The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.

Accumulation of (5 ` S)-8,5 `-cyclo-2 `-deoxyadenosine in organs of Cockayne syndrome complementation group B gene knockout mice

Cockayne syndrome (CS) is a human genetic disorder characterized by sensitivity to UV radiation, neurodegeneration, premature aging among other phenotypes. CS complementation group B (CS-B) gene (csb) encodes the CSB protein (CSB) that is involved in base excision repair of a number of oxidatively induced lesions in genomic DNA in vivo. We hypothesized that CSB may also play a role in cellular repair of the DNA helix-distorting tandem lesion (5`S)-8,5`-cyclo-2`-deoxyadenosine (S-cdA). Among many DNA lesions. S-cdA is unique in that it represents a concomitant damage to both the sugar and base moieties of the same nucleoside. Because of the presence of the C8-C5` covalent bond, S-cdA is repaired by nucleotide excision repair unlike most of other oxidatively induced lesions in DNA, which are subject to base excision repair. To test our hypothesis, we isolated genomic DNA from brain, kidney and liver of wild type and csb knockout (csb(-/-)) mice. Animals were not exposed to any exogenous oxidative stress before the experiment. DNA samples were analysed by liquid chromatography/mass spectrometry with isotope-dilution. Statistically greater background levels of S-cdA were observed in all three organs of csb(-/-) mice than in those of wild type mice. These results suggest the in vivo accumulation of S-cdA in genomic DNA due to lack of its repair in csb(-/-) mice. Thus, this study provides, for the first time, the evidence that CSB plays a role in the repair of the DNA helix-distorting tandem lesion S-cdA. Accumulation of unrepaired S-cdA in vivo may contribute to the pathology associated with CS. Published by Elsevier B.V.

Evidence that OGG1 glycosylase protects neurons against oxidative DNA damage and cell death under ischemic conditions

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1(-/-) mice compared with OGG1(+/+) mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 680-692; doi:10.1038/jcbfm.2010.147; published online 25 August 2010

Base Excision Repair Activities Differ in Human Lung Cancer Cells and Corresponding Normal Controls

Oxidative damage to DNA is thought to play a role in carcinogenesis by causing Mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway for the repair of oxidized modifications both in nuclear and mitochondrial, DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three cell lines used. However, the specific activities and cancer versus control comparison differed significantly between the nuclear and mitochondrial compartments. OGG1 activity, as measured by 8-oxodA incision, was upregulated in cancer cell mitochondria but down-regulated in the nucleus when compared to control cells. Similarly, NTH1 activity was also up-regulated in mitochondrial extracts from cancer cells but did not change significantly in the nucleus. Together, these results support the idea that alterations in BER capacity are associated with carcinogenesis.

The mitochondrial transcription factor A functions in mitochondrial base excision repair

Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids TFAM plays an important role in mitochondrial transcription and replication TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected in mitochondria, whereas base excision repair (BER) has been comprehensively characterized in these organelles The BER proteins are associated with the inner membrane in mitochondria and thus with the mitochondrial nucleoid, where TFAM is also situated However, a function for TFAM in BER has not yet been investigated This study examines the role of TFAM in BER In vitro studies with purified recombinant TFAM indicate that it preferentially binds to DNA containing 8-oxoguanines, but not to abasic sites, uracils, or a gap in the sequence TFAM inhibited the in vitro incision activity of 8-oxoguanine DNA glycosylase (OGG1), uracil-DNA glycosylase (UDG), apurinic endonuclease 1 (APE1), and nucleotide incorporation by DNA polymerase gamma (pol gamma) On the other hand, a DNA binding-defective TFAM mutant, L58A, showed less inhibition of BER in vitro Characterization of TFAM knockdown (KD) cells revealed that these lysates had higher 8oxoG incision activity without changes in alpha OGG1 protein levels TFAM KD cells had mild resistance to menadione and increased damage accumulation in the mtDNA when compared to the control cells In addition, we found that the tumor suppressor p53, which has been shown to interact with and alter the DNA binding activity of TFAM, alleviates TFAM-Induced inhibition of BER proteins Together, the results suggest that TFAM modulates BER in mitochondria by virtue of its DNA binding activity and protein interactions Published by Elsevier B V

Estudo da perda de energia e da flutuação estatística da perda de energia de íons de lítio em direções aleatórias do silício

Usando a técnica de Retroespalhamento Rutherford (RBS). no presente trabalho medimos a perda de energia e a flutuação estatística da perda de energia (straggling) como função da energia para íons de Li em alvos de silicio amorfo. Através do método dos marcadores. com amostras produzidas por implantação iônica e por epitaxia de feixe molecular,o poder de freamento foi medido em um intervalo de energia entre 250keV e 9MeV enquanto que o straggling foi determinado em um intervalo que foi de 450keV a 3MeV. Os resultados experimentais foram comparados com medidas prévias e com cálculos semi-empiricos realizados por Ziegler. Biersack e Littmark e por Konac et el., sendo encontrado um razoável acordo teórico-experimental. Foram também realizados cálculos baseados nos modelos teóricos de Aproximação por Convolução Perturbativa (PCA) e Aproximação por Convolução Unitária (UCA) propostos por Grande e Schiwielz O modelo PCA apresentou resultados aceitáveis para energias acima de 6MeV enquanto que os valores apresentados pelo divergem dos valores experimentais para energias abaixo de 2MeV. As medidas de straggling quando comparados com as predições da teoria de Bohr mostraram discrepâncias que foram entre 20% e 60%. Cálculos feitos com a aproximação para encontros binários (BEA) usando-se a secção de choque de Thompsom e a secção de choque proposta no trabalho de Vriens foram comparados com as medidas experimentais. Os resultados usando-se a secção de choque de Thompsom divergem por até 50%, enquanto que os valores calculados a secção de choque proposta por Vriens mostram uma concordância razoável para energias abaixo de 1MeV.

Prototexto, narrativa poética da ciência: uma estratégia de construção do conhecimento e religação de saberes no ensino de física

In this thesis I present the prototexto notion and to I base as a complex system while strategy of knowledge construction and reconexion of you know in physics teaching. Prototexto is a poetic narrative of the science, proposal and used initially as "instrument of creative" learning for students apprentice of science of the medium and technical teaching of the Vitória da Conquista's CEFET-BA, in the period of 1997-2004. Later become pregnant as a strategy of knowledge construction, in the Universidade Estadual da Bahia - UESB, the prototexto notion configures a complement to the mathematical formulation. The proposal of a poetic narrative of the science is that the apprentice of science starts to organize in an aesthetic-literary way your knowledge, dispersed in disciplines, starting from a theme of the physics. The prototexto emerges of my reflections concerning the classic science, identified for Edgar Morin as tends a thought excessively numeric, and that it has been reproduced in physics teaching, in most of the schools, limiting him/it to an order pattern with the mistake absence. They are operations of the prototexto: the poetic language, the pedagogic stamp, the unfinished of the argument, the system character and the apprentice's of science inclusion as subject implicated in the construction of the knowledge. the theoretical foundations are based Morin's proposition of the method as strategy, the beginning of the complementarity of Niels Bohr in conceiving excluding categories as face of a same phenomenon and the conception of creative time of Ilya Prigogine that enunciates the alliance among the nature and the man that it describes her

Seasonal changes in blood oxygen transport and acid-base status in the tegu lizard, Tupinambis merianae

Oxygen-binding properties, blood gases, and acid-base parameters were studied in tegu lizards, Tupinambis merianae, at different seasons and temperatures. Independent of temperature and pH, blood oxygen affinity was higher in dormant lizards than in those active during the summer. Haematocrit (Hct) and hemoglobin content ([Hb]) were greater in active lizards resulting in a higher oxygen-carrying capacity. Nucleoside triphosphate content ([NTP]) was reduced during dormancy, but the ratio between [NTP] and [Hb] remained unchanged. Dormancy was accompanied by an increase in plasma bicarbonate ([HCO(3)(-)]PI) and an elevation of arterial CO(2) partial pressure (P(aCO2)) and CO(2) content in the plasma (C(PlCO2)). These changes in acid-base parameters persist over a broad range of body temperatures. In vivo, arterial O(2) partial pressure (Pa(O2)) and O(2) content (Ca(O2)) were not affected by season and tended to increase with temperature. Arterial pH (pH(a)) of dormant animals is reduced compared to active lizards at body temperatures below 15 degreesC, while no significant difference was noticed at higher temperatures. (C) 2003 Elsevier B.V. All rights reserved.

Fish hemoglobins

Vertebrate hemoglobin, contained in erythrocytes, is a globular protein with a quaternary structure composed of 4 globin chains (2 alpha and 2 beta) and a prosthetic group named heme bound to each one. Having myoglobin as an ancestor, hemoglobin acquired the capacity to respond to chemical stimuli that modulate its function according to tissue requirements for oxygen. Fish are generally submitted to spatial and temporal O2 variations and have developed anatomical, physiological and biochemical strategies to adapt to the changing environmental gas availability. Structurally, most fish hemoglobins are tetrameric; however, those from some species such as lamprey and hagfish dissociate, being monomeric when oxygenated and oligomeric when deoxygenated. Fish blood frequently possesses several hemoglobins; the primary origin of this finding lies in the polymorphism that occurs in the globin loci, an aspect that may occasionally confer advantages to its carriers or even be a harmless evolutionary remnant. on the other hand, the functional properties exhibit different behaviors, ranging from a total absence of responses to allosteric regulation to drastic ones, such as the Root effect.

On asymptotic solutions of integrable wave equations

Asymptotic 'soliton train' solutions of integrable wave equations described by inverse scattering transform method with second-order scalar eigenvalue problem are considered. It is shown that if asymptotic solution can be presented as a modulated one-phase nonlinear periodic wavetrain, then the corresponding Baker-Akhiezer function transforms into quasiclassical eigenfunction of the linear spectral problem in weak dispersion limit for initially smooth pulses. In this quasiclassical limit the corresponding eigenvalues can be calculated with the use of the Bohr Sommerfeld quantization rule. The asymptotic distributions of solitons parameters obtained in this way specify the solution of the Whitham equations. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Quantization rules for bound states in quantum wells

An algebraic reformulation of the Bohr-Sommerfeld (BS) quantization rule is suggested and applied to the study of bound states in one-dimensional quantum wells. The energies obtained with the present quantization rule are compared to those obtained with the usual BS and WKB quantization rules and with the exact solution of the Schrodinger equation. We find that, in diverse cases of physical interest in molecular physics, the present quantization rule not only yields a good approximation to the exact solution of the Schrodinger equation, but yields more precise energies than those obtained with the usual BS and/or WKB quantization rules. Among the examples considered numerically are the Poeschl-Teller potential and several anharmonic oscillator potentials. which simulate molecular vibrational spectra and the problem of an isolated quantum well structure subject to an external electric field.

Reply to comment on 'Quantization rules for bound states in quantum wells'

In this reply to the comment on 'Quantization rules for bound states in quantum wells' we point out some interesting differences between the supersymmetric Wentzel-Kramers-Brillouin (WKB) quantization rule and a matrix generalization of usual WKB (mWKB) and Bohr-Sommerfeld (mBS) quantization rules suggested by us. There are certain advantages in each of the supersymmetric WKB (SWKB), mWKB and mBS quantization rules. Depending on the quantum well, one of these could be more useful than the other and it is premature to claim unconditional superiority of SWKB over mWKB and mBS quantization rules.

Asymptotic soliton train solutions of Kaup-Boussinesq equations

Asymptotic soliton trains arising from a 'large and smooth' enough initial pulse are investigated by the use of the quasiclassical quantization method for the case of Kaup-Boussinesq shallow water equations. The parameter varying along the soliton train is determined by the Bohr-Sommerfeld quantization rule which generalizes the usual rule to the case of 'two potentials' h(0)(x) and u(0)(x) representing initial distributions of height and velocity, respectively. The influence of the initial velocity u(0)(x) on the asymptotic stage of the evolution is determined. Excellent agreement of numerical solutions of the Kaup-Boussinesq equations with predictions of the asymptotic theory is found. (C) 2003 Elsevier B.V. All rights reserved.

Asymptotic soliton train solutions of the defocusing nonlinear Schrodinger equation

Asymptotic behavior of initially large and smooth pulses is investigated at two typical stages of their evolution governed by the defocusing nonlinear Schrodinger equation. At first, wave breaking phenomenon is studied in the limit of small dispersion. A solution of the Whitham modulational equations is found for the case of dissipationless shock wave arising after the wave breaking point. Then, asymptotic soliton trains arising eventually from a large and smooth initial pulse are studied by means of a semiclassical method. The parameter varying along the soliton train is calculated from the generalized Bohr-Sommerfeld quantization rule, so that the distribution of eigenvalues depends on two functions-intensity rho(0)(x) of the initial pulse and its initial chirp v(0)(x). The influence of the initial chirp on the asymptotic state is investigated. Excellent agreement of the numerical solution of the defocusing NLS equation with predictions of the asymptotic theory is found.

Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A(0) fully stripped of any anions

In this work, initial crystallographic studies of human haemoglobin (Hb) crystallized in isoionic and oxygen-free PEG solution are presented. Under these conditions, functional measurements of the O-2-linked binding of water molecules and release of protons have evidenced that Hb assumes an unforeseen new allosteric conformation. The determination of the high-resolution structure of the crystal of human deoxy-Hb fully stripped of anions may provide a structural explanation for the role of anions in the allosteric properties of Hb and, particularly, for the influence of chloride on the Bohr effect, the mechanism by which Hb oxygen affinity is regulated by pH. X-ray diffraction data were collected to 1.87 Angstrom resolution using a synchrotron-radiation source. Crystals belong to the space group P2(1)2(1)2 and preliminary analysis revealed the presence of one tetramer in the asymmetric unit. The structure is currently being refined using maximum-likelihood protocols.