levels was determined by separating red cells into several fractions by a gravity sedimentation procedure. The younger cells showed higher levels of CA I and CA II than the older cells; however, the extensive overlap in the variability between the cells from the different fractions indicated that although cell age was contributing to the overall heterogeneity, its influence was not pronounced.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44126/1/10528_2004_Article_BF00483984.pd