RESUMEN
Natural compounds originated from plants and microorganisms and their combinations are currently being investigated as a possible treatment for several diseases including cancer. Hypericin (photodynamically-active pigment from Hypericum perforatum L.) and manumycin A (inhibitor of farnesyltransferase from Streptomyces parvulus) belong to the chemicals potentially applicable in clinical practice. In this study we evaluated potential cytotoxic (via trypan blue exclusion test), genotoxic (via DNA-topology and comet assays), and mutagenic effects (via bacterial reverse mutation test) of these compounds and their combinations considering the molecular mechanism of their action in cell-free and cellular systems. Our results did not reveal neither cytotoxic nor mutagenic activities of tested compounds and their combinations. Regarding the genotoxic potential, no damage of plasmid DNA in cell-free system was detected. On the other hand, photoactivated hypericin and manumycin A were able to induce primary DNA damage in human lymphocytes analyzed by comet assay. The possible antagonistic interactions between these two metabolites were estimated using SynergyFinder software analysis and experimental data obtained from comet assay. Our findings indicate that not only the presence of substances, but also their ratio plays an important role in resulting effects of the combined treatment in cellular system.
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Antineoplásicos , Mutágenos , Humanos , Mutágenos/toxicidad , Pruebas de Mutagenicidad , Daño del ADN , Ensayo CometaRESUMEN
Secondary metabolites as a potential source of anticancer therapeutics have been the subject of many studies. Since hypericin, a metabolite isolated from Hypericum perforatum L., shows several biomedical properties applicable in oncology, the aim of our study was to investigate its potential precursor skyrin in terms of genotoxic and DNA-protective effects. These skyrin effects were analyzed by cell-free methods, and cytotoxicity was estimated by an MTT assay and by a trypan blue exclusion test, while the genotoxic/antigenotoxic potential was examined by comet assay using non-cancerous human lymphocytes and the HepG2 cancer cell line. Skyrin did not show DNA-damaging effects but rather exhibited DNA-protectivity using a DNA-topology assay. However, we observed only weak antioxidant and chelating skyrin properties in other cell-free methods. Regarding the cytotoxic activity of skyrin, HepG2 cells were more prone to skyrin-induced death in comparison to human lymphocytes. Skyrin in non-cytotoxic concentrations did not exhibit elevated genotoxicity in both cell types. On the other hand, skyrin displayed moderate DNA-protective effects that were more noticeable in the case of non-cancerous human lymphocytes. The potential genotoxic effects of skyrin were not observed, and its DNA-protective capacity was more prominent in non-cancerous cells. Therefore, skyrin might be a promising agent used in anticancer therapy.
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Antineoplásicos , Daño del ADN , Antraquinonas , ADN , HumanosRESUMEN
Bisphenol A (BPA), as a major component of some plastic products, is abundant environmental pollutant. Due to its ability to bind to several types of estrogen receptors, it can trigger multiple cellular responses, which can contribute to various manifestations at the organism level. The most studied effect of BPA is endocrine disruption, but recently its prooxidative potential has been confirmed. BPA ability to induce oxidative stress through increased ROS production, altered activity of antioxidant enzymes, or accumulation of oxidation products of biomacromolecules is observed in a wide range of organisms - estrogen receptor-positive and -negative. Subsequently, increased intracellular oxidation can lead to DNA damage induction, represented by oxidative damage, single- and double-strand DNA breaks. Importantly, BPA shows several mechanisms of action and can trigger adverse effects on all organisms inhabiting a wide variety of ecosystem types. Therefore, the main aim of this review is to summarize the genotoxic effects of BPA on organisms across all taxa.
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Disruptores Endocrinos , Compuestos de Bencidrilo/toxicidad , Daño del ADN , Ecosistema , Disruptores Endocrinos/toxicidad , Estrés Oxidativo , FenolesRESUMEN
CRISPR-Cas9 is a genome-editing technique that has been widely adopted thanks to its simplicity, efficiency, and broad application potential. Due to its advantages and pervasive use, there have been attempts to include this method in the existing curricula for students majoring in various disciplines of biology. In this perspective, we summarize the existing CRISPR-Cas courses that harness a well-established model organism: baker's yeast, Saccharomyces cerevisiae. As an example, we present a detailed description of a fully hands-on, flexible, robust, and cost-efficient practical CRISPR-Cas9 course, where students participate in yeast genome editing at every stage-from the bioinformatic design of single-guide RNA, through molecular cloning and yeast transformation, to the final confirmation of the introduced mutation. Finally, we emphasize that in addition to providing experimental skills and theoretical knowledge, the practical courses on CRISPR-Cas represent ideal platforms for discussing the ethical implications of the democratization of biology.
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Sistemas CRISPR-Cas , Saccharomyces cerevisiae , Biología , Sistemas CRISPR-Cas/genética , Curriculum , Edición Génica/métodos , Humanos , Saccharomyces cerevisiae/genéticaRESUMEN
Tyrosol (T) and hydroxytyrosol (HOT) and their glycosides are promising candidates for applications in functional food products or in complementary therapy. A series of phenylethanoid glycofuranosides (PEGFs) were synthesized to compare some of their biochemical and biological activities with T and HOT. The optimization of glycosylation promoted by environmentally benign basic zinc carbonate was performed to prepare HOT α-L-arabino-, ß-D-apio-, and ß-D-ribofuranosides. T and HOT ß-D-fructofuranosides, prepared by enzymatic transfructosylation of T and HOT, were also included in the comparative study. The antioxidant capacity and DNA-protective potential of T, HOT, and PEGFs on plasmid DNA were determined using cell-free assays. The DNA-damaging potential of the studied compounds for human hepatoma HepG2 cells and their DNA-protective potential on HepG2 cells against hydrogen peroxide were evaluated using the comet assay. Experiments revealed a spectrum of different activities of the studied compounds. HOT and HOT ß-D-fructofuranoside appear to be the best-performing scavengers and protectants of plasmid DNA and HepG2 cells. T and T ß-D-fructofuranoside display almost zero or low scavenging/antioxidant activity and protective effects on plasmid DNA or HepG2 cells. The results imply that especially HOT ß-D-fructofuranoside and ß-D-apiofuranoside could be considered as prospective molecules for the subsequent design of supplements with potential in food and health protection.
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Depuradores de Radicales Libres , Alcohol Feniletílico/análogos & derivados , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología , Células Hep G2 , Humanos , Alcohol Feniletílico/química , Alcohol Feniletílico/farmacologíaRESUMEN
Bisphenol A (BPA) is a major component of the most commonly used plastic products, such as disposable plastics, Tetra Paks, cans, sport protective equipment, or medical devices. Due to the accumulation of excessive amounts of plastic waste and the subsequent release of BPA into the environment, BPA is classified as a pollutant that is undesirable in the environment. To date, the most interesting finding is the ability of BPA to act as an endocrine disrupting compound due to its binding to estrogen receptors (ERs), and adverse physiological effects on living organisms may result from this action. Since evidence of the potential pro-oxidizing effects of BPA has accumulated over the last years, herein, we focus on the detection of oxidative stress and its origin following BPA exposure using pulsed-field gel electrophoresis, flow cytometry, fluorescent microscopy, and Western blot analysis. Saccharomyces cerevisiae cells served as a model system, as these cells lack ERs allowing us to dissect the ER-dependent and -independent effects of BPA. Our data show that high concentrations of BPA affect cell survival and cause increased intracellular oxidation in yeast, which is primarily generated in the mitochondrion. However, an acute BPA exposure does not lead to significant oxidative damage to DNA or proteins.
RESUMEN
The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We concluded that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined.
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Daño del ADN , Reparación del ADN , Recombinación Homóloga , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Meiosis , Fosforilación , Unión Proteica , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Climate change, environmental pollution and pathogen resistance to available chemical agents are part of the problems that the food industry has to face in order to ensure healthy food for people and livestock. One of the promising solutions to these problems is the use of cold atmospheric pressure plasma (CAPP). Plasma is suitable for efficient surface decontamination of seeds and food products, germination enhancement and obtaining higher yields in agricultural production. However, the plasma effects vary due to plasma source, treatment conditions and seed type. In our study, we tried to find the proper conditions for treatment of barley grains by diffuse coplanar surface barrier discharge, in which positive effects of CAPP, such as enhanced germination or decontamination effects, would be maximized and harmful effects, such as oxidation and genotoxic potential, minimized. Besides germination parameters, we evaluated DNA damage and activities of various germination and antioxidant enzymes in barley seedlings. Plasma exposure resulted in changes in germination parameters and enzyme activities. Longer exposures had also genotoxic effects. As such, our findings indicate that appropriate plasma exposure conditions need to be carefully optimized in order to preserve germination, oxidation balance and genome stability, should CAPP be used in agricultural practice.
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Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Germinación/efectos de los fármacos , Hordeum/efectos de los fármacos , Gases em Plasma/farmacología , Plantones/efectos de los fármacos , Semillas/efectos de los fármacos , Daño del ADN , ADN de Plantas/genética , ADN de Plantas/metabolismo , Hordeum/enzimología , Hordeum/genética , Hordeum/crecimiento & desarrollo , Oxidación-Reducción , Estrés Oxidativo , Peroxidasa/genética , Peroxidasa/metabolismo , Raíces de Plantas , Brotes de la Planta , Plantones/enzimología , Plantones/genética , Plantones/crecimiento & desarrollo , Semillas/enzimología , Semillas/genética , Semillas/crecimiento & desarrollo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Since the earliest agricultural attempts, humankind has been trying to improve crop quality and yields, as well as protect them from adverse conditions. Strategies to meet these goals include breeding, the use of fertilisers, and the genetic manipulation of crops, but also an interesting phenomenon called priming or adaptive response. Priming is based on an application of mild stress to prime a plant for another, mostly stronger stress. There are many priming techniques, such as osmopriming, halopriming, or using physical agents. Non-thermal plasma (NTP) represents a physical agent that contains a mixture of charged, neutral, and radical (mostly reactive oxygen and nitrogen species) particles, and can cause oxidative stress or even the death of cells or organisms upon interaction. However, under certain conditions, NTP can have the opposite effect, which has been previously documented for many plant species. Seed surface sterilization and growth enhancement are the most-reported positive effects of NTP on plants. Moreover, some studies suggest the role of NTP as a promising priming agent. This review deals with the effects of NTP treatment on plants from interaction with seed and cell surface, influence on cellular molecular processes, up to the adaptive response caused by NTP.
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Adaptación Biológica/efectos de los fármacos , Productos Agrícolas/efectos de los fármacos , Germinación/efectos de los fármacos , Gases em Plasma/farmacología , Estrés Fisiológico/efectos de los fármacos , Adaptación Biológica/genética , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Productos Agrícolas/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Germinación/genética , Estrés Oxidativo , Gases em Plasma/efectos adversos , Gases em Plasma/química , Especies de Nitrógeno Reactivo/metabolismo , Especies de Nitrógeno Reactivo/farmacología , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Semillas , Estrés Fisiológico/genéticaRESUMEN
OBJECTIVE: Period of elderly age preordains the occurrence of many health issues. People deported during World War II to concentration camps suffered from malnutrition, lack of sleep, physical and mental exhaustion. Recently, the health condition of Holocaust survivors is often complicated as a result of physical punishments and different torture methods as well as mental hardships which they had suffered during deportation. The consequences often have psychosomatic nature thus the survivors are often receivers of health care. The topic of bibliography review is based on the need to objectivize and systematically evaluate subjective health issues of Holocaust survivors in connection with trauma related to the stay in a concentration camp. The aim is to offer a review of effects of the Holocaust on health of different body systems for survivors in concentration camps and Jewish ghettos in the course of World War II. METHODS: To map the subjective problems of Holocaust survivors, evidence-based medicine (EBM) method has been used with the help of scientific database PubMed, CINAHL Plus with full text, ProQuest and other sources with specific key words and Boole operators. Prognostic type of clinical/review questions has been selected for the bibliography review, which is trying to predict the probability of relation or output of illness/condition and based on diseases or symptoms seriousness to find out expectancy for treatment/improvement of care. RESULTS: 175 studies have been found in basic search with the use of key words both in English and in Czech language. The search has not been time-limited. The advanced search has focused on different body systems and health damage due to Nazi experiments. Fourteen studies have been used to complete the study. The research results have confirmed the significant effect of Holocaust trauma on body condition of the survivors. The reasons of this condition were insufficient nutrition, unsuitable and harmful hygienic, living and working conditions and brutality of the guards. According to the research, these factors have impacted all organ systems, mainly locomotion and cardiovascular ones. The results have shown a more frequent occurrence of osteoporosis, fractures of long bones and corresponding chronical pain of people of Jewish origin who had gone through different forms of torture during World War II. Other present symptoms include gastrointestinal problems, tumors mainly in the area of colorectum and lungs. Moreover, the stay in concentration camps had influence on women's menstrual cycle. CONCLUSION: The studies of Holocaust effects are an example of the influence of an extreme mental and physical burden on the body condition of the survivors' health. The results of the studies have shown a wide range of the effects also in mental and social areas.
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Estado de Salud , Holocausto , Sobrevivientes/estadística & datos numéricos , Bibliografías como Asunto , Humanos , JudíosRESUMEN
Meiosis is the process by which haploid gametes are produced from diploid precursor cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to characterize the meiotic proteome in the fission yeast Schizosaccharomyces pombe. We compared relative levels of proteins extracted from cells harvested around meiosis I with those of meiosis II, and proteins from premeiotic S phase with the interval between meiotic divisions, when S phase is absent. Our proteome datasets revealed peptides corresponding to short open reading frames (sORFs) that have been previously identified by ribosome profiling as new translated regions. We verified expression of selected sORFs by Western blotting and analyzed the phenotype of deletion mutants. Our data provide a resource for studying meiosis that may help understand differences between meiosis I and meiosis II and how S phase is suppressed between the two meiotic divisions.
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Meiosis , Sistemas de Lectura Abierta/genética , Proteómica , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Eliminación de Gen , Marcaje Isotópico , Meiosis/genética , Fenotipo , Proteoma/metabolismo , Reproducibilidad de los Resultados , Ribosomas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
The main objective of this study is to describe the most common childhood diseases occurring in the Theresienstadt ghetto during the Second World War as well as applied anti-epidemic measures. A partial objective is to describe medical and nursing care of sick child prisoners. The data was obtained by the method of synthesis of primary and secondary data with the highest importance after adequate external source criticism using selected monographs, memoirs, survivor diaries, Orders of the day by the Council of the Elders and Reports of the Jewish self-government of the Theresienstadt ghetto from 1941 to 1945, the Archives of the Jewish Museum in Prague, the Ghetto Museum, the Museum of the History of Polish Jews in Warsaw, Post Bellum online electronic collection of oral historical interviews, and witness accounts. The validity of the presented conclusions is ensured by comparing data from several sources. The most common infections in Theresienstadt children were enteritis, scarlet fever, infectious jaundice, measles, mumps, rubella, varicella, black cough, pneumonia, otitis media, and typhoid fever. Most of these infections had entirely atypical symptomatology or complications. Children were hospitalized in children's hospitals, in children's rooms of hospitals for adults and infirmaries in children's homes. Albeit diagnostic methods had a high standard, options of treatment were very limited. The most common treatments included bed rest, diet and cold compress. Occasionally, chemotherapeutic agents (e.g. a sulphonamide drug Prontosil) and Aspirin were available. The anti-epidemic measures in the ghetto focused on hygiene, enhancing children's immunity, vaccination and pest control.
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Enfermedades Transmisibles/epidemiología , Pediatría/métodos , Áreas de Pobreza , Adulto , Anciano , Niño , Control de Enfermedades Transmisibles , Humanos , Judíos , Polonia/epidemiologíaRESUMEN
New gallotanins, methyl 2,3,4,6-tetra-O-galloyl-α-D-glucoside (G4Glc), methyl 2,3,4,6-tetra-O-galloyl-α-D-mannoside (G4Man), and methyl 2,3,4-tri-O-galloyl-α-L-rhamnoside (G3Rham), have been synthesized in order to study the protective effects of synthetic polyphenols that are structurally related with natural compounds. Apart from spectral analysis, examination of antioxidant ability and protective efficiency showed the differences among newly prepared compounds and commercial antioxidants - gallic acid (GA), methyl gallate (MG), and octyl gallate (OG) applying radical scavenging 1,1-diphenyl-2-picryl-hydrazyl (DPPH), reducing power and iron-chelating assays. The genotoxicity and DNA-protective potential of tested compounds on human peripheral blood mononuclear cells (PBMCs) were evaluated using the single-cell gel electrophoresis (comet assay) and DNA-topology assay. Experimental data revealed that gallotannins G3Rham, G4Man, and G4Glc possess significant radical scavenging/antioxidant activities and manifest very low genotoxic effect on human PBMCs. Moreover, tested compounds considerably reduce the level of DNA damage induced by hydrogen peroxide or Fe2+-ions. The results imply that new synthetic gallotannins can be considered as nontoxic agents for subsequent design of new antioxidants with potential biomedical applications.
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Antioxidantes/farmacología , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Antioxidantes/química , Compuestos de Bifenilo/química , Células Cultivadas , Ensayo Cometa , ADN/metabolismo , Daño del ADN , Ácido Gálico/química , Humanos , Hierro/química , Leucocitos Mononucleares , Picratos/químicaRESUMEN
Bisphenol A (BPA) is used for the production of plastics and epoxy resins, which are part of packaging materials for food and beverages, and can migrate into food and the environment, thus exposing human beings to its effects. Exposure to BPA has been associated with oxidative stress, cell cycle changes, and genotoxicity, and is mediated by its known endocrine-disrupting activity. Possible BPA cytotoxicity without mediation by estrogen receptors has been reported in the literature. Here, we show the toxic effects of BPA by live-cell imaging on the fission yeast Schizosaccharomyces pombe, an experimental model lacking estrogen receptors, which were in line with data from flow cytometry on intracellular oxidation (76.4 ± 14.4 and 19.4 ± 16.1% of fluorescent cells for BPA treatment and control, respectively; p < 0.05) as well as delay in cell cycle progression (after 90 min of experiment, 48.4 ± 4.30 and 64.6 ± 5.46% of cells with a 4C DNA content for BPA treatment and control, respectively; p < 0.05) upon exposure to BPA. These results strongly support the possibilities that BPA-induced cell cycle changes can be independent of estrogen receptors and that live-cell imaging is a powerful tool for genotoxic analysis.
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Compuestos de Bencidrilo/toxicidad , Ciclo Celular/efectos de los fármacos , Fenoles/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Schizosaccharomyces/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
The study focused on protective potential of phytochemicals applicable in prevention and health protection is of great importance. Various structures of these compounds and a wide range of their biological activities have inspired organic chemists to sythesize their effective analogues in order to further increase their efficacy. The aims of our study were (i) to synthesize phenylethanoid glycopyranosides: salidroside (SALI - tyrosol ß-d-glucopyranoside), tyrosol ß-d-galactopyranoside (TYBGAL), tyrosol α-d-galactopyranoside (TYAGAL), tyrosol α-d-mannopyranoside (TYAMAN), hydroxytyrosol α-d-mannopyranoside (HOTAMA), homosyringyl ß-d-glucopyranoside (HSYGLU), hydroxytyrosol ß-d-xylopyranoside (HOTXYL) and hydroxysalidroside (HOSALI); (ii) to determine their antioxidant capacities (cell-free approaches); (iii) to evaluate their cytotoxicity (MTT test), protectivity against hydrogen peroxide (H2O2; comet assay) and effect on the intracellular glutathione level (iGSH; flow cytometry) in experimental system utilizing human hepatoma HepG2 cells. HOSALI, HOTAMA, HOTXYL and HSYGLU manifested the highest antioxidant capacity in cell-free assays and they were most active in protection of HepG2 cells against H2O2. On the other hand, pre-treatment of HepG2 cells with SALI had protective effects even though SALI displayed almost no activity in cell-free assays. Differences in the efficacy of the analogues revealed that structures of their molecules in terms of aglycone combined with sugar moiety affect their activities.
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Antioxidantes/química , Antioxidantes/farmacología , Glicósidos/química , Glicósidos/farmacología , Monosacáridos/química , Monosacáridos/farmacología , Bioensayo , Compuestos de Bifenilo/química , ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Células Hep G2 , Humanos , Peróxido de Hidrógeno/farmacología , Picratos/química , Plásmidos , Relación Estructura-ActividadRESUMEN
OBJECTIVES: Bisphenol A (BPA) is an endocrine disruptor which has been shown to be a harmful compound for living organisms. It is the main component of the most commonly used plastic products such as plastic bottles, food cans and containers or dental fillings, and other medical aids. Recently, it has become a new environmental pollutant. The current knowledge about the BPA effects (including genotoxic one) on different cells is in many cases contradictory. Thus, the aim of the paper is to study the potential genotoxic effect of BPA. METHODS: An observation of the genotoxic activity of BPA on human lymphocytes was evaluated by using the alkaline comet assay and a modified comet assay with bacterial DNA repair enzyme Fpg. The potential DNA-protective effect of BPA was tested by using the DNA-topology assay. RESULTS: The results show that rising concentrations of BPA increase the risk of DNA double-strand breaks and modified purines in human lymphocytes. Interestingly, BPA shows an ability to protect plasmid DNA from the damage of iron ions in cell-free system. CONCLUSIONS: BPA itself does not induce genotoxic effect to DNA. However, BPA treatment of human lymphocytes leads to the induction of DNA damage. The proposed mechanism of BPA action in the human lymphocytes could be mediated by cell metabolism that induces an oxidative stress and ROS formation. ROS subsequently attack DNA and thus induce DNA damage. According to our results, BPA can be included in the group of substances with dual effects involving genotoxic and DNA-protective activity.
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Compuestos de Bencidrilo/toxicidad , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN/efectos de los fármacos , Contaminantes Ambientales/toxicidad , Linfocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fenoles/toxicidad , Plásmidos/efectos de los fármacos , Ensayo Cometa , ADN/genética , Daño del ADN/genética , Disruptores Endocrinos/toxicidad , Estrógenos no Esteroides/toxicidad , Depuradores de Radicales Libres/toxicidad , Humanos , Linfocitos/metabolismo , Plásmidos/genética , Purinas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The canonical role of cohesin is to mediate sister chromatid cohesion. In addition, cohesin plays important roles in processes such as DNA repair and regulation of gene expression. Mounting evidence suggests that various post-translational modifications, including phosphorylation, acetylation and sumoylation regulate cohesin functions. Our mass spectrometry analysis of cohesin purified from Schizosaccharomyces pombe cells revealed that the cohesin subunit Psm1 is methylated on two evolutionarily conserved lysine residues, K536 and K1200. We found that mutations that prevent methylation of Psm1 K536 and K1200 render sensitivity to DNA-damaging agents and show positive genetic interactions with mutations in genes encoding the Mus81-Eme1 endonuclease. Yeast two-hybrid and co-immunoprecipitation assays showed that there were interactions between subunits of the cohesin and Mus81-Eme1 complexes. We conclude that cohesin is methylated and that mutations that prevent methylation of Psm1 K536 and K1200 show synthetic phenotypes with mutants defective in the homologous recombination DNA repair pathway.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Metilación , Mutación , Unión Proteica , Schizosaccharomyces/química , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , CohesinasRESUMEN
Natural products represent the source or the inspiration for the majority of the active ingredients of medicines because of their structural diversity and a wide range of biological effects. Our aims in this study were (i) to synthesize enzymatically salidroside (SAL), the most effective phenylethanoid glycoside in Rhodiola species; (ii) to examine its antioxidant capacity using cell-free assays (reducing power, DPPH radicals scavenging and Fe2+ -chelating assays); (iii) to assess its DNA-protective potential on plasmid DNA (DNA topology assay) and in HepG2 cells (comet assay) damaged by Fe2+ ions and hydrogen peroxide, respectively; and (iv) to investigate the effects of SAL, cisplatin (CDDP) and combined treatments of SAL + CDDP on cell viability (MTT test), level of DNA damage (comet assay), proliferation, cell cycle (flow cytometry) and the expression of signalling molecules associated with cell growth and apoptotic pathways (Western immunoblotting). We found out that SAL manifested low antioxidant and DNA-protective capacity in all assays used. In both parental A2780 and CDDP-resistant A2780/CP human ovarian carcinoma cells, SAL itself exerted in fact no impact on the viability, while in combination with CDDP it showed antagonistic effect supporting the chemopreventive activity on the CDDP-induced cell damage. These results were confirmed by the partial reversal of the cell cycle alterations and the DNA damage level, as well as with partial restoration of cell survival/signalling pathways, when the expression of these molecules partially returned to their proper levels.
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Anticarcinógenos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Glucósidos/farmacología , Hepatocitos/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Fenoles/farmacología , Antineoplásicos/efectos adversos , Antineoplásicos/química , Antioxidantes/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/efectos adversos , Cisplatino/agonistas , Cisplatino/antagonistas & inhibidores , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Antagonismo de Drogas , Sinergismo Farmacológico , Femenino , Células Hep G2 , Hepatocitos/citología , Humanos , Neoplasias Ováricas/patología , Sustancias Protectoras/farmacología , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacosRESUMEN
As future scientists, university students need to learn how to avoid making errors in their own manuscripts, as well as how to identify flaws in papers published by their peers. Here we describe a novel approach on how to promote students' ability to critically evaluate scientific articles. The exercise is based on instructing teams of students to write intentionally flawed manuscripts describing the results of simple experiments. The teams are supervised by instructors advising the students during manuscript writing, choosing the 'appropriate' errors, monitoring the identification of errors made by the other team and evaluating the strength of their arguments in support of the identified errors. We have compared the effectiveness of the method with a journal club-type seminar. Based on the results of our assessment we propose that the described seminar may effectively complement the existing approaches to teach critical scientific thinking. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):22-30, 2018.
Asunto(s)
Informe de Investigación , Ciencia/educación , Estudiantes/psicología , Enseñanza , Humanos , Informe de Investigación/normas , UniversidadesRESUMEN
Hyperforin (HF), a substance that accumulates in the leaves and flowers of Hypericum perforatum L. (St. John's wort), consists of a phloroglucinol skeleton with lipophilic isoprene chains. HF exhibits several medicinal properties and is mainly used as an antidepressant. So far, the antigenotoxicity of HF has not been investigated at the level of primary genetic damage, gene mutations, and chromosome aberrations, simultaneously. The present work is designed to investigate the potential antigenotoxic effects of HF using three different experimental test systems. The antigenotoxic effect of HF leading to the decrease of primary/transient promutagenic genetic changes was detected by the alkaline comet assay on human lymphocytes. The HF antimutagenic effect leading to the reduction of gene mutations was assessed using the Ames test on the standard Salmonella typhimurium (TA97, TA98, and TA100) bacterial strains, and the anticlastogenic effect of HF leading to the reduction of chromosome aberrations was evaluated by the in vitro mammalian chromosome aberration test on the human tumor cell line HepG2 and the non-carcinogenic cell line VH10. Our findings provided evidence that HF showed antigenotoxic effects towards oxidative mutagen zeocin in the comet assay and diagnostic mutagen (4-nitroquinoline-1-oxide) in the Ames test. Moreover, HF exhibited an anticlastogenic effect towards benzo(a)pyrene and cisplatin in the chromosome aberration test.