The DNA Damage Response and Checkpoint Adaptation in Saccharomyces cerevisiae: Distinct Roles for the Replication Protein A2 (Rfa2) N-Terminus

In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with “repairable” DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a “last-resort” mechanism for cell survival.     

Nutrient limitation of algal periphyton in streams along an acid mine drainage gradient

Metal oxyhydroxide precipitates that form from acid mine drainage (AMD) may indirectly limit periphyton by sorbing nutrients, particularly P. We examined effects of nutrient addition on periphytic algal biomass (chl a), community structure, and carbon and nitrogen content along an AMD gradient. Nutrient diffusing substrata with treatments of +P, +NP and control were placed at seven stream sites. Conductivity and SO₄ concentration ranged over an order of magnitude among sites and were used to define the AMD gradient, as they best indicate mine discharge sources of metals that create oxyhydroxide precipitates. Aqueous total phosphorous (TP) ranged from 2 to 23 μg · L^?1 and significantly decreased with increasing SO₄. Mean chl a concentrations at sites ranged from 0.2 to 8.1 μg · cm^?2. Across all sites, algal biomass was significantly higher on +NP than control treatments (Co), and significantly increased with +NP. The degree of nutrient limitation was determined by the increase in chl a concentration on +NP relative to Co (response ratio), which ranged from 0.6 to 9.7. Response to nutrient addition significantly declined with increasing aqueous TP, and significantly increased with increasing SO₄. Thus, nutrient limitation of algal biomass increased with AMD impact, indicating metal oxyhydroxides associated with AMD likely decreased P availability. Algal species composition was significantly affected by site but not nutrient treatment. Percent carbon content of periphyton on the Co significantly increased with AMD impact and corresponded to an increase in the relative abundance of Chlorophytes. Changes in periphyton biomass and cellular nutrient content associated with nutrient limitation in AMD streams may affect higher trophic levels.     

Development of a prototype wound dressing technology which can detect and report colonization by pathogenic bacteria

A new methodology for detecting the microbiological state of a wound dressing in terms of its colonization with pathogenic bacteria such as Staphylococcus aureus or Pseudomonas aeruginosa has been developed. Here we report how stabilized lipid vesicles containing self-quenched carboxyfluorescein dye are sensitive to lysis only by toxins/virulence factors from P. aeruginosa and S. aureus but not by a non-toxic Escherichia coli species. The development of the stabilized vesicles is discussed and their response to detergent (triton), bacterial toxin (α-hemolysin) and lipases (phospholipase A₂). Finally, fabrics with stabilized vesicles attached via plasma deposited maleic anhydride coupling are shown visibly responding to S. aureus (MSSA 476) and P. aeruginosa (PAO1) but not E. coli DH5α in a prototype dressing.     

Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells

Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H₂O₂ to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H₂O₂ was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.     

TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment

The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine.     

Induction of oxidative stress as a possible mechanism of the antifungal action of three phenylpropanoids

The increasing incidence of hospital-acquired infections caused by drug-resistant pathogens, host toxicity, the poor efficacy of drugs and high treatment costs has drawn attention to the potential of natural products as antifungals in mucocutaneous infections and combinational therapies. Moreover, cellular and subcellular targets for these compounds may provide better options for the development of novel antifungal therapies. Eugenol, methyl eugenol and estragole are phenylpropanoids found in essential oil. They are known to possess pharmacological properties including antimicrobial activity. Induction of oxidative stress characterized by elevated levels of free radicals and an impaired antioxidant defence system is implicated as a possible mechanism of cell death. An insight into the mechanism of action was gained by propidium iodide cell sorting and oxidative stress response to test compounds in Candida albicans. The extent of lipid peroxidation (LPO) of cytoplasmic membranes was estimated to confirm a state of oxidative stress. Activity levels of primary defence enzymes and glutathione were thus further determined. Whereas these compounds cause fungal cell death by disrupting membrane integrity at minimum inhibitory concentrations (MIC), sub-MIC doses of these compounds significantly impair the defence system in C. albicans. The study has implications for understanding microbial cell death caused by essential oil components eliciting oxidative stress in Candida. The formation of membrane lesions by these phenylpropanoids thus appears to be the result of free radical cascade-mediated LPO.