ATP-dependent chromatin remodeling and histone acetyltransferases in 5-FU cytotoxicity in Saccharomyces cerevisiae.

Chromatin is thought to modulate access of repair proteins to DNA lesions, and may be altered by chromatin remodelers to facilitate repair. We investigated the participation of chromatin remodelers and DNA repair in 5-fluorouracil (5-FU) cytotoxicity in Saccharomyces cerevisiae. 5-FU is an antineoplastic drug commonly used in clinical settings. Among the several strains tested, only those with deficiencies in ATP-dependent chromatin remodeling (CR) and some histone acetyltransferases (HAT) exhibited sensitivity to 5-FU. CR and HAT double-mutants exhibited increased resistance to 5-FU in comparison to the wild-type mutant, but were still arrested in G2/M, as were the sensitive strains. The participation of Htz1p in 5-FU toxicity was also evaluated in single- and double-mutants of CR and HAT; the most significant effect was on cell cycle distribution. 5-FU lesions are repaired by different DNA repair machineries, including homologous recombination (HR) and post-replication repair (PRR). We investigated the role of CR and HAT in these DNA repair pathways. Deficiencies in Nhp10 and CR combined with deficiencies in HR or PRR increased 5-FU sensitivity; however, combined deficiencies of HAT, HR, and PRR did not. CRs are directly recruited to DNA damage and lead to chromatin relaxation, which facilitates access of HR and PRR proteins to 5-FU lesions. Combined deficiencies in HAT with defects in HR and PRR did not potentiate 5-FU cytotoxicity, possibly because they function in a common pathway.

Mineral content is related to antioxidant and antimutagenic properties of grape juice.

Grape juices are an important source of food antioxidants. Unfortunately, there is little data about the mineral composition and the antioxidant, mutagenic and antimutagenic activities of grape juice in eukaryote cells. We evaluated the mineral contents (Mg, Ca, Mn, Fe, Cu, Zn, Si, S, Cl) of grape juices, the antioxidant, mutagenic and/or antimutagenic activities of the juices in the yeast Saccharomyces cerevisiae, and looked for a possible association between mineral content and antioxidant, mutagenic and/or antimutagenic activities of juice samples. Eight commercial grape juices, four purple (Bordo variety) and four white (Niagara variety), were evaluated. Most of the minerals were in similar concentrations in purple and white grape juices, except for calcium and copper; purple grapes had more calcium content and white grapes had more copper content. All grape juices had important antioxidant and antimutagenic activities in S. cerevisiae and prevented the oxidative damage provoked by hydrogen peroxide (P < 0.05). Positive correlations (P < 0.05) were observed between antioxidant and antimutagenic activities and mineral content. In this context, we concluded that the grape juices, white and purple, are an important mineral source, and these contents explain, in part, the important antioxidant and antimutagenic activities.

A systems pharmacology analysis of major chemotherapy combination regimens used in gastric cancer treatment: predicting potential new protein targets and drugs.

Gastric cancer is the leading cause of cancer-related death worldwide, and treatment options include surgery and chemotherapy. Because of its prevalence, chemotherapy for gastric cancer treatment represents an active area of pharmacology research, and different small compounds have been used as single treatments or in combination therapy. Unfortunately, chemoresistance is a common phenomenon in gastric cancer cells, and the current arsenal of small compounds used in chemotherapy is not effective for long periods of treatment. Thus, to understand how gastric cancer cells develop chemoresistance and also to find new protein targets and small compounds for gastric cancer treatment, a systems pharmacology-based study was performed using the proteomic and small compounds-protein interaction data available for Homo sapiens. A major physical protein-protein and chemo-protein interaction (PPPI-PCPI) network was obtained, and five subnetworks representing different biological processes were observed. Interestingly, the small compounds currently used to treat gastric cancer converge on the same biological processes, potentially resulting in the development of chemoresistance. This analysis was followed by a network centrality study, which allows for selection of protein targets and/or small compounds, termed bottlenecks, that are defined as central nodes. The bottlenecks control the flow of biological information within the network, and their disruption can break the entire network into small components. From ten major bottlenecks observed within the network, seven bottlenecks represent new protein targets that are suitable for the development of new combinatory drug regimens for gastric cancer treatment.

Phenolic content and antioxidant activities of white and purple juices manufactured with organically- or conventionally-produced grapes.

Although the beneficial effects of moderate wine intake are well-known, data on antioxidant capacity of grape juices are scarce and controversial. The purpose of this study was to quantify total polyphenols, anthocyanins, resveratrol, catechin, epicatechin, procyanidins, and ascorbic acid contents in grape juices, and to assess their possible antioxidant activity. Eight Vitis labrusca juices--white or purple, from organically- or conventionally-grown grapes, and obtained in pilot or commercial scale--were used. Organic grape juices showed statistically different (p<0.05) higher values of total polyphenols and resveratrol as compared conventional grape juices. Purple juices presented higher total polyphenol content and in vitro antioxidant activity as compared to white juices, and this activity was positively correlated (r=0.680; p<0.01) with total polyphenol content. These results indicate that white and purple grape juices can be used as antioxidants and nutritional sources.

Network of healthy communities of Rio de Janeiro--Brazil

Poor communities in Rio de Janeiro, which are known as favelas, suffer from various problems related to poor housing, poverty, unemployment, violence and organized crime, and lack of access to basic services, such as health care and education. In order to tackle these determinants, and inspired by WHO's international Healthy Communities/Cities movement, the etwork of Healthy Communities of Rio de Janeiro was formed in 2004. The Network is coordinated by the Center for Health Promotion (CEDAPS) and now includes more than 100 community groups and organizations in the state of Rio de Janeiro. Their aim is to promote health, community development and equity through community empowerment, participation, capacity building and advocacy. The paper describes the work that has been done since the Network's inception and the challenges which they face to reach their goals in the context of a country like Brazil. The Network represents an important landmark of how poor populations can organize themselves in a collective, participatory and constructive way to influence public policy and strive for better conditions of life in disadvantaged settings, like the favelas. (AU)

Psoralen-sensitive mutant pso9-1 of Saccharomyces cerevisiae contains a mutant allele of the DNA damage checkpoint gene MEC3.

Complementation analysis of the pso9-1 yeast mutant strain sensitive to photoactivated mono- and bifunctional psoralens, UV-light 254 nm, and nitrosoguanidine, with pso1 to pso8 mutants, confirmed that it contains a novel pso mutation. Molecular cloning via the reverse genetics complementation approach using a yeast genomic library suggested pso9-1 to be a mutant allele of the DNA damage checkpoint control gene MEC3. Non-complementation of several sensitivity phenotypes in pso9-1/mec3Delta diploids confirmed allelism. The pso9-1 mutant allele contains a -1 frameshift mutation (deletion of one A) at nucleotide position 802 (802delA), resulting in nine different amino acid residues from that point and a premature termination. This mutation affected the binding properties of Pso9-1p, abolishing its interactions with both Rad17p and Ddc1p. Further interaction assays employing mec3 constructions lacking the last 25 and 75 amino acid carboxyl termini were also not able to maintain stable interactions. Moreover, the pso9-1 mutant strain could no longer sense DNA damage since it continued in the cell cycle after 8-MOP + UVA treatment. Taken together, these observations allowed us to propose a model for checkpoint activation generated by photo-induced adducts.

The eukaryotic Pso2/Snm1/Artemis proteins and their function as genomic and cellular caretakers.

DNA double-strand breaks (DSBs) represent a major threat to the genomic stability of eukaryotic cells. DNA repair mechanisms such as non-homologous end joining (NHEJ) are responsible for the maintenance of eukaryotic genomes. Dysfunction of one or more of the many protein complexes that function in NHEJ can lead to sensitivity to DNA damaging agents, apoptosis, genomic instability, and severe combined immunodeficiency. One protein, Pso2p, was shown to participate in the repair of DSBs induced by DNA inter-strand cross-linking (ICL) agents such as cisplatin, nitrogen mustard or photo-activated bi-functional psoralens. The molecular function of Pso2p in DNA repair is unknown, but yeast and mammalian cell line mutants for PSO2 show the same cellular responses as strains with defects in NHEJ, e.g., sensitivity to ICLs and apoptosis. The Pso2p human homologue Artemis participates in V(D)J recombination. Mutations in Artemis induce a variety of immunological deficiencies, a predisposition to lymphomas, and an increase in chromosomal aberrations. In order to better understand the role of Pso2p in the repair of DSBs generated as repair intermediates of ICLs, an in silico approach was used to characterize the catalytic domain of Pso2p, which led to identification of novel Pso2p homologues in other organisms. Moreover, we found the catalytic core of Pso2p fused to different domains. In plants, a specific ATP-dependent DNA ligase I contains the catalytic core of Pso2p, constituting a new DNA ligase family, which was named LIG6. The possible functions of Pso2p/Artemis/Lig6p in NHEJ and V(D)J recombination and in other cellular metabolic reactions are discussed.

The eukaryotic Pso2/Snm1/Artemis proteins and their function as genomic and cellular caretakers

DNA double-strand breaks (DSBs) represent a major threat to the genomic stability of eukaryotic cells. DNA repair mechanisms such as non-homologous end joining (NHEJ) are responsible for the maintenance of eukaryotic genomes. Dysfunction of one or more of the many protein complexes that function in NHEJ can lead to sensitivity to DNA damaging agents, apoptosis, genomic instability, and severe combined immunodeficiency. One protein, Pso2p, was shown to participate in the repair of DSBs induced by DNA inter-strand cross-linking (ICL) agents such as cisplatin, nitrogen mustard or photo-activated bi-functional psoralens. The molecular function of Pso2p in DNA repair is unknown, but yeast and mammalian cell line mutants for PSO2 show the same cellular responses as strains with defects in NHEJ, e.g., sensitivity to ICLs and apoptosis. The Pso2p human homologue Artemis participates in V(D)J recombination. Mutations in Artemis induce a variety of immunological deficiencies, a predisposition to lymphomas, and an increase in chromosomal aberrations. In order to better understand the role of Pso2p in the repair of DSBs generated as repair intermediates of ICLs, an in silico approach was used to characterize the catalytic domain of Pso2p, which led to identification of novel Pso2p homologues in other organisms. Moreover, we found the catalytic core of Pso2p fused to different domains. In plants, a specific ATP-dependent DNA ligase I contains the catalytic core of Pso2p, constituting a new DNA ligase family, which was named LIG6. The possible functions of Pso2p/Artemis/Lig6p in NHEJ and V(D)J recombination and in other cellular metabolic reactions are discussed.

Thermoconditional modulation of the pleiotropic sensitivity phenotype by the Saccharomyces cerevisiae PRP19 mutant allele pso4-1.

The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene's influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT-PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2Delta, rlf2Delta and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2Delta pso4-1 and rlf2Delta pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae.

Diauxic shift-induced stress resistance against hydroperoxides in Saccharomyces cerevisiae is not an adaptive stress response and does not depend on functional mitochondria.

Respiring Saccharomyces cerevisiae cells grown on a non-fermentable carbon source are intrinsically more resistant to several stresses, including oxidative stress. The mechanisms leading to increased stress resistance are not yet well understood. Active mitochondria are the major source of intracellular reactive oxygen species (ROS), which could cause the up-regulation of the antioxidant defense systems. We investigated the role of mitochondria in the intrinsic stress resistance against the hydroperoxides H2O2 and tert-butylhydroperoxide 4 h after a shift in carbon source. We found that, independently of functional mitochondria, the yeast acquired the intrinsic resistance of respiring cells against hydroperoxides solely as a response to a change of carbon source in the growth medium. Furthermore, utilizing reporter gene fusion constructs, we monitored the expression of the gamma-glutamylcysteinyl synthetase (encoded by GSH1) and the two superoxide dismutases (encoded by SOD1 and SOD2) during the metabolic transition from fermentation to respiration; and we detected an up-regulation of all three genes during the diauxic shift. Overall available data allowed us to propose that the antioxidant system of S. cerevisiae could be considered as a class of genes under glucose/carbon catabolite regulation. This control system is different from the well-known adaptive response to oxidative stress.