Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.     

OX40 ligand-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-Apc signaling to boost the host T cell antitumor response

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.     

The mitochondrial ornithine transporter. Bacterial expression,reconstitution, functional characterization,and tissue distribution of two human isoforms

Two isoforms of the human ornithine carrier, ORC1 and ORC2, have been identified by overexpression of the proteins in bacteria and by study of the transport properties of the purified proteins reconstituted into liposomes. Both transport L-isomers of ornithine, lysine, arginine, and citrulline by exchange and by unidirectional mechanisms, and they are inactivated by the same inhibitors. ORC2 has a broader specificity than ORC1, and L- and D-histidine, L-homoarginine, and D-isomers of ornithine, lysine, and ornithine are all substrates. Both proteins are expressed in a wide range of human tissues, but ORC1 is the predominant form. The highest levels of expression of both isoforms are in the liver. Five mutant forms of ORC1 associated with the human disease hyperornithinemia-hyperammonemia-homocitrullinuria were also made. The mutations abolish the transport properties of the protein. In patients with hyperornithinemia-hyperammonemia-homocitrullinuria, isoform ORC2 is unmodified, and its presence compensates partially for defective ORC1.     

Mutagenesis of putative catalytic and regulatory residues of Streptomyces chromofuscus phospholipase D differentially modifies phosphatase and phosphodiesterase activities

Phospholipase D from Streptomyces chromofuscus (sc-PLD) is a member of the diverse family of metallo-phosphodiesterase/phosphatase enzymes that also includes purple acid phosphatases, protein phosphatases, and nucleotide phosphodiesterases. Whereas iron is an essential cofactor for scPLD activity, Mn2+ is also found in the enzyme. A third metal ion, Ca2+, has been shown to enhance scPLD catalytic activity although it is not an essential cofactor. Sequence alignment of scPLD with known phosphodiesterases and phosphatases requiring metal ions suggested that His-212, Glu-213, and Asp-389 could be involved in Mn2+ binding. H212A, E213A, and D389A were prepared to test this hypothesis. These three mutant enzymes and wild type scPLD show similar metal content but considerably different catalytic properties, suggesting different roles for each residue. His-212 appears involved in binding the phosphate group of substrates, whereas Glu-213 acts as a ligand for Ca2+. D389A showed a greatly reduced phosphodiesterase activity but almost unaltered ability to hydrolyze the phosphate group in p-nitrophenyl phosphate suggesting it had a critical role in aligning groups at the active site to control phosphodiesterase versus phosphatase activities. We propose a model for substrate and cofactor binding to the catalytic site of scPLD based on these results and on sequence alignment to purple acid phosphatases of known structure.     

Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerability of polymorphonuclear leukocytes

Since oxidative stress is thought to play an important role in the pathogenesis and complications of diabetes, we used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as FPG (formamidopyrimidine DNA glycosylase)-sensitive sites, in peripheral blood cells (PBC) from type 2 diabetes patients and healthy controls. Oxidative DNA damage in leukocytes was increased in diabetic compared to normal subjects. However, no differences in the levels of DNA damage in isolated lymphocytes were found between the two groups. These data indicate a higher vulnerability to oxidative damage of polymorphonuclear as compared to mononuclear leukocytes in type 2 diabetes. Thus, the measurement of oxidative DNA damage in leukocytes by means of the comet assay is a suitable marker for the evaluation of systemic oxidative stress in diabetic patients.     

Table of Contents of Volume 51 - 2003

Table of Contents

Table of Contents of Volume 51 - 2003     

Carotenoid cleavage products modify respiratory burst and induce apoptosis of human neutrophils

Carotenoid supplementation in the treatment of diseases associated with oxidative stress has been recently questioned because of the cell damage and the increased risk of lung cancer in male smokers. Because of the complex role of neutrophils in lung diseases, we investigated whether carotenoid derivatives could affect respiratory burst and apoptosis of human neutrophils purified from peripheral blood. Stimulation of superoxide production was induced by nanomolar and micromolar concentrations of carotenoid cleavage products with aliphatic chains of different length, but not by carotenoids lacking the carbonyl moiety. The stimulatory effect of carotenoid cleavage products was observed in cells activated by phorbol myristate acetate (PMA), while a slight inhibition of superoxide production was noticed with cells activated by the chemotactic tripeptide N-formyl-Met-Leu-Phe (f-MLP). At higher concentrations, carotenoid cleavage products inhibited superoxide production in the presence of both PMA and f-MLP. In the presence of 20 microM carotenoid cleavage products, inhibition of superoxide production was accompanied by DNA fragmentation and increased level of intracellular caspase-3 activity.     

Multiple effects of paclitaxel are modulated by a high c-myc amplification level

Paclitaxel affects microtubule stability by binding to beta-tubulin, thus leading to cell accumulation in the G(2)/M phase, polyploidization, and apoptosis. Because both cell proliferation and apoptosis could be somehow regulated by the protooncogene c-myc, in this work we have investigated whether the c-myc amplification level could modulate the multiple effects of paclitaxel. To this aim, paclitaxel was administered to SW613-12A1 and -B3 human colon carcinoma cell lines (which are characterized by a high and low c-myc endogenous amplification level, respectively), and to the B3mycC5 cell line, with an enforced exogenous expression of c-myc copies. In this experimental system, we previously demonstrated that a high endogenous/exogenous level of amplification of c-myc enhances serum deprivation- and DNA damage-induced apoptosis. Accordingly, the present results indicate that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype, and promotes apoptosis to a great extent, thus suggesting that c-myc expression level is relevant in modulating the cellular responses to paclitaxel. We have recently shown in HeLa cells that the phosphorylated form of c-Myc accumulates in the nucleus, as distinct nucleolar and extranucleolar spots; here, we demonstrated that, after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm.     

Preparation and functional characterization of thylakoids from Arabidopsis thaliana

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O₂ and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate. This revised version was published online in June 2006 with corrections to the Cover Date.