The effects of oxidative stress on the retinal structure and function

Des études antérieures ont démontré que le métabolisme de la rétine, son apport sanguin et sa consommation de l'oxygène sont plus élevés dans le noir (Riva C.E. et al. 1983, Wang L. et al. 1996, Tam B.M. and Moritz O.L. 2007). Les stimuli physiologiques jouent supposément un rôle important dans le développement des différents systèmes nerveux (Arthur W. Spira, David Parkinson 1991). La privation de la rétine de son stimulus physiologique, la lumière, est un moyen valable de démontrer la validité de ce concept. D'autres études ont affirmé que les injections de dichlorure de paraquat dans la cavité vitréenne causent une sévère rétinopathie (Rétinopathie induite par paraquat: RIP). Cette rétinopathie est provoquée par les dérivés réactifs de l'oxygène (DRO) générés par le paraquat (Cingolani C. et al. 2006, Lu L. et al. 2006). Le but de notre premier projet (''Dark rearing project'') était de déterminer si les conséquences nocives de l'hyperoxie postnatale chez les rats albinos SD pourraient être amoindries en élevant une portée de rats au noir. Nos résultats suggèrent qu'une augmentation du métabolisme de la rétine causée par la déprivation de lumière chez les ratons, pourrait protéger ou masquer certains effets néfastes de l'hyperoxie postnatale. Le but de notre deuxième étude (''Paraquat project'') était d'examiner les possibles points de similitude entre RIP et d'autres modèles de rétinopathies oxydatives étudiés présentement par notre équipe, à savoir: Rétinopathie induite par l'oxygène (RIO) et Rétinopathie induite par la lumière (RIL). Nos résultats suggèrent que l'injection de dichlorure de paraquat dans la cavité vitréenne cause des changements sévères de la fonction de la rétine, tandis que sa structure semble intacte. La sévérité de ces changements dépend inversement de la maturité de la rétine au moment de l'injection.

Les R-loops et leurs conséquences sur l'expression génique chez Escherichia coli.

Des variations importantes du surenroulement de l’ADN peuvent être générées durant la phase d’élongation de la transcription selon le modèle du « twin supercoiled domain ». Selon ce modèle, le déplacement du complexe de transcription génère du surenroulement positif à l’avant, et du surenroulement négatif à l’arrière de l’ARN polymérase. Le rôle essentiel de la topoisomérase I chez Escherichia coli est de prévenir l’accumulation de ce surenroulement négatif générée durant la transcription. En absence de topoisomérase I, l’accumulation de ce surenroulement négatif favorise la formation de R-loops qui ont pour conséquence d’inhiber la croissance bactérienne. Les R-loops sont des hybrides ARN-ADN qui se forment entre l’ARN nouvellement synthétisé et le simple brin d’ADN complémentaire. Dans les cellules déficientes en topoisomérase I, des mutations compensatoires s’accumulent dans les gènes qui codent pour la gyrase, réduisant le niveau de surenroulement négatif du chromosome et favorisant la croissance. Une des ces mutations est une gyrase thermosensible qui s’exprime à 37 °C. La RNase HI, une enzyme qui dégrade la partie ARN d’un R-loop, peut aussi restaurer la croissance en absence de topoisomérase I lorsqu’elle est produite en très grande quantité par rapport à sa concentration physiologique. En présence de topoisomérase I, des R-loops peuvent aussi se former lorsque la RNase HI est inactive. Dans ces souches mutantes, les R-loops induisent la réponse SOS et la réplication constitutive de l’ADN (cSDR). Dans notre étude, nous montrons comment les R-loops formés en absence de topoisomérase I ou RNase HI peuvent affecter négativement la croissance des cellules. Lorsque la topoisomérase I est inactivée, l’accumulation d’hypersurenroulement négatif conduit à la formation de nombreux R-loops, ce qui déclenche la dégradation de l’ARN synthétisé. Issus de la dégradation de l’ARNm de pleine longueur, des ARNm incomplets et traductibles s’accumulent et causent l’inhibition de la synthèse protéique et de la croissance. Le processus par lequel l’ARN est dégradé n’est pas encore complètement élucidé, mais nos résultats soutiennent fortement que la RNase HI présente en concentration physiologique est responsable de ce phénotype. Chose importante, la RNase E qui est l’endoribonuclease majeure de la cellule n’est pas impliquée dans ce processus, et la dégradation de l’ARN survient avant son action. Nous montrons aussi qu’une corrélation parfaite existe entre la concentration de RNase HI, l’accumulation d’hypersurenroulement négatif et l’inhibition de la croissance bactérienne. Lorsque la RNase HI est en excès, l’accumulation de surenroulement négatif est inhibée et la croissance n’est pas affectée. L’inverse se produit Lorsque la RNase HI est en concentration physiologique. En limitant l’accumulation d’hypersurenroulement négatif, la surproduction de la RNase HI prévient alors la dégradation de l’ARN et permet la croissance. Quand la RNase HI est inactivée en présence de topoisomérase I, les R-loops réduisent le niveau d’expression de nombreux gènes, incluant des gènes de résistance aux stress comme rpoH et grpE. Cette inhibition de l’expression génique n’est pas accompagnée de la dégradation de l’ARN contrairement à ce qui se produit en absence de topoisomérase I. Dans le mutant déficient en RNase HI, la diminution de l’expression génique réduit la concentration cellulaire de différentes protéines, ce qui altère négativement le taux de croissance et affecte dramatiquement la survie des cellules exposées aux stress de hautes températures et oxydatifs. Une inactivation de RecA, le facteur essentiel qui déclenche la réponse SOS et le cSDR, ne restaure pas l’expression génique. Ceci démontre que la réponse SOS et le cSDR ne sont pas impliqués dans l’inhibition de l’expression génique en absence de RNase HI. La croissance bactérienne qui est inhibée en absence de topoisomérase I, reprend lorsque l’excès de surenroulement négatif est éliminé. En absence de RNase HI et de topoisomérase I, le surenroulement négatif est très relaxé. Il semble que la réponse cellulaire suite à la formation de R-loops, soit la relaxation du surenroulement négatif. Selon le même principe, des mutations compensatoires dans la gyrase apparaissent en absence de topoisomérase I et réduisent l’accumulation de surenroulement négatif. Ceci supporte fortement l’idée que le surenroulement négatif joue un rôle primordial dans la formation de R-loop. La régulation du surenroulement négatif de l’ADN est donc une tâche essentielle pour la cellule. Elle favorise notamment l’expression génique optimale durant la croissance et l’exposition aux stress, en limitant la formation de R-loops. La topoisomérase I et la RNase HI jouent un rôle important et complémentaire dans ce processus.

Évaluation de la cytogénotoxicité humaine induite par l’exposition à de faibles doses de benzo-a-pyrène, à l’aide de biomarqueurs précoces

Le benzo-a-pyrène (BaP) est un hydrocarbure aromatique polycyclique (HAP) cancérogène pour l’homme, qui contamine toutes les sphères de notre environnement. Son métabolite, le BaP-7,8-diol-9,10-époxyde (BPDE) est considéré comme son cancérogène ultime. Le BPDE se lie à l’ADN, formant des adduits qui doivent être réparés et qui seraient responsables des dommages à l’ADN et de la cancérogenèse induite par le BaP. Les adduits BPDE-ADN et les dommages à l’ADN (bris simple-brin [BSB] à l’ADN, aberrations chromosomiques [AC], échanges entre chromatides-sœurs [ÉCS] et micronoyaux [MN]) ont été mesurés dans les lymphocytes humains exposés à de faibles concentrations de BaP, provenant de jeunes volontaires non-fumeurs et en santé. Suite à l’exposition au BaP, le niveau d’adduits BPDE-ADN et la fréquence des AC et des MN augmentent significativement, puis diminuent aux concentrations les plus élevées de BaP testées, suggérant une induction du métabolisme de phase II du BaP. Lors de la mesure des ÉCS, nous obtenons une courbe dose-réponse linéaire, indiquant la production d’un autre type de lésions devant être réparées par le système de réparation par recombinaison homologue. Ces lésions pourraient être des bris à l’ADN ou des bases oxydées (8-OH-dG), ce qui est suggéré par l’analyse des corrélations existant entre nos biomarqueurs. Par ailleurs, la comparaison de la courbe dose-réponse des hommes et des femmes montre que des différences existent entre les sexes. Ainsi, les ÉCS, les AC et les MN sont significativement augmentés chez les hommes à la plus faible concentration de BaP, alors que chez les femmes cette augmentation, quoique présente, est non significative. Des différences interindividuelles sont également observées et sont plus importantes pour les adduits BPDE-ADN, les MN et les AC, alors que pour les ÉCS elles sont minimes. Les analyses statistiques effectuées ont permis d’établir que quatre facteurs (niveau d’exposition au BaP, adduits BPDE-ADN, fréquence des AC et nombre de MN par cellule micronucléée) expliquent jusqu’à 59 % de la variabilité observée dans le test des ÉCS, alors qu’aucun facteur significatif n’a pu être identifié dans le test des AC et des MN. L’analyse du mécanisme de formation de nos biomarqueurs précoces permet de suggérer que les bris à l’ADN et les bases oxydées devraient être classées comme biomarqueurs de dose biologique efficace, au sein des biomarqueurs d’exposition, dans le continuum exposition-maladie du BaP, étant donné qu’ils causent la formation des biomarqueurs de génotoxicité (ÉCS, AC et MN). Par ailleurs, le test des AC et des MN ont permis de confirmer l’action clastogénique du BaP en plus de mettre en évidence des effets aneugènes affectant surtout la ségrégation des chromosomes lors de la division cellulaire. Ces effets aneugènes, reliés à l’étape de progression dans la cancérogenèse, pourraient être particulièrement importants puisque l’exposition au BaP et aux HAP est chronique et dure plusieurs années, voire des décennies. La compréhension des mécanismes régissant la formation des biomarqueurs étudiés dans cette étude, ainsi que des relations existant entre eux, peut être appliquée à de nombreux contaminants connus et émergents de notre environnement et contribuer à en évaluer le mode d’action.

Selective inhibition of inducible nitric oxide synthase prevents lipid peroxidation in cartilage from patients with osteoarthritis.

INTRODUCTION: Emerging evidence indicates that nitric oxide (NO), which is increased in osteoarthritic (OA) cartilage, plays a role in 4-hydroxynonenal (HNE) generation through peroxynitrite formation. HNE is considered as the most reactive product of lipid peroxidation (LPO). We have previously reported that HNE levels in synovial fluids are more elevated in knees of OA patients compared to healthy individuals. We also demonstrated that HNE induces a panoply of inflammatory and catabolic mediators known for their implication in OA cartilage degradation. The aim of the present study was to investigate the ability of inducible NO synthase (iNOS) inhibitor, L-NIL (L-N6-(L-Iminoethyl)Lysine), to prevent HNE generation through NO inhibition in human OA chondrocytes. METHOD: Cells and cartilage explants were treated with or without either an NO generator (SIN or interleukin 1beta (IL-1β)) or HNE in absence or presence of L-NIL. Protein expression of both iNOS and free-radical-generating NOX subunit p47 (phox) were investigated by western blot. iNOS mRNA detection was measured by real-time RT-PCR. HNE production was analysed by ELISA, Western blot and immunohistochemistry. S-nitrosylated proteins were evaluated by Western Blot. Prostaglandin E2 (PGE2) and metalloproteinase 13 (MMP-13) levels as well as glutathione S-transferase (GST) activity were each assessed with commercial kits. NO release was determined using improved Griess method. Reactive oxygen species (ROS) generation was revealed using fluorescent microscopy with the use of commercial kits. RESULTS: L-NIL prevented IL-1β-induced NO release, iNOS expression at protein and mRNA levels, S-nitrosylated proteins and HNE in a dose dependent manner after 24h of incubation. Interestingly, we revealed that L-NIL abolished IL-1β-induced NOX component p47phox as well as ROS release. The HNE-induced PGE2 release and both cyclooxygenase-2 (COX-2) and MMP-13 expression were significantly reduced by L-NIL addition. Furthermore, L-NIL blocked the IL-1β induced inactivation of GST, an HNE-metabolizing enzyme. Also, L-NIL prevented HNE induced cell death at cytotoxic levels. CONCLUSION: Altogether, our findings support a beneficial effect of L-NIL in OA by preventing LPO process in NO-dependent and/or independent mechanisms.

Molecular and functional characterization of the chemotactic genes in the PCBs-degrader Pseudomonas pseudoalcaligenes KF707

Although bacteria represent the simplest form of life on Earth, they have a great impact on all living beings. For example the degrader bacterium Pseudomonas pseudoalcaligenes KF707 is used in bioremediation procedures for the recovery of polluted sites. Indeed, KF707 strain is know for its ability to degrade biphenyl and polychlorinated biphenyls - to which is chemotactically attracted - and to tolerate the oxydative stress due to toxic metal oxyanions such as tellurite and selenite. Moreover, in bioremediation processes, target compounds can be easily accessible to KF707 through biofilm formation. All these considerations suggest that KF707 is such a unique microorganism and this Thesis work has been focused on determining the molecular nature of some of the peculiar physiological traits of this strain. The genome project provided a large set of informations: putative genes involved in the degradation of aromatic and toxic compounds and associated to stress response were identified. Notably, multiple chemotactic operons and cheA genes were also found. Deleted mutants in the cheA genes were constructed and their role in motility, chemotaxis and biofilm formation were assessed and compared to those previously attributed to a cheA1 gene in a KF707 mutant constructed by a mini-Tn5 transposon insertion and which was impaired in motility and biofilm development. The results of this present Thesis work, taken together, were interpreted to suggest that in Pseudomonas pseudoalcaligenes KF707 strain, multiple factors are involved in these networks and they might play different roles depending on the environmental conditions. The ability of KF707 strain to produce signal molecules possibly involved in cell-to-cell communication, was also investigated: lack of a lux-like QS system - which is conversely widely present in Gram negative bacteria – keeps open the question about the actual molecular nature of KF707 quorum sensing mechanism.

Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.

Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.

Effect Of Platform Connection And Abutment Material On Stress Distribution In Single Anterior Implant-supported Restorations: A Nonlinear 3-dimensional Finite Element Analysis.

Although various abutment connections and materials have recently been introduced, insufficient data exist regarding the effect of stress distribution on their mechanical performance. The purpose of this study was to investigate the effect of different abutment materials and platform connections on stress distribution in single anterior implant-supported restorations with the finite element method. Nine experimental groups were modeled from the combination of 3 platform connections (external hexagon, internal hexagon, and Morse tapered) and 3 abutment materials (titanium, zirconia, and hybrid) as follows: external hexagon-titanium, external hexagon-zirconia, external hexagon-hybrid, internal hexagon-titanium, internal hexagon-zirconia, internal hexagon-hybrid, Morse tapered-titanium, Morse tapered-zirconia, and Morse tapered-hybrid. Finite element models consisted of a 4×13-mm implant, anatomic abutment, and lithium disilicate central incisor crown cemented over the abutment. The 49 N occlusal loading was applied in 6 steps to simulate the incisal guidance. Equivalent von Mises stress (σvM) was used for both the qualitative and quantitative evaluation of the implant and abutment in all the groups and the maximum (σmax) and minimum (σmin) principal stresses for the numerical comparison of the zirconia parts. The highest abutment σvM occurred in the Morse-tapered groups and the lowest in the external hexagon-hybrid, internal hexagon-titanium, and internal hexagon-hybrid groups. The σmax and σmin values were lower in the hybrid groups than in the zirconia groups. The stress distribution concentrated in the abutment-implant interface in all the groups, regardless of the platform connection or abutment material. The platform connection influenced the stress on abutments more than the abutment material. The stress values for implants were similar among different platform connections, but greater stress concentrations were observed in internal connections.

Diet Carotenoid Lutein Modulates The Expression Of Genes Related To Oxygen Transporters And Decreases Dna Damage And Oxidative Stress In Mice.

Lutein (LT) is a carotenoid obtained by diet and despite its antioxidant activity had been biochemically reported, few studies are available concerning its influence on the expression of antioxidant genes. The expression of 84 genes implicated in antioxidant defense was quantified using quantitative reverse transcription polymerase chain reaction array. DNA damage was measured by comet assay and glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were quantified as biochemical parameters of oxidative stress in mouse kidney and liver. cDDP treatment reduced concentration of GSH and increased TBARS, parameters that were ameliorated in treatment associated with LT. cDDP altered the expression of 32 genes, increasing the expression of GPx2, APC, Nqo1 and CCs. LT changed the expression of 37 genes with an induction of 13 mainly oxygen transporters. In treatments associating cDDP and LT, 30 genes had their expression changed with a increase of the same genes of the cDDP treatment alone. These results suggest that LT might act scavenging reactive species and also inducing the expression of genes related to a better antioxidant response, highlighting the improvement of oxygen transport. This improved redox state of the cell through LT treatment could be related to the antigenotoxic and antioxidant effects observed.

Overexpression Of Ucp1 In Tobacco Induces Mitochondrial Biogenesis And Amplifies A Broad Stress Response.

Uncoupling protein one (UCP1) is a mitochondrial inner membrane protein capable of uncoupling the electrochemical gradient from adenosine-5'-triphosphate (ATP) synthesis, dissipating energy as heat. UCP1 plays a central role in nonshivering thermogenesis in the brown adipose tissue (BAT) of hibernating animals and small rodents. A UCP1 ortholog also occurs in plants, and aside from its role in uncoupling respiration from ATP synthesis, thereby wasting energy, it plays a beneficial role in the plant response to several abiotic stresses, possibly by decreasing the production of reactive oxygen species (ROS) and regulating cellular redox homeostasis. However, the molecular mechanisms by which UCP1 is associated with stress tolerance remain unknown. Here, we report that the overexpression of UCP1 increases mitochondrial biogenesis, increases the uncoupled respiration of isolated mitochondria, and decreases cellular ATP concentration. We observed that the overexpression of UCP1 alters mitochondrial bioenergetics and modulates mitochondrial-nuclear communication, inducing the upregulation of hundreds of nuclear- and mitochondrial-encoded mitochondrial proteins. Electron microscopy analysis showed that these metabolic changes were associated with alterations in mitochondrial number, area and morphology. Surprisingly, UCP1 overexpression also induces the upregulation of hundreds of stress-responsive genes, including some involved in the antioxidant defense system, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione-S-transferase (GST). As a consequence of the increased UCP1 activity and increased expression of oxidative stress-responsive genes, the UCP1-overexpressing plants showed reduced ROS accumulation. These beneficial metabolic effects may be responsible for the better performance of UCP1-overexpressing lines in low pH, high salt, high osmolarity, low temperature, and oxidative stress conditions. Overexpression of UCP1 in the mitochondrial inner membrane induced increased uncoupling respiration, decreased ROS accumulation under abiotic stresses, and diminished cellular ATP content. These events may have triggered the expression of mitochondrial and stress-responsive genes in a coordinated manner. Because these metabolic alterations did not impair plant growth and development, UCP1 overexpression can potentially be used to create crops better adapted to abiotic stress conditions.

Taurine Supplementation Reduces Blood Pressure And Prevents Endothelial Dysfunction And Oxidative Stress In Post-weaning Protein-restricted Rats.

Taurine is a sulfur-containing amino acid that exerts protective effects on vascular function and structure in several models of cardiovascular diseases through its antioxidant and anti-inflammatory properties. Early protein malnutrition reprograms the cardiovascular system and is linked to hypertension in adulthood. This study assessed the effects of taurine supplementation in vascular alterations induced by protein restriction in post-weaning rats. Weaned male Wistar rats were fed normal- (12%, NP) or low-protein (6%, LP) diets for 90 days. Half of the NP and LP rats concomitantly received 2.5% taurine supplementation in the drinking water (NPT and LPT, respectively). LP rats showed elevated systolic, diastolic and mean arterial blood pressure versus NP rats; taurine supplementation partially prevented this increase. There was a reduced relaxation response to acetylcholine in isolated thoracic aortic rings from the LP group that was reversed by superoxide dismutase (SOD) or apocynin incubation. Protein expression of p47phox NADPH oxidase subunit was enhanced, whereas extracellular (EC)-SOD and endothelial nitric oxide synthase phosphorylation at Ser 1177 (p-eNOS) were reduced in aortas from LP rats. Furthermore, ROS production was enhanced while acetylcholine-induced NO release was reduced in aortas from the LP group. Taurine supplementation improved the relaxation response to acetylcholine and eNOS-derived NO production, increased EC-SOD and p-eNOS protein expression, as well as reduced ROS generation and p47phox expression in the aortas from LPT rats. LP rats showed an increased aortic wall/lumen ratio and taurine prevented this remodeling through a reduction in wall media thickness. Our data indicate a protective role of taurine supplementation on the high blood pressure, endothelial dysfunction and vascular remodeling induced by post-weaning protein restriction. The beneficial vascular effect of taurine was associated with restoration of vascular redox homeostasis and improvement of NO bioavailability.

Ki-1/57 And Cgi-55 Ectopic Expression Impact Cellular Pathways Involved In Proliferation And Stress Response Regulation.

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

Oxidative Stress And Susceptibility To Mitochondrial Permeability Transition Precedes The Onset Of Diabetes In Autoimmune Non-obese Diabetic Mice.

Beta cell destruction in type 1 diabetes (TID) is associated with cellular oxidative stress and mitochondrial pathway of cell death. The aim of this study was to determine whether oxidative stress and mitochondrial dysfunction are present in T1D model (non-obese diabetic mouse, NOD) and if they are related to the stages of disease development. NOD mice were studied at three stages: non-diabetic, pre-diabetic, and diabetic and compared with age-matched Balb/c mice. Mitochondria respiration rates measured at phosphorylating and resting states in liver and soleus biopsies and in isolated liver mitochondria were similar in NOD and Balb/c mice at the three disease stages. However, NOD liver mitochondria were more susceptible to calcium-induced mitochondrial permeability transition as determined by cyclosporine-A-sensitive swelling and by decreased calcium retention capacity in all three stages of diabetes development. Mitochondria H2O2 production rate was higher in non-diabetic, but unaltered in pre-diabetic and diabetic NOD mice. The global cell reactive oxygen species (ROS), but not specific mitochondria ROS production, was significantly increased in NOD lymphomononuclear and stem cells in all disease stages. In addition, marked elevated rates of 2',7'-dichlorodihydrofluorescein (H2DCF) oxidation were observed in pancreatic islets from non-diabetic NOD mice. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and lipidomic approach, we identified oxidized lipid markers in NOD liver mitochondria for each disease stage, most of them being derivatives of diacylglycerols and phospholipids. These results suggest that the cellular oxidative stress precedes the establishment of diabetes and may be the cause of mitochondrial dysfunction that is involved in beta cell death.

Leaf-, Panel- And Latex-expressed Sequenced Tags From The Rubber Tree (hevea Brasiliensis) Under Cold-stressed And Suboptimal Growing Conditions: The Development Of Gene-targeted Functional Markers For Stress Response.

Hevea brasiliensis is a native species of the Amazon Basin of South America and the primary source of natural rubber worldwide. Due to the occurrence of South American Leaf Blight disease in this area, rubber plantations have been extended to suboptimal regions. Rubber tree breeding is time-consuming and expensive, but molecular markers can serve as a tool for early evaluation, thus reducing time and costs. In this work, we constructed six different cDNA libraries with the aim of developing gene-targeted molecular markers for the rubber tree. A total of 8,263 reads were assembled, generating 5,025 unigenes that were analyzed; 912 expressed sequence tags (ESTs) represented new transcripts, and two sequences were highly up-regulated by cold stress. These unigenes were scanned for microsatellite (SSR) regions and single nucleotide polymorphisms (SNPs). In total, 169 novel EST-SSR markers were developed; 138 loci were polymorphic in the rubber tree, and 98 % presented transferability to six other Hevea species. Locus duplication was observed in H. brasiliensis and other species. Additionally, 43 SNP markers in 13 sequences that showed similarity to proteins involved in stress response, latex biosynthesis and developmental processes were characterized. cDNA libraries are a rich source of SSR and SNP markers and enable the identification of new transcripts. The new markers developed here will be a valuable resource for linkage mapping, QTL identification and other studies in the rubber tree and can also be used to evaluate the genetic variability of other Hevea species, which are valuable assets in rubber tree breeding.

Photoelastic Analysis Of Fixed Partial Prosthesis Crown Height And Implant Length On Distribution Of Stress In Two Dental Implant Systems.

The aim of this study was to evaluate by photoelastic analysis stress distribution on short and long implants of two dental implant systems with 2-unit implant-supported fixed partial prostheses of 8 mm and 13 mm heights. Sixteen photoelastic models were divided into 4 groups: I: long implant (5 × 11 mm) (Neodent), II: long implant (5 × 11 mm) (Bicon), III: short implant (5 × 6 mm) (Neodent), and IV: short implants (5 × 6 mm) (Bicon). The models were positioned in a circular polariscope associated with a cell load and static axial (0.5 Kgf) and nonaxial load (15°, 0.5 Kgf) were applied to each group for both prosthetic crown heights. Three-way ANOVA was used to compare the factors implant length, crown height, and implant system (α = 0.05). The results showed that implant length was a statistically significant factor for both axial and nonaxial loading. The 13 mm prosthetic crown did not result in statistically significant differences in stress distribution between the implant systems and implant lengths studied, regardless of load type (P > 0.05). It can be concluded that short implants showed higher stress levels than long implants. Implant system and length was not relevant factors when prosthetic crown height were increased.