Deletion of OGG1 Results in a Differential Signature of Oxidized Purine Base Damage in mtDNA Regions

Mitochondrial oxidative stress accumulates with aging and age-related diseases and induces alterations in mitochondrial DNA (mtDNA) content. Since mtDNA qualitative alterations are also associated with aging, repair of mtDNA damage is of great importance. The most relevant form of DNA repair in this context is base excision repair (BER), which removes oxidized bases such as 8-oxoguanine (8-oxoG) and thymine glycol through the action of the mitochondrial isoform of the specific 8-oxoG DNA glycosylase/apurinic or apyrimidinic (AP) lyase (OGG1) or the endonuclease III homolog (NTH1). Mouse strains lacking OGG1 (OGG1/) or NTH1 (NTH1/) were analyzed for mtDNA alterations. Interestingly, both knockout strains presented a significant increase in mtDNA content, suggestive of a compensatory mtDNA replication. The mtDNA “common deletion” was not detected in either knockout mouse strain, likely because of the young age of the mice. Formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites accumulated in mtDNA from OGG1/ but not from NTH1/ mice. Interestingly, the D-loop region was most severely aected by the absence of OGG1, suggesting that this region may be a hotspot for oxidative damage. Thus, we speculate that mtDNA alterations may send a stress message to evoke cell changes through a retrograde mitochondrial–nucleus communication

Sustained kidney biochemical derangement in treated experimental diabetes : a clue to metabolic memory.

The occurrence of biochemical alterations that last for a long period of time in diabetic individuals even after adequate handling of glycemia is an intriguing phenomenon named metabolic memory. In this study, we show that a kidney pathway is gradually altered during the course of diabetes and remains persistently changed after late glycemic control in streptozotocin-induced diabetic rats. This pathway comprises an early decline of uric acid clearance and pAMPK expression followed by fumarate accumulation, increased TGF-? expression, reduced PGC-1? expression, and downregulation of methylation and hydroxymethylation of mitochondrial DNA. The sustained decrease of uric acid clearance in treated diabetes may support the prolonged kidney biochemical alterations observed after tight glycemic control, and this regulation is likely mediated by the sustained decrease of AMPK activity and the induction of inflammation. This manuscript proposes the first consideration of the possible role of hyperuricemia and the underlying biochemical changes as part of metabolic memory in diabetic nephropathy development after glycemic control

Envolvimento do calcio no mecanismo de citoxicidade do pro-oxidante tetra hidroxibenzoquinona (THQ)

Orientadores : Maria Edwiges Hoffmann, Anibal Eugenio VercesiDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A THO é uma hidroxiquinona que auto-oxida rapidamente em solução gerando radicais semiquinona e espécies ativadas de oxigênio, o que produz uma situação de estresse oxidativo. Neste trabalho, investigamos a participação~do íon Ca2+ no mecanismo de citotoxicidade da THO, e analisamos o possível papel do DNA e da mitocôndria como alvos celulares da ação~da THO. Demonstramos que a reação de auto-oxidação da THO causa efeitos tóxicos em células de mamíferos (linhagem V79), como i) a inibição da proliferação celular; ii) a fragmentação do DNA celular; e iii) a alteração da membrana mitocondrial interna. Estudos de proteção evidenciaram que a espécie oxidante responsável pelo efeito citostático e pelas alterações mitocondriais é o peróxido de hidrogênio, e que esses efeitos são provavelmente causados pela reação do H202 com os radicais semiquinona, gerando radicais hidroxila. Mostramos também que o íon Ca2+ tem um papel fundamental na citotoxicidade da THO, participando dos 3 mecanismos acima descritos. Enquanto que nos dois primeiros processos se observa uma potencialização dos efeitos da THO na presença de Ca2+ , a produção das alterações mitocondriais é completamente dependente de sua presença. Finalmente, evidenciamos que células V79 possuem mecanismos para reverter tanto as quebras de DNA quanto as lesões mitocondriais Isto sugere que a indução dessas lesões deverá interferir com o crescimento celular somente quando os sistemas celulares responsáveis por sua reversão estiverem saturados, caracterizando uma condição de irreversibilidade que conduz a morte celularAbstract: THO is a hydroxyquinone wich autoxidases readily in aqueous solution producing semiquinone radicaIs and active oxygen species, that leeds to a condition of oxidative stress. In this work we describe the involvement of Ca2+ in the mechanism of THO induced Cytotoxicity and analyse the possible roles of DNA and mitochondria as critical targets for THO toxicity. Our studies show that during THO autoxidation deleterious effects are generated in mammalian fybroblasts (V79 line), such as i) inhibition of cell proliferationi ii) induction of DNA breaksi and iii) alteration of lnner mitochondrial membrane permeability. Protection experiments with antioxidants showed that both, growth inhibition and mitochondrial damage results from hydrogen peroxide dependent and metal independent reactions, probably via hydroxyl radical generation. We also report here the crucial role of Ca2+ on THO Cytotoxicity, increasing the inhibition of cell proliferation and the levels of DNA breakage. In addition, our results evidenciated that mitochondrial membrane THO results from a Ca2+ dependent mechanism. permeability by It is also showed that V79 cells are able to repair the THO induced DNA breaks and restore mitochondrial functions. It is hypothesized that irreversible oxidant injury occurs when the cellular antioxidant mechanisms ocerwhelmed and the cell is no longer able to surviveMestradoMestre em Ciências Biológica

Guidelines to develop interactive tutorials on 3D biomolecules: the case of DNA repair by photolyase

INTRODUCTION: This work presents the following guidelines for developing interactive learning objects on the 3D biomolecules: i- identify a relevant educational need; ii-select an appropriate subject; iii- employ interactive 3D molecular structures; iv- simultaneously display animation with related textual information or 2D diagrams; v- integrating different modes of representation of chemical phenomena. Based on these, was constructed the "Photolyase Tutorial". Photolyase is an enzyme that repairs DNA. It recognizes the cyclobutane pyrimidine dimer lesion (CPD) caused by exposure to ultraviolet light. Understanding the repair mechanism requires understanding the structure-activity relationships of the molecules involved. Therefore, this topic is a convenient subject for teaching key aspects of the structure of protein and nucleic acids. There are few interactive 3D DNA repair tutorials available and most of these are in english, therefore not acessible for students that do not domain this language. Besides, these tutorials generally focus only on displaying structural features without make foster correlations between the structure and chemical aspects of repair mechanisms. OBJECTIVES: In order to construct a interactive tutorial that fills these gaps, the 3D structures of a DNA molecule, a DNA with CPD lesion and the photolyase enzyme were manipulated focusing on structural and chemical explanation. MATERIALS AND METHODS: These structures were obtained from PDB (http://www.rcsb.org/pdb/home/home.do) and manipulated to show biochemical and chemical concepts by using the resources from the LABIQ Platform (http://labiq.is.usp.br). DISCUSSION AND RESULTS: Several concepts are covered, i.e., H-bonding, active site, co-factors etc. The tutorial is organized in small conceptual blocks presenting, in a stepwise fashion, how the enzyme recognizes the DNA lesion and proceeds to repair. Each block comprises a trigger button that controls the interactive molecular viewer from the short text information or 2D diagram. CONCLUSION: This way, we developed a interactive tutorial that combines different modes of representation of chemical and structural concepts of biomolecules in a single interface

Tutorial Estrutura e Estabilidade do DNA: animações interativas da estrutura tridimensional do DNA

The tutorial "DNA Structure and Stability" was developed articulating Ausubel's Theory of Meaningful Learning and Mayer's Multimedia principles, in order to favor the DNA structure significant learning through the guided and gradual exploration of the DNA three-dimensional structure interactive animations. In this sense, conceptual units with auxiliary medias have been established, such as texts, diagrams and tables, which assist in the explanation of animations. Several chemical and biochemical concepts are explained in order to favor understanding of DNA as a biopolymer determined by its intra/intermolecular chemical interactions that consequently establish the structure/stability relationships and structure/biological activity. In this way, was developed an interactive tutorial that can be used in several teaching and learning situations in order to favor the construction of knowledge by the learner.O tutorial “Estrutura e estabilidade do DNA” foi desenvolvido articulando a Teoria de Aprendizagem Significativa de Ausubel e princípios de Multimídia de Mayer, no intuito de favorecer a aprendizagem significativa da estrutura do DNA por meio da exploração guiada e paulatina de animações interativas da estrutura tridimensional do DNA. Para tal, foram estabelecidas unidades conceituais que possuem mídias acessórias, como textos, diagramas e tabelas, que auxiliam na explicação das animações. Diversos conceitos químicos e bioquímicos são explicados no intuito de favorecer a compreensão do DNA como um biopolímero determinado pelas interações químicas intra/intermoleculares que, consequentemente, estabelecem as relações estrutura/estabilidade e estrutura/atividade biológica. Desta forma, foi desenvolvido um tutorial interativo que pode ser usado em diversas situações de ensino e aprendizagem no intuito de favorecer a construção do conhecimento pelo aprendiz