Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21- Mediated Early Senescence Signalling

Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level.     

Regulatory effects of endogenous protease inhibitors in acute lung inflammatory injury

Inflammatory lung injury is probably regulated by the balance between proteases and protease inhibitors together with oxidants and antioxidants, and proinflammatory and anti-inflammatory cytokines. Rat tissue inhibitor of metalloprotease-2 (TIMP-2) and secreted leukoprotease inhibitor (SLPI) were cloned, expressed, and shown to be up-regulated at the levels of mRNA and protein during lung inflammation in rats induced by deposition of IgG immune complexes. Using immunoaffinity techniques, endogenous TIMP-2 in the inflamed lung was shown to exist as a complex with 72- and 92-kDa metalloproteinases (MMP-2 and MMP-9). In inflamed lung both TIMP-2 and SLPI appeared to exist as enzyme inhibitor complexes. Lung expression of both TIMP-2 and SLPI appeared to involve endothelial and epithelial cells as well as macrophages. To assess how these endogenous inhibitors might affect the lung inflammatory response, animals were treated with polyclonal rabbit Abs to rat TIMP-2 or SLPI. This intervention resulted in significant intensification of lung injury (as revealed by extravascular leak of albumin) and substantially increased neutrophil accumulation, as determined by cell content in bronchoalveolar lavage (BAL) fluids. These events were correlated with increased levels of C5a-related chemotactic activity in BAL fluids, while BAL levels of TNF-alpha and chemokines were not affected by treatment with anti-TIMP-2 or anti-SLPI. The data suggest that endogenous TIMP-2 and SLPI dynamically regulate the intensity of lung inflammatory injury, doing so at least in part by affecting the generation of the inflammatory mediator, C5a.     

The cellular immune response to Mycobacterium tuberculosis infection in the guinea pig

Pulmonary tuberculosis in guinea pigs is an extremely useful model for drug and vaccine testing due to the fact that its pathological disease process is similar to that present in humans. Progress in this field has been hindered because the tools necessary to undertake a complete immunological analysis of the guinea pig cellular immune response against Mycobacterium tuberculosis have been lacking. In this study, we combined a new flow cytometric gating strategy with immunohistochemistry to track T cells, B cells, and the MIL4 Ab, which detects both guinea pig heterophils (neutrophils) and eosinophils, to provide the first documentation of the kinetics of influx and positioning of these cell populations. The results show that the responding T cells are mostly CD4 cells and that after day 30 of the infection numbers of these cells in the lungs drops dramatically. These appear to be replaced by a steady increase in B cells and granulocytes which was associated with worsening lung pathology. These data reveal new information about the cellular phenotypes which mediate protective immunity or host immunopathogenesis during M. tuberculosis infection in this key animal model.     

A Cross-Site Comparison of Factors Influencing Soil Nitrification Rates in Northeastern USA Forested Watersheds

Elevated N deposition is continuing on many forested landscapes around the world and our understanding of ecosystem response is incomplete. Soil processes, especially nitrification, are critical. Many studies of soil N transformations have focused on identifying relationships within a single watershed but these results are often not transferable. We studied 10 small forested research watersheds in the northeastern USA to determine if there were common factors related to soil ammonification and nitrification. Vegetation varied between mixed northern hardwoods and mixed conifers. Watershed surface soils (Oa or A horizons) were sampled at grid or transect points and analyzed for a suite of chemical characteristics. At each sampling point, vegetation and topographic metrics (field and GIS-based) were also obtained. Results were examined by watershed averages (n = 10), seasonal/watershed averages (n = 28), and individual sampling points (n = 608). Using both linear and tree regression techniques, the proportion of conifer species was the single best predictor of nitrification rates, with lower rates at higher conifer dominance. Similar to other studies, the soil C/N ratio was also a good predictor and was well correlated with conifer dominance. Unlike other studies, the presence of Acer saccharum was not by itself a strong predictor, but was when combined with the presence of Betula alleghaniensis. Topographic metrics (slope, aspect, relative elevation, and the topographic index) were not related to N transformation rates across the watersheds. Although found to be significant in other studies, neither soil pH, Ca nor Al was related to nitrification. Results showed a strong relationship between dominant vegetation, soil C, and soil C/N. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Long-range regulatory synergy is required to allow control of the TAC1 locus by MEK/ERK signalling in sensory neurones

Changes in the expression of the neuropeptide substance P (SP) in different populations of sensory neurones are associated with the progression of chronic inflammatory disease. Thus, understanding the genomic and cellular mechanisms driving the expression of the TAC1 gene, which encodes SP, in sensory neurones is essential to understanding its role in inflammatory disease. We used a novel combination of computational genomics, primary-cell culture and mouse transgenics to determine the genomic and cellular mechanisms that control the expression of TAC1 in sensory neurones. Intriguingly, we demonstrated that the promoter of the TAC1 gene must act in synergy with a remote enhancer, identified using comparative genomics, to respond to MAPK signalling that modulates the expression of TAC1 in sensory neurones. We also reveal that noxious stimulation of sensory neurones triggers this synergy in larger diameter sensory neurones--an expression of SP associated with hyperalgesia. This noxious stimulation of TAC1 enhancer-promotor synergy could be strongly blocked by antagonism of the MEK pathway. This study provides a unique insight into the role of long-range enhancer-promoter synergy and selectivity in the tissue-specific response of promoters to specific signal transduction pathways and suggests a possible new avenue for the development of novel anti-inflammatory therapies.     

The plastic fly: the effect of sustained fluctuations in adult food supply on life‐history traits

Many adult traits in Drosophila melanogaster show phenotypic plasticity, and the effects of diet on traits such as lifespan and reproduction are well explored. Although plasticity in response to food is still present in older flies, it is unknown how sustained environmental variation affects life‐history traits. Here, we explore how such life‐long fluctuations of food supply affect weight and survival in groups of flies and affect weight, survival and reproduction in individual flies. In both experiments, we kept adults on constant high or low food and compared these to flies that experienced fluctuations of food either once or twice a week. For these ‘yoyo’ groups, the initial food level and the duration of the dietary variation differed during adulthood, creating four ‘yoyo’ fly groups. In groups of flies, survival and weight were affected by adult food. However, for individuals, survival and reproduction, but not weight, were affected by adult food, indicating that single and group housing of female flies affects life‐history trajectories. Remarkably, both the manner and extent to which life‐history traits varied in relation to food depended on whether flies initially experienced high or low food after eclosion. We therefore conclude that the expression of life‐history traits in adult life is affected not only by adult plasticity, but also by early adult life experiences. This is an important but often overlooked factor in studies of life‐history evolution and may explain variation in life‐history experiments.     

Effects of ethanol on the lipid composition of bovine vascular smooth muscle cells in culture

Ethanol (50 mM) had no effect on the growth rate or viability of arterial smooth muscle cells over 3.5 days. The cholesterol:phospholipid ratio of the cells was unchanged after 7 days exposure. The major phospholipid components phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were unchanged by ethanol exposure. Sphingomyelin content fell significantly within 12 hr. There were major changes in the fatty acid composition of the phospholipids with a reduction in saturated fatty acids and an increase in unsaturated fatty acids.     

An endogenous inhibitor of indoleamine-N-methyltransferase in cerebrospinal fluid

Human cerebrospinal fluid (CSF) has been found to contain a potent inhibitor of rabbit lung indoleamine-N-methyltransferase. Inhibition is non-competitive with respect to both the methyl donor, S-adenosylmethionine and the methyl acceptor, N-methyltryptamine. The inhibitor is dialyzable and heat stable. The inhibitor is not specific for INMT, and can inhibit the activity of hydroxyindole-O-methyltransferase and phenylethanolamine-N-methyltransferase. The molecular nature of the inhibitor is unknown. Data presented indicate that it is not one of the reaction products, another indoleamine or a peptide.