Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products

Epidemiological studies testing the effect of β-carotene in humans have found a relative risk for lung cancer in smokers supplemented with β-carotene. We investigated the reactions of retinal and β-apo-8′-carotenal, two β-carotene oxidation products, with 2′-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N²-etheno-2′-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with β-carotene or β-carotene oxidation products, significantly increased levels of 1,N²-etheno-2′-deoxyguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of β-carotene oxidation products.     

Calcium inhibits bap-dependent multicellular behavior in Staphylococcus aureus

Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.     

Cigarette smoke concentrate increases 8-epi-PGF2alpha and TGFbeta1 secretion in rat mesangial cells

Epidemiological studies have shown that cigarette smoke, an oxidant agent, is a risk factor for the development of diabetic nephropathy (DN), in which pathogenesis transforming growth factor beta(1) (TGFbeta(1)) plays a key role. In our experimental model we exposed mesangial cell cultures to cigarette smoke concentrate (CSC) to study the effect of smoking on the pathogenesis of DN. Thus, we analyzed the effect of CSC on TGFbeta(1) and lipid peroxidation (8-epi-PGF(2alpha)) in rat mesangial cells. Furthermore, since the protein kinase C (PKC) pathway appears to be a key factor for the enhanced production of TGFbeta(1), we also analyzed the effect of the selective PKCbeta inhibitor LY379196 on TGFbeta(1) response to CSC. CSC induced an increase of both TGFbeta(1) and 8-epi-PGF(2) compared to basal conditions (5 mM glucose). The CSC-induced increase in TGFbeta(1) secretion was significantly suppressed by LY379196. These data suggest that smoking could increase TGFbeta(1) production, probably due to oxidative stress and PKCbeta activation. This finding supports the concept that smoking is a risk factor for DN development.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Evidence for the sucrose-binding protein role in carbohydrate metabolism and transport at early developmental stage

Despite the large amount of data regarding sucrose-binding proteins (SBP), their functions remain largely unknown and controversial. In this investigation we performed a detailed temporal and spatial characterization of the phenotypes related to photosynthesis, sucrose exudation and carbohydrate metabolism in SBP antisense plants to gain insights into the physiological role of SBP. Significant reductions in net photosynthesis and in stomatal conductance were observed in the SBP antisense lines but were restricted to the vegetative phase, and persisted during a daily time course at this phase. Photosynthesis was saturated at a substantially lower irradiance in source leaves of the antisense lines, suggesting that light utilization is decreased in these plants. A slight reduction in soluble sugars was observed throughout the development of source leaves, partially overlapping a decrease in sucrose synthase activity (EC 2.4.1.13); whereas a transient increase in starch and adinosine diphosphate (ADP)-glucose pyrophosphorylase activity (EC 2.7.7.27) as well as decreased leaf sucrose exudation were detected in the beginning of the vegetative phase. These changes in source leaves were accompanied by reductions in sucrose and starch in sink leaves, hexoses and sucrose in roots and hexoses in shoot apex, which were observed before the occurrence of a significant reduction in height and in leaf number in the transgenic lines. These alterations in growth parameters did not persist throughout the development, but were associated with a delay in flowering time and leaf senescence in the SBP antisense lines. A likely involvement of SBP in sink strength is discussed.     

beta-lactam antibiotics induce the SOS response and horizontal transfer of virulence factors in Staphylococcus aureus

Antibiotics that interfere with DNA replication and cell viability activate the SOS response. In Staphylococcus aureus, the antibiotic-induced SOS response promotes replication and high-frequency horizontal transfer of pathogenicity island-encoded virulence factors. Here we report that β-lactams induce a bona fide SOS response in S. aureus, characterized by the activation of the RecA and LexA proteins, the two master regulators of the SOS response. Moreover, we show that β-lactams are capable of triggering staphylococcal prophage induction in S. aureus lysogens. Consequently, and as previously described for SOS induction by commonly used fluoroquinolone antibiotics, β-lactam-mediated phage induction also resulted in replication and high-frequency transfer of the staphylococcal pathogenicity islands, showing that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors.     

Phylogeography of Pseudacris regilla (Anura: Hylidae) in western North America, with a proposal for a new taxonomic rearrangement

The Baja California populations of Pseudacris regilla, a widespread species in Western North America ranging from British Columbia to southern Baja California, are characterized by extensive geographic fragmentation. We performed phylogeographic and historical demographic analyses on 609 bp of the cytochrome b mitochondrial gene of 110 individuals representing 28 populations to determine the relative influences of current and historical processes in shaping the present distribution of genetic diversity on the Baja California Peninsula. Haplotypes from this area were nested in a clade with three well-differentiated groups. Two of these groups are from Baja California Sur and another is from California and Baja California. The estimated date for the split of these groups, between 0.9-1 Ma, fits with previously proposed hypotheses of vicariance due to different transpeninsular seaways, although successive population fragmentation and expansion due to climatic oscillations during Pleistocene glaciations cannot be discarded. Historical demographic analyses detected signs of past population expansions, especially in the southernmost group. With respect to populations north of this region, two older clades were identified, one with haplotypes mainly distributed in central California, and the other corresponding to the northern half of the species range, in what apparently is a recurrent pattern in the Pacific coast of North America. Based on the concordance between mt-DNA and available allozyme data indicating that these species have a long independent evolutionary history, we propose to consider the three major clades as distinct species: P. regilla, P. pacifica, and P. hypochondriaca.     

Peculiar H+ Homeostasis of Saccharomyces cerevisiae during the Late Stages of Wine Fermentation

Intracellular pH (pH(in)) is a tightly regulated physiological parameter, which controls cell performance in all living systems. The purpose of this work was to evaluate if and how H(+) homeostasis is accomplished by an industrial wine strain of Saccharomyces cerevisiae while fermenting real must under the harsh winery conditions prevalent in the late stages of the fermentation process, in particular low pH and high ethanol concentrations and temperature. Cells grown at 15, 25, and 30°C were harvested in exponential and early and late stationary phases. Intracellular pH remained in the range of 6.0 to 6.4, decreasing significantly only by the end of glucose fermentation, in particular at lower temperatures (pH(in) 5.2 at 15°C), although the cells remained viable and metabolically active. The cell capability of extruding H(+) via H(+)-ATPase and of keeping H(+) out by means of an impermeable membrane were evaluated as potential mechanisms of H(+) homeostasis. At 30°C, H(+) efflux was higher in all stages. The most striking observation was that cells in late stationary phase became almost impermeable to H(+). Even when these cells were challenged with high ethanol concentrations (up to 20%) added in the assay, their permeability to H(+) remained very low, being almost undetectable at 15°C. Comparatively, ethanol significantly increased the H(+) permeability of cells in exponential phase. Understanding the molecular and physiological events underlying yeast H(+) homeostasis at late stages of fermentations may contribute to the development of more robust strains suitable to efficiently produce a high-quality wine.     

APOBEC3 proteins and genomic stability: The high cost of a good defense

The human APOBEC3 family of cytidine deaminases constitutes a cellular intrinsic defense mechanism that is effective against a range of viruses and retro-elements. While it is well-established that these enzymes are powerful mutators of viral DNA, the possibility that their activity could threaten the integrity of the host genome has only recently begun to be investigated. Here, we discuss the implications of new evidence suggesting that APOBEC3 proteins can mediate the deamination of cellular DNA. The maintenance of genomic integrity in the face of this potential off-target activity must require high-fidelity DNA repair and strict regulation of APOBEC3 gene expression and enzyme activity. Conversely, the ability of specific members of the APOBEC3 family to activate DNA damage signaling pathways might also reflect another way that these proteins contribute to the host immune response.Key words: APOBEC3, cytidine deaminase, AID, uracil-DNA glycosylase, DNA damage, hypermutation, genomic instability, cancer     

Enrichment experiments and primary production at Sagres (SW Portugal)

Water was collected from the Sagres station (SW Portugal) in September 2002, at a site adjacent to the upwelling centre of Cabo São Vicente, during relaxation of upwelling conditions. Surface and depth samples were enriched with inorganic nutrients in order to evaluate their relative influence on the microalgal assemblage. Small-scale, short-term bioassays involved separate in vitro additions of nitrogen and phosphorus. Enrichments with nitrogen led to a general increase of primary production, suggesting nitrogen as the primary potential nutrient limiting microalgal growth during this period, as well as altering the relative microplanktonic composition in favour of diatoms.