SummaryCaloric restriction is the most effective non-genetic intervention to enhance lifespan known to date. A major research interest has been the development of therapeutic strategies capable of promoting the beneficial results of this dietary regimen. In this sense, we propose that compounds that decrease the efficiency of energy conversion, such as mitochondrial uncouplers, can be caloric restriction mimetics. Treatment of mice with low doses of the protonophore 2,4-dinitrophenol promotes enhanced tissue respiratory rates, improved serological glucose, triglyceride and insulin levels, decrease of reactive oxygen species levels and tissue DNA and protein oxidation, as well as reduced body weight. Importantly, 2,4-dinitrophenol-treated animals also presented enhanced longevity. Our results demonstrate that mild mitochondrial uncoupling is a highly effective in vivo antioxidant strategy, and describe the first therapeutic intervention capable of effectively reproducing the physiological, metabolic and lifespan effects of caloric restriction in healthy mammals.

No abstract

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93)-->Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.

Our study shows that new anthracene and naphthalene derivatives function as compounds for trapping and chemically generating singlet molecular oxygen [O2(1Δg)], respectively. The syntheses of these derivatives are described, as well as some localization testing in cells.

. Prof. Lineu Prestes 748, 05508-900, São Paulo -SP Recebido em 6/6/05; aceito em 5/1/06; publicado na web em 11/8/06 OXIDATIVE DAMAGE AND NEURODEGENERATION. WHAT HAVE WE LEARNED FROM TRANSGENIC AND KNOCKOUT ANIMALS? Accumulated evidence indicates that oxidative stress plays a role in neurodegenerative diseases, such as Alzheimer, Parkinson and Amyotrophic Lateral Sclerosis. Here, we emphasize the results provided by the technology of genetically modified animals. Studies with transgenic and knockout mice have allowed great advances in the research of oxidative stress in general and in the central nervous system, and are pointing to potential targets for the development of new drugs and therapies to disrupt the cycle of events that lead to neuronal death. Thus, genetically modified animals are a valuable tool for the comprehension of human diseases, including neurodegenerative ones.Keywords: oxidative damage; neurodegeneration; transgenic and knockout mice. INTRODUÇÃOAtualmente, devido ao aumento da expectativa de vida da população, existe um crescente interesse no estudo de doenças neurodegenerativas como Alzheimer, Parkinson, Huntington e Esclerose Lateral Amiotrófica (ELA). Uma vertente destes estudos demonstra que o estresse oxidativo tem um papel importante e pode até mesmo desencadear o processo de neurodegeneração. Esta vertente é fortalecida pelo fato de que neurônios são altamente propensos a situações de estresse oxidativo.O estresse oxidativo é definido como a situação na qual a formação de espécies reativas excede significativamente a capacidade de defesa antioxidante e de reparo do organismo, tendo como conseqüência o aumento de danos a biomoléculas (DNA, lipídios, proteí-nas . Neste contexto, a investigação de situações de estresse oxidativo no sistema nervoso central (SNC) é primordial para compreender como espécies oxidativas contribuem para a patologia de doenças neurodegenerativas. Grandes avanços nesta área foram alcançados graças a uma valiosa ferramenta: a tecnologia de camundongos geneticamente modificados. As contribuições feitas por esta tecnologia são enfatizadas neste trabalho através de uma revisão dos resultados obtidos com grande número de camundongos transgênicos e nocautes no estudo da regulação do estresse oxidativo no SNC. Espécies reativas e defesas antioxidantes

The uptake of ascorbate by neuroblastoma cells using a ruthenium oxide hexacyanoferrate (RuOHCF)-modified carbon fiber disc (CFD) microelectrode (r = 14.5 microm) was investigated. By use of the proposed electrochemical sensor the amperometric determination of ascorbate was performed at 0.0 V in minimum essential medium (MEM, pH = 7.2) with a limit of detection of 25 micromol L(-1). Under the optimum experimental conditions, no interference from MEM constituents and reduced glutathione (used to prevent the oxidation of ascorbate during the experiments) was noticed. The stability of the RuOHCF-modified electrode response was studied by measuring the sensitivity over an extended period of time (120 h), a decrease of around 10% being noticed at the end of the experiment. The rate of ascorbate uptake by control human neuroblastoma SH-SY5Y cells, and cells transfected with wild-type Cu,Zn-superoxide dismutase (SOD WT) or with a mutant typical of familial amyotrophic lateral sclerosis (SOD G93A), was in agreement with the level of oxidative stress in these cells. The usefulness of the RuOHCF-modified microelectrode for in vivo monitoring of ascorbate inside neuroblastoma cells was also demonstrated.

Biomolecule oxidation promoted by Cu, Zn-superoxide dismutase (SOD1) has been studied because of its potential role in neurodegenerative diseases. We studied the mechanism of DNA damage promoted by the SOD1-H2O2 system. The system promoted the formation of strand breaks in plasmid DNA and the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in calf thymus DNA. We were also able to detect, for the first time, 1,N2-etheno-2'-deoxyguanosine (1,N'-epsilondGuo) in calf thymus DNA exposed to SOD1-H2O2. The addition of a copper chelator caused a decrease in the frequency of 8-oxodGuo and 1,N2-epsilondGuo, indicating the participation of copper ions lost from SOD1 active sites. The addition of bicarbonate increased the levels of both DNA lesions. We conclude that copper liberated from SODI active sites has a central role in the mechanism of DNA damage promoted by SOD1 in the presence of H2O2, and that bicarbonate can modulate the reactivity of released copper.

ERRATA Página 18, linha 19: A referência original da informação "...em condições basais, cerca de 1% deste oxigênio é convertido em radical superóxido pela semi-ubiquinona..." é Boveris e Chance, 1973. (Boveris, A. e B. Chance. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J, v.134, p.707-16. 1973). Página 24, linha 6: Desconsiderar o termo "in vitro". Página 24, linha 20: Desconsiderar as quebras de fita simples. Página 25, linha 11: MYH, um homólogo da DNA glicosilase mutY de Escherichia coli, também faz parte do sistema de reparo da lesão 8-oxodGuo em eucariotos. Ela retira a adenina pareada com 8-oxodGuo. (Michaels, M.L. e J.H. Miller. The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8oxoguanine). J Bacteriol , v.174, p. 6321-25. 1992). Página 30, linha 3: As endonucleases somente clivam o DNA, e não retiram nucleotídeos. (de Souza-Pinto, N.C., D.M. Wilson III, T.V. Stevnsnerb e V.A. Bohr. Mitochondrial DNA, base excision repair and neurodegeneration. Dna Repair, v.7, p.1098-109. 2008).