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.

Recombinant ß-1,3-1,4-glucanase from Theobroma cacao impairs Moniliophthora perniciosa mycelial growth.

In this work, we identified a gene from Theobroma cacao L. genome and cDNA libraries, named TcGlu2, that encodes a ß-1,3-1,4-glucanase. The TcGlu2 ORF was 720 bp in length and encoded a polypeptide of 239 amino acids with a molecular mass of 25.58 kDa. TcGlu2 contains a conserved domain characteristic of ß-1,3-1,4-glucanases and presented high protein identity with ß-1,3-1,4-glucanases from other plant species. Molecular modeling of TcGlu2 showed an active site of 13 amino acids typical of glucanase with ß-1,3 and 1,4 action mode. The recombinant cDNA TcGlu2 obtained by heterologous expression in Escherichia coli and whose sequence was confirmed by mass spectrometry, has a molecular mass of about 22 kDa (with His-Tag) and showed antifungal activity against the fungus Moniliophthora perniciosa, causal agent of the witches' broom disease in cacao. The integrity of the hyphae membranes of M. perniciosa, incubated with protein TcGlu2, was analyzed with propidium iodide. After 1 h of incubation, a strong fluorescence emitted by the hyphae indicating the hydrolysis of the membrane by TcGlu2, was observed. To our knowledge, this is the first study of a cacao ß-1,3-1,4-glucanase expression in heterologous system and the first analysis showing the antifungal activity of a ß-1,3-1,4-glucanase, in particular against M. perniciosa.

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.

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.

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.

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.

Role of PSO genes in repair of DNA damage of Saccharomyces cerevisiae.

Photoactivated psoralens used in treatment of skin diseases like Psoriasis and Vitiligo cause DNA damage, the repair of which may lead to mutations and thus to higher risk to have skin cancer. The simple eukaryote Saccharomyces cerevisiae was chosen to investigate the cells' genetic endowment with repair mechanisms for this type of DNA damage and to study the genetic consequences of such repair. Genetic studies on yeast mutants sensitive to photoactivated psoralens, named pso mutants, showed their allocation to 10 distinct loci. Cloning and molecular characterization allowed their grouping into three functional classes: (I) the largest group comprises seven PSO genes that are either generally or specifically involved in error-prone DNA repair and thus affect induced mutability and recombination; (II) one PSO gene that represents error-free excision repair, and (III) two PSO genes encoding proteins not influencing DNA repair but physiological processes unrelated to nucleic acid metabolism. Of the seven DNA repair genes involved in induced mutagenesis three PSO loci [PSO1/REV3, PSO8/RAD6, PSO9/MEC3] were allelic to already known repair genes, whereas three, PSO2/SNM1, PSO3/RNR4, and PSO4/PRP19 represent new genes involved in DNA repair and nucleic acid metabolism in S. cerevisiae. Gene PSO2 encodes a protein indispensable for repair of interstrand cross-link (ICL) that are produced in DNA by a variety of bi- and polyfunctional mutagens and that appears to be important for a likewise repair function in humans as well. In silico analysis predicts a putative endonucleolytic activity for Pso2p/Snm1p in removing hairpins generated as repair intermediates. The absence of induced mutation in pso3/rnr4 mutants indicates an important role of this subunit of ribonucleotide reductase (RNR) in regulation of translesion polymerase zeta in error-prone repair. Prp19p/Pso4p influences efficiency of DNA repair via splicing of pre-mRNAs of intron-containing repair genes but also may function in the stability of the nuclear scaffold that might influence DNA repair capacity. The seventh gene, PSO10 which controls an unknown step in induced mutagenesis is not yet cloned. Two genes, PSO6/ERG3 and PSO7/COX11, are responsible for structural elements of the membrane and for a functional respiratory chain (RC), respectively, and their function thus indirectly influences sensitivity to photoactivated psoralens.

Further phenotypic characterization of pso mutants of Saccharomyces cerevisiae with respect to DNA repair and response to oxidative stress.

The sensitivity responses of seven pso mutants of Saccharomyces cerevisiae towards the mutagens N-nitrosodiethylamine (NDEA), 1,2:7,8-diepoxyoctane (DEO), and 8-hydroxyquinoline (8HQ) further substantiated their allocation into two distinct groups: genes PSO1 (allelic to REV3), PSO2 (SNM1), PSO4 (PRP19), and PSO5 (RAD16) constitute one group in that they are involved in repair of damaged DNA or in RNA processing whereas genes PSO6 (ERG3) and PSO7 (COX11) are related to metabolic steps protecting from oxidative stress and thus form a second group, not responsible for DNA repair. PSO3 has not yet been molecularly characterized but its pleiotropic phenotype would allow its integration into either group. The first three PSO genes of the DNA repair group and PSO3, apart from being sensitive to photo-activated psoralens, have another common phenotype: they are also involved in error-prone DNA repair. While all mutants of the DNA repair group and pso3 were sensitive to DEO and NDEA the pso6 mutant revealed WT or near WT resistance to these mutagens. As expected, the repair-proficient pso7-1 and cox11-Delta mutant alleles conferred high sensitivity to NDEA, a chemical known to be metabolized via redox cycling that yields hydroxylamine radicals and reactive oxygen species. All pso mutants exhibited some sensitivity to 8HQ and again pso7-1 and cox11-Delta conferred the highest sensitivity to this drug. Double mutant snm1-Delta cox11-Delta exhibited additivity of 8HQ and NDEA sensitivities of the single mutants, indicating that two different repair/recovery systems are involved in survival. DEO sensitivity of the double mutant was equal or less than that of the single snm1-Delta mutant. In order to determine if there was oxidative damage to nucleotide bases by these drugs we employed an established bacterial test with and without metabolic activation. After S9-mix biotransformation, NDEA and to a lesser extent 8HQ, lead to significantly higher mutagenesis in an Escherichia coli tester strain WP2-IC203 as compared to WP2, whereas DEO-induced mutagenicity remained unchanged.