Viral transport of DNA damage that mimics a stalled replication fork.

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.

Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage: A challenge for neuroprotection.

There is currently no approved neuroprotective pharmacotherapy for acute conditions such as stroke and cerebral asphyxia. One of the reasons for this may be the multiplicity of cell death mechanisms, because inhibition of a particular mechanism leaves the brain vulnerable to alternative ones. It is therefore essential to understand the different cell death mechanisms and their interactions. We here review the multiple signaling pathways underlying each of the three main morphological types of cell death - apoptosis, autophagic cell death and necrosis - emphasizing their importance in the neuronal death that occurs during cerebral ischemia and hypoxia-ischemia, and we analyze the interactions between the different mechanisms. Finally, we discuss the implications of the multiplicity of cell death mechanisms for the design of neuroprotective strategies.

Oxidative folding of synthetic polypeptides S-protected as tert-butylthio derivatives.

A new method for oxidative folding of synthetic polypeptides assembled by stepwise solid phase synthesis is introduced. Folding is obtained in excellent yields by reacting S-tert-butylthiolated polypeptides with a 100-fold molar excess of cysteine at 37 degrees C in a slightly alkaline buffer containing chaotropic salts, and in the presence of air-oxygen. This novel protocol has been applied to the folding of S-tert-butylthiolated human thymus and activation-regulated chemokine (hu-TARC) derivatives as well as to larger segments of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Folded P. falciparum polypeptides have been used as substrates of endoproteinase Glu-C (Glu-C) and endoproteinase Asp-N (Asp-N) in an attempt to identify their disulfide connectivities. Particular practical advantages of the present method are (i) easy purification and storage of the S-protected peptide derivatives, (ii) elimination of the risk of cysteine alkylation during the acidolytic cleavage deprotection and resin cleavage steps, (iii) possibility to precisely evaluate the extent of folding and disulfide bond formation by mass spectrometry, and (iv) facile recovery of the final folded product.

The impact of pre- and post-natal contexts on immunity, glucocorticoids and oxidative stress resistance in wild and domesticated grey partridges

Genetic background, prenatal and post-natal early-life conditions influence the development of interconnected physiological systems and thereby shape the phenotype. Certain combinations of genotypes and pre- and post-natal conditions may provide higher fitness in a specific environmental context. Here, we investigated how grey partridges Perdix perdix of two strains (wild and domesticated) cope physiologically with pre- and post-natal predictable vs. unpredictable food supply. Food unpredictability occurs frequently in wild environments and requires physiological and behavioural adjustments. Well-orchestrated and efficient physiological systems are presumably more vital in a wild environment as compared to captivity. We thus predicted that wild-strain grey partridges have a stronger immunity, glucocorticoid (GC) stress response and oxidative stress resistance (OSR) than domesticated birds, which have undergone adaptations to captivity. We also predicted that wild-strain birds react more strongly to environmental stimuli and, when faced with harsh prenatal conditions, are better able to prepare their offspring for similarly poor post-natal conditions than birds of domesticated origin. We found that wild-strain offspring were physiologically better prepared for stressful situations as compared to the domesticated strain. They had a high GC stress response and a high OSR when kept under predictable food supply. Wild-strain parents reacted to prenatal unpredictable food supply by lowering their offspring's GC stress response, which potentially lowered GC-induced oxidative pressure. No such pattern was evident in the domesticated birds. Irrespective of strain and prenatal feeding scheme, post-natal unpredictable food supply boosted immune indices, and GC stress response was negatively related to antibody response in females and to mitochondrial superoxide production. Wild-strain grey partridge showed fitness-relevant physiological advantages and appeared to prepare their offspring for the prospective environment. Negative relationships between GC stress response, immunity and oxidative indices imply a pivotal role of an organism's oxidative balance and support the importance of considering multiple physiological systems simultaneously.

Evaluation of oxidative stress parameters and metabolic activities of nurses working day and night shifts

The aim of this study was to evaluate the oxidative stress and metabolic activities of nurses working day and night shifts. Intensive care unit (ICU) (n=70) and ordinary service (OS) nurses (n=70) were enrolled in the study. Just before and the end of the shifts, blood samples were obtained to measure the participants' oxidative stress parameters. Metabolic activities were analyzed using the SenseWear Armband. Oxidative stress parameters were increased at the end of the shifts for all OS and ICU nurses compared to the beginning of the shifts. Compared to the OS nurses, the ICU nurses' TAS, TOS, and OSI levels were not significantly different at the end of the day and night shifts. The metabolic activities of the OS and ICU nurses were found to be similar. As a result, the OS and ICU nurses' oxidative stress parameters and metabolic activities were not different, and all of the nurses experienced similar effects from both the day and night shifts.

Discounting Spotted Apples: Investigating Consumers’ Willingness to Accept Cosmetic Damage in an Organic Product, November 2006

Organic producers have limited methods of avoiding plant diseases that result in cosmetic damage to produce. Therefore, the appearance of organic produce is often less than perfect. We use an experimental auction to investigate how cosmetic damage affects consumers’ willingness to pay for organic apples. We find that 75% of the participants are willing to pay more for organic than for conventional apples given identical appearance. However, at the first sight of any imperfection in the appearance of the organic apples, this segment is significantly reduced. Furthermore, we find that there is a significant effect of interaction between cosmetic damage and product methods. Even though most consumers say they buy organic products to avoid pesticides, we find that cosmetic damage has a larger impact on the willingness to pay for organic apples than for conventional apples.

Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia.

BACKGROUND: Acute exposure to high altitude stimulates free radical formation in lowlanders, yet whether this persists during chronic exposure in healthy, well-adapted and maladapted highlanders suffering from chronic mountain sickness (CMS) remains to be established. METHODS: Oxidative-nitrosative stress (as determined by the presence of the biomarkers ascorbate radical [A •- ], via electron paramagnetic resonance spectroscopy, and nitrite [NO 2 2 ], via ozone-based chemiluminescence) was assessed in venous blood of 25 male highlanders in Bolivia living at 3,600 m with CMS (n 5 13, CMS 1 ) and without CMS (n 5 12, CMS 2 ). Twelve age- and activity-matched, healthy, male lowlanders were examined at sea level and during acute hypoxia. We also measured fl ow-mediated dilatation (FMD), arterial stiffness defined by augmentation index normalized for a heart rate of 75 beats/min (AIx-75), and carotid intima-media thickness (IMT). RESULTS: Compared with normoxic lowlanders, oxidative-nitrosative stress was moderately increased in the CMS 2 group ( P , .05), as indicated by elevated A •- (3,191 457 arbitrary units [AU] vs 2,640 445 AU) and lower NO 2 2 (206 55 nM vs 420 128 nM), whereas vascular function remained preserved. This was comparable to that observed during acute hypoxia in lowlanders in whom vascular dysfunction is typically observed. In contrast, this response was markedly exaggerated in CMS 1 group (A •- , 3,765 429 AU; NO 2 2 , 148 50 nM) compared with both the CMS 2 group and lowlanders ( P , .05). This was associated with systemic vascular dysfunction as indicated by lower ( P , .05 vs CMS 2 ) FMD (4.2% 0.7% vs 7.6% 1.7%) and increased AIx-75 (23% 8% vs 12% 7%) and carotid IMT (714 127 m M vs 588 94 m M). CONCLUSIONS: Healthy highlanders display a moderate, sustained elevation in oxidative-nitrosative stress that, unlike the equivalent increase evoked by acute hypoxia in healthy lowlanders, failed to affect vascular function. Its more marked elevation in patients with CMS may contribute to systemic vascular dysfunction.

Assessment of diesel exhaust particulate exposure and surface characteristics in association with levels of oxidative stress biomarkers

Exposure to PM10 and PM2.5 (particulate matter with aerodynamic diameter smaller than 10 μm and 2.5 μm, respectively) is associated with a range of adverse health effects, including cancer, pulmonary and cardiovascular diseases. Surface characteristics (chemical reactivity, surface area) are considered of prime importance to understand the mechanisms which lead to harmful effects. A hypothetical mechanism to explain these adverse effects is the ability of components (organics, metal ions) adsorbed on these particles to generate Reactive Oxygen Species (ROS), and thereby to cause oxidative stress in biological systems (Donaldson et al., 2003). ROS can attack almost any cellular structure, like DNA or cellular membrane, leading to the formation of a wide variety of degradation products which can be used as a biomarker of oxidative stress. The aim of the present research project is to test whether there is a correlation between the exposure to Diesel Exhaust Particulate (DEP) and the oxidative stress status. For that purpose, a survey has been conducted in real occupational situations where workers were exposed to DEP (bus depots). Different exposure variables have been considered: - particulate number, size distribution and surface area (SMPS); - particulate mass - PM2.5 and PM4 (gravimetry); - elemental and organic carbon (coulometry); - total adsorbed heavy metals - iron, copper, manganese (atomic adsorption); - surface functional groups present on aerosols (Knudsen flow reactor). (Demirdjian et al., 2005). Several biomarkers of oxidative stress (8-hydroxy-2'-deoxyguanosine and several aldehydes) have been determined either in urine or serum of volunteers. Results obtained during the sampling campaign in several bus depots indicated that the occupational exposure to particulates in these places was rather low (40-50 μg/m3 for PM4). Size distributions indicated that particles are within the nanometric range. Surface characteristics of sampled particles varied strongly, depending on the bus depot. They were usually characterized by high carbonyl and low acidic sites content. Among the different biomarkers which have been analyzed within the framework of this study, mean levels of 8- hydroxy-2'-deoxyguanosine and several aldehydes (hexanal, heptanal, octanal, nonanal) increased during two consecutive days of exposure for non-smokers. In order to bring some insight into the relation between the particulate characteristics and the formation of ROS by-products, biomarkers levels will be discussed in relation with exposure variables.

Ammonia toxicity to the brain.

Hyperammonemia can be caused by various acquired or inherited disorders such as urea cycle defects. The brain is much more susceptible to the deleterious effects of ammonium in childhood than in adulthood. Hyperammonemia provokes irreversible damage to the developing central nervous system: cortical atrophy, ventricular enlargement and demyelination lead to cognitive impairment, seizures and cerebral palsy. The mechanisms leading to these severe brain lesions are still not well understood, but recent studies show that ammonium exposure alters several amino acid pathways and neurotransmitter systems, cerebral energy metabolism, nitric oxide synthesis, oxidative stress and signal transduction pathways. All in all, at the cellular level, these are associated with alterations in neuronal differentiation and patterns of cell death. Recent advances in imaging techniques are increasing our understanding of these processes through detailed in vivo longitudinal analysis of neurobiochemical changes associated with hyperammonemia. Further, several potential neuroprotective strategies have been put forward recently, including the use of NMDA receptor antagonists, nitric oxide inhibitors, creatine, acetyl-L-carnitine, CNTF or inhibitors of MAPKs and glutamine synthetase. Magnetic resonance imaging and spectroscopy will ultimately be a powerful tool to measure the effects of these neuroprotective approaches.

Oxidative and glycogenolytic cCapacities within the developing chick heart.

Cardiac morphogenesis and function are known to depend on both aerobic and anaerobic energy-producing pathways. However, the relative contribution of mitochondrial oxidation and glycogenolysis, as well as the determining factors of oxygen demand in the distinct chambers of the embryonic heart, remains to be investigated. Spontaneously beating hearts isolated from stage 11, 20, and 24HH chick embryos were maintained in vitro under controlled metabolic conditions. O(2) uptake and glycogenolytic rate were determined in atrium, ventricle, and conotruncus in the absence or presence of glucose. Oxidative capacity ranged from 0.2 to 0.5 nmol O(2)/(h.microg protein), did not depend on exogenous glucose, and was the highest in atria at stage 20HH. However, the highest reserves of oxidative capacity, assessed by mitochondrial uncoupling, were found at the youngest stage and in conotruncus, representing 75 to 130% of the control values. At stage 24HH, glycogenolysis in glucose-free medium was 0.22, 0.17, and 0.04 nmol glucose U(h.microg protein) in atrium, ventricle, and conotruncus, respectively. Mechanical loading of the ventricle increased its oxidative capacity by 62% without altering glycogenolysis or lactate production. Blockade of glycolysis by iodoacetate suppressed lactate production but modified neither O(2) nor glycogen consumption in substrate-free medium. These findings indicate that atrium is the cardiac chamber that best utilizes its oxidative and glycogenolytic capacities and that ventricular wall stretch represents an early and major determinant of the O(2) uptake. Moreover, the fact that O(2) and glycogen consumptions were not affected by inhibition of glyceraldehyde-3-phosphate dehydrogenase provides indirect evidence for an active glycerol-phosphate shuttle in the embryonic cardiomyocytes.

Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells.

Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly.

Toxic effects of brake wear particles on epithelial lung cells in vitro

Background: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. Results: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. Conclusion: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress. [Authors]

Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, and inflammatory and cell death signaling pathways in diabetic cardiomyopathy.

Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose. Background Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans. Methods Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy, and flow cytometry. Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrative stress, nuclear factor-kappa B and mitogen-activated protein kinase (c-Jun N-terminal kinase, p-38, p38 alpha) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), tumor necrosis factor-alpha, markers of fibrosis (transforming growth factor-beta, connective tissue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 and poly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyl transferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclear factor-kappa B activation, and cell death in primary human cardiomyocytes. Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death and fibrosis. (J Am Coll Cardiol 2010;56:2115-25) (C) 2010 by the American College of Cardiology Foundation.

Development of the Crohn's disease digestive damage score, the Lémann score.

Crohn's disease (CD) is a chronic progressive destructive disease. Currently available instruments measure disease activity at a specific point in time. An instrument to measure cumulative structural damage to the bowel, which may predict long-term disability, is needed. The aim of this article is to outline the methods to develop an instrument that can measure cumulative bowel damage. The project is being conducted by the International Program to develop New Indexes in Crohn's disease (IPNIC) group. This instrument, called the Crohn's Disease Digestive Damage Score (the Lémann score), should take into account damage location, severity, extent, progression, and reversibility, as measured by diagnostic imaging modalities and the history of surgical resection. It should not be "diagnostic modality driven": for each lesion and location, a modality appropriate for the anatomic site (for example: computed tomography or magnetic resonance imaging enterography, and colonoscopy) will be used. A total of 24 centers from 15 countries will be involved in a cross-sectional study, which will include up to 240 patients with stratification according to disease location and duration. At least 120 additional patients will be included in the study to validate the score. The Lémann score is expected to be able to portray a patient's disease course on a double-axis graph, with time as the x-axis, bowel damage severity as the y-axis, and the slope of the line connecting data points as a measure of disease progression. This instrument could be used to assess the effect of various medical therapies on the progression of bowel damage. (Inflamm Bowel Dis 2011).