Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria

The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.     

Bowers on the savanna: display courts and mate choice in a lekking widowbird

Individual display courts, constructed by lekking male Jackson'swidowbirds, Euplectes jacksoni, were studied with respect tofemale mate choice. The court is a shaped tuft of grass, surroundedby a circular track on which displays and mating are performed.At the base of the tuft there are two cup-shaped recesses. Femalessit in front of, and often pluck at, one ofthese recesses whilemales display at the opposite side for about 1 min before theyapproach and attempt to copulate. The vast majority of femalesvisiting courts with experimentally destroyed or impaired recessesstayed shorter than the average time of male approach, whereasvisits to improved courts lasted longer than visits to intactcourts. All recorded copulations occurred at intact or improvedcourts. The display court is a weak predictor of male matingsuccess compared to other choice cues such as display rate andtail length, but females may require the court quality to exceeda certain stimulus threshold to accept the male. Court qualitycorrelates positively with male lek attendance and tail length.This paper discusses the implications of the results for sexualselection and considers the possibility that display courtsare ‘external sexual ornaments.’ From comparativedata on breeding behavior in related species, the display courtand its associated behaviors are suggested to originate frommale nest-initiation and female nest-inspection within an ancestralstate of resource-defense polygyny.     

Growth response of altitudinal ecotypes of mountain birch to temperature and fertilisation

High- and low-altitude ecotypes of mountain birch (Betula pubescens ssp. czerepanovii) showed clear differences in their responses to various experimental conditions, including two temperature regimes and four fertilisation rates. There was, however, no simple way to characterise the elevational ecotypes in terms of relative growth rate, nitrogen (N) productivity, or root N uptake rate. The leaf N concentration was generally higher in the high-altitude seedlings than in the low-altitude seedlings. At low temperature, high-altitude mountain birch maintained a relatively high growth rate by combining high root N uptake rate and high leaf N concentration with high N productivity. An increase in temperature and/or fertiliser rate resulted in a marginal increase in N productivity in the high-altitude seedlings but resulted in a strong increase in N productivity in the low-altitude seedlings. In parallel, increased temperature resulted in a pronounced decrease in leaf N concentration only in the low-altitude seedlings. Our results suggest that the weak growth response to increased temperature in high-altitude mountain birch is functionally related to high leaf N concentration. The high leaf N concentration of high-altitude mountain birch is genetically determined and has an adaptive value in a cold environment. This suggests that there is a trade-off between high N productivity at low temperature and a strong response of N productivity to temperature. Received: 21 March 1998 / Accepted: 1 December 1998     

Polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite Giardia

The polymeric Ig receptor (pIgR) is conserved in mammals and has an avian homologue, suggesting evolutionarily important functions in vertebrates. It transports multimeric IgA and IgM across polarized epithelia and is highly expressed in the intestine, yet little direct evidence exists for its importance in defense against common enteric pathogens. In this study, we demonstrate that pIgR can play a critical role in intestinal defense against the lumen-dwelling protozoan parasite Giardia, a leading cause of diarrheal disease. The receptor was essential for the eradication of Giardia when high luminal IgA levels were required. Clearance of Giardia muris, in which IgA plays a dominant role, was severely compromised in pIgR-deficient mice despite significant fecal IgA output at 10% of normal levels. In contrast, eradication of the human strain Giardia lamblia GS/M, for which adaptive immunity is less IgA dependent in mice, was unaffected by pIgR deficiency, indicating that pIgR had no physiologic role when lower luminal IgA levels were sufficient for parasite elimination. Immune IgA was greatly increased in the serum of pIgR-deficient mice, conferred passive protection against Giardia, and recognized several conserved giardial Ags, including ornithine carbamoyltransferase, arginine deiminase, alpha-enolase, and alpha- and beta-giardins, that are also detected in human giardiasis. Corroborative observations were made in mice lacking the J chain, which is required for pIgR-dependent transepithelial IgA transport. These results, together with prior data on pIgR-mediated immune neutralization of luminal cholera toxin, suggest that pIgR is essential in intestinal defense against pathogenic microbes with high-level and persistent luminal presence.     

Docking simulation with a purine nucleoside specific homology model of deoxycytidine kinase, a target enzyme for anticancer and antiviral therapy

5'-Phosphorylation, catalyzed by human deoxycytidine kinase (dCK), is a crucial step in the metabolic activation of anticancer and antiviral nucleoside antimetabolites, such as cytarabine (AraC), gemcitabine, cladribine (CdA), and lamivudine. Recently, crystal structures of dCK (dCKc) with various pyrimidine nucleosides as substrates have been reported. However, there is no crystal structure of dCK with a bound purine nucleoside, although purines are good substrates for dCK. We have developed a model of dCK (dCKm) specific for purine nucleosides based on the crystal structure of purine nucleoside bound deoxyguanosine kinase (dGKc) as the template. dCKm is essential for computer aided molecular design (CAMD) of novel anticancer and antiviral drugs that are based on purine nucleosides since these did not bind to dCKc in our docking experiments. The active site of dCKm was larger than that of dCKc and the amino acid (aa) residues of dCKm and dCKc, in particular Y86, Q97, D133, R104, R128, and E197, were not in identical positions. Comparative docking simulations of deoxycytidine (dC), cytidine (Cyd), AraC, CdA, deoxyadenosine (dA), and deoxyguanosine (dG) with dCKm and dCKc were carried out using the FlexX docking program. Only dC (pyrimidine nucleoside) docked into the active site of dCKc but not the purine nucleosides dG and dA. As expected, the active site of dCKm appeared to be more adapted to bind purine nucleosides than the pyrimidine nucleosides. While water molecules were essential for docking experiments using dCKc, the absence of water molecules in dCKm did not affect the ability to correctly dock various purine nucleosides.     

Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.     

Environmental impacts of forest production and supply of pulpwood: Spanish and Swedish case studies

Background, aim and scope  Forest operations use large amounts of energy, which must be considered when life cycle assessment (LCA) methodology is applied to forest products. Forest management practices differ considerably between countries and may also differ within a country. This paper aims to identify and compare the environmental burdens from forest operations in Sweden and Spain focused on pulpwood production and supply to pulp mills. Materials and methods  To perform the analysis, the main forest plantations were investigated as well as the most important tree species used in pulp mills in both countries: eucalyptus and, Norway spruce and Scots pine, were taken into account for the Spanish and Swedish case studies, respectively. Energy requirements for pulpwood production and supply to Spanish and Swedish pulp mills are evaluated in this paper. All forest operations from site preparation to extraction of felled wood to the delivery point at the roadside are included within the system boundaries as well as wood transport from forest landing to the pulp mill gate. Seedling and machinery production are excluded from the system boundaries due to lack of field data. The impact assessment phase was carried out according to the Swedish Environmental Management Council and, in particular, the impact categories assessed in forest and agricultural LCAs (global warming, acidification, eutrophication and photochemical oxidant formation) were analysed. SimaPro 7.10 software was used to perform the impact assessment stage. Results  Different types of wood are produced in both case studies: softwood in Sweden and hardwood in Spain, with higher production of round wood and biomass per hectare in Sweden. Total energy use for pulpwood production and supply are in a similar order of magnitude, up to 395 MJ and 370 MJ/m³ solid under bark in Spain and Sweden, respectively. Field operations, such as silviculture and logging, are more energy-intensive in the Spanish case study. However, secondary hauling of pulpwood to pulp mill requires more energy in the Swedish case study. These important differences are related to different forest management practices as well as to pulpwood supply to the pulp mill. The eventual imports of pulpwood, application of pesticides, thinning step or final felling considerably affects energy requirements, which are reflected on the environmental results. Discussion  Although differences between both case studies were observed, several stages were investigated: wood delivery to the pulp mill by road, harvesting and forwarding, contribute considerably to acidification, eutrophication and global warming potential in both cases. The type of wood, the machines used in forest operations (mechanised or motor-manual), the use of fossil fuels and the amount of wood produced influence the results. These differences must be kept in mind in comparative studies between such different countries. Conclusions  The results obtained in this work allow one to forecast the importance of forest operations in LCA of forest products (in this case, wood pulp) and the influence of energy use in the results. Special attention has been paid in the inventory analysis stage for both case studies. It is possible to gain a better environmental performance in both case studies if alternative practices are considered, mainly focused on site preparation and stand tending in the Spanish system and on pulpwood supply in the Swedish one. Recommendations and perspectives  This study provides useful information that can assist forest-based industries in the aim of increasing their sustainability. Future work will focus on the study of several transport alternatives of pulpwood supply including railway, road and ship. In addition, pulpwood processing in Spanish and Swedish paper pulp mills considered to be representative of the “state of art” will be carried out in order to get a complete picture of this kind of forest-based industry. In addition, the use of biofuels (such as forest biomass) instead of fossil fuels and CO₂ uptake of wood via photosynthesis will be carried out in order to have a complete perspective of forest ecosystems.