Sensitivity of Yeast Mutants Deficient in Mitochondrial or Vacuolar ABC Transporters to Pathogenesis-Related Protein TcPR-10 of Theobroma cacao.

Pathogenesis-related proteins (PRs) are induced in plants after infection by pathogens and/or abiotic stress. Among these proteins, the family 10 (PR-10) influences the biosynthesis of secondary metabolites and shows antimicrobial ribonuclease activity. TcPR-10p (Pathogenesis-related Protein 10 of Theobroma cacao) was isolated from resistant and susceptible Moniliophthora perniciosa cacao cultivars. Cell survival with Saccharomyces cerevisiae mutant lines deficient in ATP-binding cassette (ABC) transporter proteins indicated the influence on resistance to TcPR-10p. Proteins of the ABC transport type are considered important in the process of resistance to antimicrobials and toxins. Thus, the objective of this work was to observe the sensitivity of ABC transporter yeast mutants in the presence of the TcPR-10p. Chronic exposure of S. cerevisiae mitochondrial (BYatm1Δ and BYmdl1Δ) and vacuole (BYnft1Δ, BYvmr1Δ, BYybt1Δ, BYycf1Δ and BYbpt1Δ) ABC transporter mutants to TcPR-10p (3 μg/mL, 0, 6, 12 and 24 h) was performed. Two TcPR-10p sensitive strains (BYmdl1Δ and BYnft1Δ) were submitted to a fluorescence test with the fluorogenic dihydroethidium (DHE), to visualize the presence of oxidative stress in the cells. Oxidative stress-increased sensitivity was confirmed by flow cytometry indicating induced cell death either via apoptosis or necrosis. This yeast data combined with previous data of literature (of M. perniciosa sensitivity to TcPR-10p) show that increased sensitivity to TcPR-10p in these mutants could be due to the TcPR10p-generated higher levels of intracellular reactive oxygen species (ROS), leading to increased cell death either via necrosis or apoptosis.

Changes in cellular infrastructure after induced endoplasmic reticulum stress in Moniliophthora perniciosa.

Moniliophthora perniciosa is a basidiomycete fungus that causes witches' broom disease in Theobroma cacao We analyzed the morphology and survival of fungal hyphae and endoplasmic reticulum (ER) remodeling in either glucose- or glycerol-grown M. perniciosa after treatment with ER stress-inducing chemicals dithiothreitol (DTT) or tunicamycin (TM). Changes in intracellular redox potential can cause endoplasmic reticulum (ER) stress due to diminished efficiency in protein folding that could in turn reduce cell survival. Such stress diminishes protein-folding efficiency that could in turn reduce cell survival. Light microscopy revealed morphological changes in hyphae after TM but not after DTT treatment, regardless of the media carbon source. Decrease in fungal survival, after both TM and DTT treatments, was dose-dependent and glycerol-grown cells showed a higher resistance to both chemicals compared to glucose-grown cells. Electron microscopy showed TM and DDT-induced ER stress in M. perniciosa as evidenced by structural alterations of the organelle. The volume of ER structures increased as a typical consequence of unfolded protein stress, and the number of autophagosomes was higher. In glycerol-grown fungus DTT treatment slightly induced expression of molecular chaperone BiP. The TM exposure-induced expression of gene MpIRE1, involved in signaling of the unfolded protein response, was higher in glycerol than glucose-grown cells. Such difference was not observable with expression of gene MpATG8, encoding a key protein in autosome formation, that was induced 1.4-fold and 1.2-fold in glucose or glycerol-grown cells, respectively. DHE-based fluorescence assay showed M. perniciosa oxidative stress induced by H2O2, and treated cells had a higher level of oxidative stress compared to control. A comprehensive study of remodeling of ER is important in understanding M. perniciosa fungus resistance to oxidative stress and its ability to implement a successful infection in T. cacao.

DNA Protection against Oxidative Damage Using the Hydroalcoholic Extract of Garcinia mangostana and Alpha-Mangostin.

Garcinia mangostana, popularly known as "mangosteen fruit," originates from Southeast Asia and came to Brazil about 80 years ago where it mainly grows in the states of Pará and Bahia. Although mangosteen or its extracts have been used for ages in Asian folk medicine, data on its potential genotoxicity is missing. We, therefore, evaluated genotoxicity/mutagenicity of hydroethanolic mangosteen extract [HEGM, 10 to 640 µg/mL] in established test assays (Comet assay, micronucleus test, and Salmonella/microsome test). In the Comet assay, HEGM-exposed human leukocytes showed no DNA damage. No significant HEGM-induced mutation in TA98 and TA100 strains of Salmonella typhimurium (with or without metabolic activation) was observed and HEGM-exposed human lymphocytes had no increase of micronuclei. However, HEGM suggested exposure concentration-dependent antigenotoxic potential in leukocytes and antioxidant potential in the yeast Saccharomyces cerevisiae. HEGM preloading effectively protected against H2O2-induced DNA damage in leukocytes (Comet assay). Preloading of yeast with HEGM for up to 4 h significantly protected the cells from lethality of chronic H2O2-exposure, as expressed in better survival. Absence of genotoxicity and demonstration of an antigenotoxic and antioxidant potential suggest that HEGM or some substances contained in it may hold promise for pharmaceutical or nutraceutical application.

Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene.

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet-C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

Sak1 kinase interacts with Pso2 nuclease in response to DNA damage induced by interstrand crosslink-inducing agents in Saccharomyces cerevisiae.

By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal ß-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells.

Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation.

Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI < 3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications.

Astragalin from Cassia alata induces DNA adducts in vitro and repairable DNA damage in the yeast Saccharomyces cerevisiae.

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC(50) value of 2.27 µg mL(-1) and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-ß-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation.

Study of DNA-emodin interaction by FTIR and UV-vis spectroscopy.

Emodin, a plant- and fungus-derived anthraquinone, exerts genotoxic and antioxidative effects and shows promise in antitumor and antibacterial therapies. The aim of this study was to examine the molecular interactions of emodin with DNA in aqueous solution at physiological pH using spectroscopic methods. Fourier Transform Infrared (FTIR) Spectroscopy and UV absorption spectra were used to determine the structural features, the binding mode and the association constants. Our UV-spectroscopic results indicate that emodin interacts with DNA by intercalation and by external binding. FTIR results suggest that emodin interaction occurs preferably via adenine and thymine base pairs and also weakly with the phosphate backbone of the DNA double helix. The binding constant for emodin-DNA complex formation is estimated to be K=5.59×10(3)M(-1). No significant changes of DNA conformation were observed upon emodin-DNA complexation.

Enhanced resistance of yeast mutants deficient in low-affinity iron and zinc transporters to stannous-induced toxicity.

Tin or stannous (Sn(2+)) compounds are used as catalysts, stabilizers in plastic industries, wood preservatives, agricultural biocides and nuclear medicine. In order to verify the Sn(2+) up-take and toxicity in yeast cells we utilized a multi-elemental analysis known as particle-induced X-ray emission (PIXE) along with cell survival assays and quantitative real-time PCR. The detection of Sn(2+) by PIXE was possible only in yeast cells in stationary phase of growth (STAT cells) that survive at 25mM Sn(2+) concentration. Yeast cells in exponential phase of growth (LOG cells) tolerate only micro-molar Sn(2+) concentrations that result in intracellular concentration below of the method detection limit. Our PIXE analysis showed that STAT XV185-14c yeast cells demonstrate a significant loss of intracellular elements such as Mg, Zn, S, Fe and an increase in P levels after 1h exposure to SnCl(2). The survival assay showed enhanced tolerance of LOG yeast cells lacking the low-affinity iron and zinc transporters to stannous treatment, suggesting the possible involvement in Sn(2+) uptake. Moreover, our qRT-PCR data showed that Sn(2+) treatment could generate reactive oxygen species as it induces activation of many stress-response genes, including SOD1, YAP1, and APN1.

Early development of Moniliophthora perniciosa basidiomata and developmentally regulated genes.

BACKGROUND: The hemibiotrophic fungus Moniliophthora perniciosa is the causal agent of Witches' broom, a disease of Theobroma cacao. The pathogen life cycle ends with the production of basidiocarps in dead tissues of the infected host. This structure generates millions of basidiospores that reinfect young tissues of the same or other plants. A deeper understanding of the mechanisms underlying the sexual phase of this fungus may help develop chemical, biological or genetic strategies to control the disease. RESULTS: Mycelium was morphologically analyzed prior to emergence of basidiomata by stereomicroscopy, light microscopy and scanning electron microscopy. The morphological changes in the mycelium before fructification show a pattern similar to other members of the order Agaricales. Changes and appearance of hyphae forming a surface layer by fusion were correlated with primordia emergence. The stages of hyphal nodules, aggregation, initial primordium and differentiated primordium were detected. The morphological analysis also allowed conclusions on morphogenetic aspects. To analyze the genes involved in basidiomata development, the expression of some selected EST genes from a non-normalized cDNA library, representative of the fruiting stage of M. perniciosa, was evaluated. A macroarray analysis was performed with 192 selected clones and hybridized with two distinct RNA pools extracted from mycelium in different phases of basidiomata formation. This analysis showed two groups of up and down-regulated genes in primordial phases of mycelia. Hydrophobin coding, glucose transporter, Rho-GEF, Rheb, extensin precursor and cytochrome p450 monooxygenase genes were grouped among the up-regulated. In the down-regulated group relevant genes clustered coding calmodulin, lanosterol 14 alpha demethylase and PIM1. In addition, 12 genes with more detailed expression profiles were analyzed by RT-qPCR. One aegerolysin gene had a peak of expression in mycelium with primordia and a second in basidiomata, confirming their distinctiveness. The number of transcripts of the gene for plerototolysin B increased in reddish-pink mycelium and indicated an activation of the initial basidiomata production even at this culturing stage. Expression of the glucose transporter gene increased in mycelium after the stress, coinciding with a decrease of adenylate cyclase gene transcription. This indicated that nutrient uptake can be an important signal to trigger fruiting in this fungus. CONCLUSION: The identification of genes with increased expression in this phase of the life cycle of M. perniciosa opens up new possibilities of controlling fungus spread as well as of genetic studies of biological processes that lead to basidiomycete fruiting. This is the first comparative morphologic study of the early development both in vivo and in vitro of M. perniciosa basidiomata and the first description of genes expressed at this stage of the fungal life cycle.

High-affinity copper transport and Snq2 export permease of saccharomyces cerevisiae modulate cytotoxicity of PR-10 from Theobroma cacao.

A pathogenesis-related (PR) protein from Theobroma cacao (TcPR-10) was identified from a cacao-Moniliophthora perniciosa interaction cDNA library. Nucleotide and amino acid sequences showed homology with other PR-10 proteins having P loop motif and Betv1 domain. Recombinant TcPR-10 showed in vitro and in vivo ribonuclease activity, and antifungal activity against the basidiomycete cacao pathogen M. perniciosa and the yeast Saccharomyces cerevisiae. Fluorescein isothiocyanate-labeled TcPR-10 was internalized by M. perniciosa hyphae and S. cerevisiae cells and inhibited growth of both fungi. Energy and temperature-dependent internalization of the TcPR-10 suggested an active importation into the fungal cells. Chronical exposure to TcPR-10 of 29 yeast mutants with single gene defects in DNA repair, general membrane transport, metal transport, and antioxidant defenses was tested. Two yeast mutants were hyperresistant compared with their respective isogenic wild type: ctr3Delta mutant, lacking the high-affinity plasma membrane copper transporter and mac1Delta, the copper-sensing transcription factor involved in regulation of high-affinity copper transport. Acute exposure of exponentially growing yeast cells revealed that TcPR-10 resistance is also enhanced in the Snq2 export permease-lacking mutant which has reduced intracellular presence of TcPR-10.

Green tea extract-patents and diversity of uses.

Green tea is one of the most consumed beverages in the world. Presently, Camellia sinensis has become a source not only for the development of several food extracts but also nutraceutical, cosmetic and medicinal purposes. The technology developed to produce these extracts aims to improve the organoleptic characteristics of the products as taste and smell, and their shelf life. But it also searches to demonstrate some medicinal attributes like antioxidant, anti-aging, anti-tumor and anti-viral activities in relation to the chemical composition of the green tea catechins, especially (-)-epigallocatechin gallate (EGCG). The target of this review is to present the various patents related to the extraction methods and their claims, and to discuss the evidence found in the literature about the pharmacological activities of green tea. It summarizes the recent progress in technology to obtain the green tea extract and in clinical studies on its applications. Health-promoting products and disease-preventing applications of green tea extract or compounds isolated from it also take part of this text.

Allelism of Saccharomyces cerevisiae gene PSO10, involved in error-prone repair of psoralen-induced DNA damage, with SUMO ligase-encoding MMS21.

In order to extend the understanding of the genetical and biochemical basis of photo-activated psoralen-induced DNA repair in the yeast Saccharomyces cerevisiae we have identified and cloned 10 pso mutants. Here, we describe the phenotypic characterization and molecular cloning of the pso10-1 mutant which is highly sensitive to photoactivated psoralens, UV(254) (nm) radiation and the alkylating agent methylmethane sulphonate. The pso10-1 mutant allele also confers a block in the mutagenic response to photoactivated psoralens and UV(254) (nm) radiation, and homoallelic diploids do not sporulate. Molecular cloning using a yeast genomic library, sequence analysis and genetic complementation experiments proved pso10-1 to be a mutant allele of gene MMS21 that encodes a SUMO ligase involved in the sumoylation of several DNA repair proteins. The ORF of pso10-1 contains a single nucleotide C-->T transition at position 758, which leads to a change in amino acid sequence from serine to phenylalanine [S253F]. Pso10-1p defines a leaky mutant phenotype of the essential MMS21 gene, and as member of the Smc5-Smc6 complex, still has some essential functions that allow survival of the mutant. DNA repair via translesion synthesis is severely impaired as the pso10-1 mutant allele confers severely blocked induced forward and reverse mutagenesis and shows epistatic interaction with a rev3Delta mutant allele. By identifying the allelism of PSO10 and MMS21 we demonstrate the need of a fully functional Smc5-Smc6 complex for a WT-like adequate repair of photoactivated psoralen-induced DNA damage in yeast.

RNR4 mutant alleles pso3-1 and rnr4Delta block induced mutation in Saccharomyces cerevisiae.

The PSO3 gene of Saccharomyces cerevisiae was molecularly cloned by complementing the cold-sensitivity phenotype of a pso3-1 mutant and was found to be allelic to RNR4, encoding one of the two DNA damage-inducible small subunits of the ribonucleotide reductase (RNR) complex. Compared to a rnr4Delta mutant that allows only very little mutation induction at very low doses of 254(nm) ultraviolet light (UVC), the pso3-1 mutant allele confers leakiness in that it permits some DNA damage-induced mutagenesis at low doses of UVC. Similarly, the pso3 mutant is slightly less sensitive to UVC than an rnr4Delta mutant. Cloning and sequencing of the RNR4 locus of the pso3-1 mutant revealed that its intermediate phenotype is attributable to a G --> A transition at nucleotide 352, leading to replacement of glycine by arginine [G118R] in the mutant's protein. Both RNR4 mutant alleles confer significantly less sensitivity to UVC than mutant alleles of non-UVC-mutable REV3, indicating that, apart from nucleotide excision repair, RAD6-dependent error-free DNA repair may still be functional. The phenotype of a strongly reduced UVC-induced mutagenesis for rnr4 mutant alleles has not yet been described; it suggests the importance of this gene for a fully functional RNR providing correct amounts of DNA precursor molecules, thereby, allowing translesion synthesis (error-prone) of UVC-damaged DNA. Stationary phase cells of the rnr4Delta mutant, but not of the original pso3-1 mutant, are swollen with a fourfold to eightfold increase in volume. The central role of RNR in DNA precursor metabolism and its complex regulation allow for several modes of suppression that may influence the phenotypes of RNR4 mutants, especially those containing the leaky pso3-1 mutant allele.

Broken hyphae of the basidiomycete Crinipellis perniciosa allow quantitative assay of toxicity.

Breaking of hyphae derived from growth of the phytopathogenic fungus Crinipellis perniciosa in liquid media yielded cell aggregates that performed as "quasi single cell" in toxicity assays. When treated with the chemical mutagens 4-nitroquinoleine-1-oxide (4NQO), hydrogen peroxide (H2O2), or paraquat (PAQ) as well as with ultraviolet light (UVC), broken hyphae of C. perniciosa gave a single cell-like response, i.e., survival curves similar to those obtained when treating single-cell suspensions. C. perniciosa had significantly higher UVC resistance than haploid bakers yeast but was more sensitive to 4NQO and extremely sensitive to PAQ and H2O2 when compared to likewise-treated yeast. Haploid C. perniciosa basidiospores (monokaryotic) were significantly more UVC resistant than C. perniciosa broken hyphae and than haploid and diploid yeast wild-type strains. This suggests a high capacity in C. perniciosa for repair of UVC-induced DNA lesions or, alternatively, an efficient protection from UVC irradiation, especially in basidiospores. The pronounced sensitivity of the dikaryotic form of C. perniciosa to PAQ and H2O2 points to a weak protection from oxidative stress-inducing agents.

In silico identification and analysis of new Artemis/Artemis-like sequences from fungal and metazoan species.

The Artemis Group comprises mammalian proteins with important functions in the repair of ionizing radiation-induced DNA double-strand breaks and in the cleavage of DNA hairpin extremities generated during V(D)J recombination. Little is known about the presence of Artemis/Artemis-like proteins in non-mammalian species. We have characterized new Artemis/Artemis-like sequences from the genomes of some fungi and from non-mammalian metazoan species. An in-depth phylogenetic analysis of these new Artemis/Artemis-like sequences showed that they form a distinct clade within the Pso2p/Snm1p A and B Groups. Hydrophobic cluster analysis and three-dimensional modeling allowed to map and to compare conserved regions in these Artemis/Artemis-like proteins. The results indicate that Artemis probably belongs to an ancient DNA recombination mechanism that diversified with the evolution of multi-cellular eukaryotic lineage.

The eukaryotic Pso2p/Snm1p family revisited: in silico analyses of Pso2p A, B and Plasmodium groups.

The eukaryotic family of Pso2/Snm1 exo/endonuclease proteins has important functions in repair of DNA damages induced by chemical interstrand cross-linking agents and ionizing radiation. These exo/endonucleases are also necessary for V(D)J recombination and genomic caretaking. However, despite the growing biochemical data about this family, little is known about the number of orthologous/paralogous Pso2p/Snm1p sequences in eukaryotes and how they are phylogenetically organized. In this work we have characterized new Pso2p/Snm1p sequences from the finished and unfinished eukaryotic genomes and performed an in-depth phylogenetic analysis. The results indicate that four phylogenetically related groups compose the Pso2p/Snm1p family: (i) the Artemis/Artemis-like group, (ii) the Pso2p A group, (iii) the Pso2p B group and (iv) the Pso2p Plasmodium group. Using the available biochemical and genomic information about Pso2p/Snm1p family, we concentrate our research in the study of Pso2p A, B and Plasmodium groups. The phylogenetic results showed that A and B groups can be organized in specific subgroups with different functions in DNA metabolism. Moreover, we subjected selected Pso2p A, B and Plasmodium proteins to hydrophobic cluster analysis (HCA) in order to map and to compare conserved regions within these sequences. Four conserved regions could be detected by HCA, which are distributed along the metallo-beta-lactamase and beta-CASP motifs. Interestingly, both Pso2p A and B proteins are structurally similar, while Pso2p Plasmodium proteins have a unique domain organization. The possible functions of A, B and Plasmodium groups are discussed.

Pro-oxidant action of diphenyl diselenide in the yeast Saccharomyces cerevisiae exposed to ROS-generating conditions.

Organoselenium compounds have a potential thiol peroxidase-like activity. Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. Using TRAP assay of chemiluminescense we have shown that diphenyl diselenide clearly possesses a pro-oxidant property. For an investigation on the mechanisms of this property, we used mutant strains of Saccharomyces cerevisiae defective in antioxidant defenses, i.e. in superoxide dismutase, in biosynthesis of glutathione, and the transcription factor yAP-1-lacking yap 1 mutant that cannot activate genes of the oxidative stress response. Exposure of growing cultures to the drug increased cell sensitivity to oxidizing agents. The pro-oxidant effect was independent of the metabolic condition or of the oxidative mutagen tested. N-acetylcysteine, a precursor of glutathione biosynthesis, could neutralize the pro-oxidant effects of diphenyl diselenide by stimulating an increase of endogenous glutathione biosynthesis or by directly binding to the drug. Vitamin E (Trolox), a known antioxidant, was also able to protect S. cerevisiae against the pro-oxidant effect of diphenyl diselenide. In vitro assays showed that diphenyl diselenide interacts non-enzymatically with the thiol group of glutathione.

The function of Alr1p of Saccharomyces cerevisiae in cadmium detoxification: insights from phylogenetic studies and particle-induced X-ray emission.

Two genes in Saccharomyces cerevisiae, ALR1 and ALR2, encode transmembrane proteins involved in Mg2+ uptake. The present study investigates the phylogenetic relationship of Alr1p/Alr2p with bacterial CorA proteins and some proteins related to Mg2+ influx/efflux transport in mitochondrial and bacterial zinc transporters; including hydrophobic cluster analysis (HCA). The phylogenetic results indicate that the Alrp sequences of S. cerevisiae share a common carboxy-terminus with proteins related to zinc efflux transport. We also analyse the intracellular metal content by particle-induced X-ray emission (PIXE) after cell exposure to cadmium. The PIXE analysis of cadmium-exposed ALR mutants and wild-type yeast cells suggests that Alrp has a central role in cell survival in a cadmium-rich environment.

A new group of plant-specific ATP-dependent DNA ligases identified by protein phylogeny, hydrophobic cluster analysis and 3-dimensional modelling.

The eukaryotic ATP-dependent DNA ligases comprise a group of orthologous proteins that have distinct roles in DNA metabolism. In contrast with the well-known DNA ligases of animal cells, the DNA ligases of plant cells are poorly described. Until now, only two DNA ligases (I and IV) genes of Arabidopsis thaliana (L.) Heynh were isolated and characterised. Use of the complete genomic sequences of Oryza sativa L. and A. thaliana, as well as the partially assembled genomic data of Medicago truncatula L. and Brassica spp., allowed us to identify a new family of ATP-dependent DNA ligases that are found only in the Viridiplantae kingdom. An in-depth phylogenetic analysis of protein sequences showed that this family composes a distinct clade, which shares a last universal common ancestor with DNA ligases I. In silico sequence studies indicate that these proteins have distinct physico-chemical properties when compared with those of animal and fungal DNA ligases. Moreover, hydrophobic cluster analysis and 3-dimensional modelling allowed us to map two conserved domains within these DNA ligases I-like proteins. Additional data of microsynteny analysis indicate that these DNA ligases I-like genes are linked to the S and SLL2 loci of Brassica spp. and A. thaliana, respectively. Combining the results of all analyses, we propose the creation of the DNA ligases VI (LIG6) family, which is composed by plant-specific DNA ligases.