Clonagem e caracterização molecular do mutante PSO10-1 de Saccharomyces cerevisiae

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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 rad2{Delta}, rlf2{Delta} and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2{Delta} pso4-1 and rlf2{Delta} pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae

Genotoxicidade do diseleneto de difenila em uma linhagem celular de adenocarcinoma de mama - MCF7

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Extração e caracterização do óleo essencial das inflorescências de Piper gaudichaudianum Kunth

Piper gaudichaudianum Kunth, popularmente conhecida como Jaborandi, Iaborandi ou Pariparoba, éuma espécie nativa da Mata Atlântica. Ensaios fitoquímicos anteriores, utilizando-se o teste de Sudam, demonstraramresultado positivo para a presença de óleos e graxas. Em função disto, o objetivo deste trabalho foirealizar a extração e identificação dos constituintes do óleo essencial das inflorescências de Piper gaudichaudianum.A extração ocorreu em aparelho tipo Clevenger modificado por um tempo de 6 horas. A análise do óleo foirealizada em equipamento de CG-EM. O rendimento de óleo essencial foi de 0,37% e os principais constituintesidentificados foram: a-humuleno (23,05%), ß-cariofileno (10,40%) e o ß-pineno (7,00%).Palavras-chave: Piper gaudichaudianum, óleo essencial, clevenger modificado

Avaliação da atividade mutagênica e antimutagênica induzidas pelo extrato vegetal da Costus spicatus

A Costus spicatus Swartz, que popularmente é chamada de “Cana-do-Brejo”, é uma espécie nativada América Latina habitando em locais úmidos. É utilizada popularmente para patologias do sistemaurinário. Entretanto, o uso desta planta para fins terapêuticos ainda carece de estudos de toxicidade e desegurança. Em função disto, o objetivo desse trabalho foi verificar as atividades mutagênica e antimutagênicado extrato bruto hidroalcoólico, preparado das mesmas condições em que ele é consumido pela população,utilizando a levedura Saccharomyces cerevisiae como modelo experimental. Os experimentos sugeremque este extrato não apresenta atividade mutagênica e possa ter atividade antimutagênica dependenteda dose utilizada e tratamento.Palavras-chave: cana-do-brejo, Costus spicatus, mutagênese, antimutagênese, Saccharomycescerevisiae

Structural Aspects of Antioxidant and Genotoxic Activities of Two Flavonoids Obtained from Ethanolic Extract of Combretum leprosum

Combretum leprosum Mart., a member of the Combretaceae family, is a traditionally used Brazilian medicinal plant, although no evidence in the literature substantiates its antioxidant action and the safety of its use. We evaluated the antioxidant properties of the ethanolic extract (EE) from flowers of C. leprosum and its isolated products 5,3′-dihydroxy-3,7,4′-trimethoxyflavone (FCL2) and 5,3′,4′-trihydroxy-3,7-dimethoxyflavone (FCL5) in Saccharomyces cerevisiae strains proficient and deficient in antioxidant defenses. Their mutagenic activity was also assayed in S. cerevisiae, whereas cytotoxic and genotoxic properties were evaluated by MTT and Comet Assays, respectively, in V79 cells. We show that the EE, FCL2, and FCL5 have a significant protective effect against H2O2. FCL2 showed a better antioxidant action, which can be related to the activation of the 3′-OH in the presence of a methoxyl group at 4′ position in the B-ring of the molecule, while flavonoids did not induce mutagenesis in yeast, and the EE was mutagenic at high concentrations. The toxicity of these compounds in V79 cells increases from FCL2 = FCL5 < EE; although not cytotoxic, FCL5 induced an increase in DNA damage. The antioxidant effect, along with the lower toxicity and the absence of genotoxicity, suggests that FCL2 could be suitable for pharmacological use

Aplicação de uma metodologia de HPCL para avaliação do potencial antioxidante in vitro da planta Croton cajucara Benth a base da Xantina Oxidase

A espécie Croton cajucara Benth (Sacaca) vem sendo utilizada popularmente como recurso medicinalno tratamento de várias doenças como Hipercolesterolemia e o Diabete Melittus, indicando umaprovável ação antioxidante. O objetivo deste estudo é a aplicação de uma metodologia a base da cromatografialíquida de alta eficiência (HPLC) para determinar o potencial antioxidante in vitro da Sacaca através dosistema enzimático da Xantina Oxidase, um método rápido, eficaz e sensível. Os radicais hidroxila produzidosno teste, espécies reativas de oxigênio, são derivatizados em produtos estáveis 2,3- e 2,5-DHBA, quesão detectados via HPLC. Observa-se uma atividade antioxidante in vitro do chá da casca da Sacaca que é dependente da concentração e volume do chá. Estes resultados preliminares demonstram um eminentepotencial antioxidante do Croton cajucara Benth.Palavras-chave: Croton cajucara Benth, atividade antioxidante in vitro, xantina oxidase, HPLC

Both XPA and DNA polymerase eta are necessary for the repair of doxorubicin-induced DNA lesions

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. in fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA. (C) 2011 Elsevier Ireland Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)USP-COFECUB (São Paulo, Brazil)Univ São Paulo, Dept Microbiol, Inst Biomed Sci, São Paulo, BrazilUniv Paris Sud, Inst Gustave Roussy, Ctr Natl Rech Sci, UMR8200, Villejuif, FranceFed Univ São Paulo UNIFESP, Dept Biol Sci, Diadema, SP, BrazilUniv Fed Rio Grande do Sul, Ctr Biotechnol, Dept Biophys, Porto Alegre, RS, BrazilFed Univ Hlth Sci Porto Alegre UFCSPA, Dept Basic Hlth Sci, Porto Alegre, RS, BrazilFed Univ São Paulo UNIFESP, Dept Biol Sci, Diadema, SP, BrazilWeb of Scienc

DNA damage in homocystinuria: 8-oxo‑,8‑dihydro‑2’-deoxyguanosine levels in cystathionine-β-synthase deficient patients and the in vitro protective effect of N-acetyl‑L‑cysteine

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine β-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS‑deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2’- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 μM and 200 μM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria. Keywords: Cystathionine-β-synthase deficiency; oxidative stress; 8-oxo-7,8-dihydro- 2’-deoxyguanosine; homocysteine; DNA damage; N-acetyl-L-cystein