Firefly Luciferase and Rluc8 Exhibit Differential Sensitivity to Oxidative Stress in Apoptotic Cells

Over the past decade, firefly Luciferase (fLuc) has been used in a wide range of biological assays, providing insight into gene regulation, protein-protein interactions, cell proliferation, and cell migration. However, it has also been well established that fLuc activity can be highly sensitive to its surrounding environment. In this study, we found that when various cancer cell lines (HeLa, MCF-7, and 293T) stably expressing fLuc were treated with staurosporine (STS), there was a rapid loss in bioluminescence. In contrast, a stable variant of Renilla luciferase (RLuc), RLuc8, exhibited significantly prolonged functionality under the same conditions. To identify the specific underlying mechanism(s) responsible for the disparate sensitivity of RLuc8 and fLuc to cellular stress, we conducted a series of inhibition studies that targeted known intracellular protein degradation/modification pathways associated with cell death. Interestingly, these studies suggested that reactive oxygen species, particularly hydrogen peroxide (H2O2), was responsible for the diminution of fLuc activity. Consistent with these findings, the direct application of H2O2 to HeLa cells also led to a reduction in fLuc bioluminescence, while H2O2 scavengers stabilized fLuc activity. Comparatively, RLuc8 was far less sensitive to ROS. These observations suggest that fLuc activity can be substantially altered in studies where ROS levels become elevated and can potentially lead to ambiguous or misleading findings

Hydrogen peroxide-induced cell and tissue injury: Protective effects of Mn 2+

Recent evidence indicates that under in vitro conditions, superoxide anion and hydrogen peroxide (H 2 O 2 ) are unstable in the presence of manganese ion (Mn 2+ ). The current studies snow that in the presence of Mn 2+ , H 2 O 2 -mediated injury of endothelial cells is greatly attenuated. A source of bicarbonate ion and amino acid is required for Mn 2+ to exert its protective effects. Injury by phorbol ester-activated neutrophils is also attenuated under the same conditions. EDTA reverses the protective effects. Acute lung injury produced in vivo in rats by intratracheal instillation of glucose-glucose oxidase is almost completely blocked in rats treated with Mn 2+ and glycine. Conversely, treatment of rats with EDTA, a chelator of Mn 2+ , markedly accentuates lung injury caused by glucose-glucose oxidase. These data are consistent with the findings of others that Mn 2+ can facilitate direct oxidation of amino acids with concomitant H 2 O 2 disproportionation. This could form the basis of a new therapeutic approach against oxygen radical-mediated tissue injury.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44505/1/10753_2004_Article_BF00917314.pd

Energy Reallocation to Breeding Performance through Improved Nest Building in Laboratory Mice.

Mice are housed at temperatures (20-26°C) that increase their basal metabolic rates and impose high energy demands to maintain core temperatures. Therefore, energy must be reallocated from other biological processes to increase heat production to offset heat loss. Supplying laboratory mice with nesting material may provide sufficient insulation to reduce heat loss and improve both feed conversion and breeding performance. Naïve C57BL/6, BALB/c, and CD-1breeding pairs were provided with bedding alone, or bedding supplemented with either 8g of Enviro-Dri, 8g of Nestlets, for 6 months. Mice provided with either nesting material built more dome-like nests than controls. Nesting material improved feed efficiency per pup weaned as well as pup weaning weight. The breeding index (pups weaned/dam/week) was higher when either nesting material was provided. Thus, the sparing of energy for thermoregulation of mice given additional nesting material may have been responsible for the improved breeding and growth of offspring

The Long Life of Birds: The Rat-Pigeon Comparison Revisited

The most studied comparison of aging and maximum lifespan potential (MLSP) among endotherms involves the 7-fold longevity difference between rats (MLSP 5y) and pigeons (MLSP 35y). A widely accepted theory explaining MLSP differences between species is the oxidative stress theory, which purports that reactive oxygen species (ROS) produced during mitochondrial respiration damage bio-molecules and eventually lead to the breakdown of regulatory systems and consequent death. Previous rat-pigeon studies compared only aspects of the oxidative stress theory and most concluded that the lower mitochondrial superoxide production of pigeons compared to rats was responsible for their much greater longevity. This conclusion is based mainly on data from one tissue (the heart) using one mitochondrial substrate (succinate). Studies on heart mitochondria using pyruvate as a mitochondrial substrate gave contradictory results. We believe the conclusion that birds produce less mitochondrial superoxide than mammals is unwarranted

Rules Governing Selective Protein Carbonylation

BACKGROUND:Carbonyl derivatives are mainly formed by direct metal-catalysed oxidation (MCO) attacks on the amino-acid side chains of proline, arginine, lysine and threonine residues. For reasons unknown, only some proteins are prone to carbonylation. METHODOLOGY/PRINCIPAL FINDINGS:we used mass spectrometry analysis to identify carbonylated sites in: BSA that had undergone in vitro MCO, and 23 carbonylated proteins in Escherichia coli. The presence of a carbonylated site rendered the neighbouring carbonylatable site more prone to carbonylation. Most carbonylated sites were present within hot spots of carbonylation. These observations led us to suggest rules for identifying sites more prone to carbonylation. We used these rules to design an in silico model (available at http://www.lcb.cnrs-mrs.fr/CSPD/), allowing an effective and accurate prediction of sites and of proteins more prone to carbonylation in the E. coli proteome. CONCLUSIONS/SIGNIFICANCE:We observed that proteins evolve to either selectively maintain or lose predicted hot spots of carbonylation depending on their biological function. As our predictive model also allows efficient detection of carbonylated proteins in Bacillus subtilis, we believe that our model may be extended to direct MCO attacks in all organisms

Rule-Based Cell Systems Model of Aging using Feedback Loop Motifs Mediated by Stress Responses

Investigating the complex systems dynamics of the aging process requires integration of a broad range of cellular processes describing damage and functional decline co-existing with adaptive and protective regulatory mechanisms. We evolve an integrated generic cell network to represent the connectivity of key cellular mechanisms structured into positive and negative feedback loop motifs centrally important for aging. The conceptual network is casted into a fuzzy-logic, hybrid-intelligent framework based on interaction rules assembled from a priori knowledge. Based upon a classical homeostatic representation of cellular energy metabolism, we first demonstrate how positive-feedback loops accelerate damage and decline consistent with a vicious cycle. This model is iteratively extended towards an adaptive response model by incorporating protective negative-feedback loop circuits. Time-lapse simulations of the adaptive response model uncover how transcriptional and translational changes, mediated by stress sensors NF-κB and mTOR, counteract accumulating damage and dysfunction by modulating mitochondrial respiration, metabolic fluxes, biosynthesis, and autophagy, crucial for cellular survival. The model allows consideration of lifespan optimization scenarios with respect to fitness criteria using a sensitivity analysis. Our work establishes a novel extendable and scalable computational approach capable to connect tractable molecular mechanisms with cellular network dynamics underlying the emerging aging phenotype

Oxidative Modifications of Rat Liver Cell Components During Fasciola hepatica Infection

The aim of this paper was to assess the influence of Fasciola hepatica infection on oxidative modifications of rat liver cell components such as proteins and lipids. Wistar rats were infected per os with 30 metacercariae of F. hepatica. Activities and concentrations of liver damage markers were determined in the 4th, 7th, and 10th week postinfection (wpi). A decrease in antioxidant capacity of the host liver, manifested by a decrease in total antioxidant status (TAS), was observed. Diminution of antioxidant abilities resulted in enhanced oxidative modifications of lipids and proteins. F. hepatica infection enhanced lipid peroxidation, which was visible in the statistically significant increase in the level of different lipid peroxidation products such as conjugated dienes (CDs), lipid hydroperoxides (LOOHs), malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). The level of protein modification markers in the rat liver was also significantly changed and the most intensified changes were observed at seventh week postinfection. Concentration of carbonyl groups and dityrosine was significantly increased, whereas the level of tryptophan and sulfhydryl and amino groups was decreased. Changes in the antioxidant abilities of the liver and in the lipid and protein structure of the cell components resulted in destruction of the function of the liver. F. hepatica infection was accompanied by raising serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as markers of liver damage. A significant decrease in lysosomal as well as in the total activity of cathepsin B during fasciolosis was also observed

Oxidative protein labeling in mass-spectrometry-based proteomics

Oxidation of proteins and peptides is a common phenomenon, and can be employed as a labeling technique for mass-spectrometry-based proteomics. Nonspecific oxidative labeling methods can modify almost any amino acid residue in a protein or only surface-exposed regions. Specific agents may label reactive functional groups in amino acids, primarily cysteine, methionine, tyrosine, and tryptophan. Nonspecific radical intermediates (reactive oxygen, nitrogen, or halogen species) can be produced by chemical, photochemical, electrochemical, or enzymatic methods. More targeted oxidation can be achieved by chemical reagents but also by direct electrochemical oxidation, which opens the way to instrumental labeling methods. Oxidative labeling of amino acids in the context of liquid chromatography(LC)–mass spectrometry (MS) based proteomics allows for differential LC separation, improved MS ionization, and label-specific fragmentation and detection. Oxidation of proteins can create new reactive groups which are useful for secondary, more conventional derivatization reactions with, e.g., fluorescent labels. This review summarizes reactions of oxidizing agents with peptides and proteins, the corresponding methodologies and instrumentation, and the major, innovative applications of oxidative protein labeling described in selected literature from the last decade