Exploring trophic niches and parasite communities of sympatric Arctic charr and brown trout populations of southern Norway

Hydrobiologia - Catchment-scale variation between lake habitats has the potential to simultaneously influence the trophic niche and parasite community of fish hosts. In this study, we investigated...     

Residues within the N-terminal domain of human topoisomerase I play a direct role in relaxation

All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, strand rotation, and ligation. A comparison with previously published studies showing no effect of deleting the first 174 or 190 amino acids of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux, J. J. (1999) J. Biol. Chem. 274, 32950-32960; Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F., Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G., Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme.     

The importance of structural transitions of the switch II region for the functions of elongation factor Tu on the ribosome

Elongation factor Tu (EF-Tu) undergoes a large conformational transition when switching from the GTP to GDP forms. Structural changes in the switch I and II regions in the G domain are particularly important for this rearrangement. In the switch II region, helix alpha2 is flanked by two glycine residues: Gly(83) in the consensus element DXXG at the N terminus and Gly(94) at the C terminus. The role of helix alpha2 was studied by pre-steady-state kinetic experiments using Escherichia coli EF-Tu mutants where either Gly(83), Gly(94), or both were replaced with alanine. The G83A mutation slows down the association of the ternary complex EF-Tu.GTP.aminoacyl-tRNA with the ribosome and abolishes the ribosome-induced GTPase activity of EF-Tu. The G94A mutation strongly impairs the conformational change of EF-Tu from the GTP- to the GDP-bound form and decelerates the dissociation of EF-Tu.GDP from the ribosome. The behavior of the double mutant is dominated by the G83A mutation. The results directly relate structural transitions in the switch II region to specific functions of EF-Tu on the ribosome.     

Use of cryopreserved peripheral mononuclear blood cells in biomonitoring

This study was performed to investigate the effect of storing blood samples by freezing on selected biomarkers and possible implications for biomonitoring. Comparative measurements were performed in order to investigate the use of cryopreserved vs. freshly separated peripheral mononuclear blood cells (PMBC) obtained from donor blood. Measurements of DNA-repair, mutant frequency, and subcell content were included. Samples for large biomonitoring studies are usually taken from study groups within a short time period of days/weeks and storing of study material for later analysis can be necessary. We measured the DNA repair activity as dimethylsulfate induced unscheduled DNA synthesis (UDS) in PMBC incubated with either autologous plasma or fetal bovine serum (FBS). Comparison of the hprt mutant frequency by the T cell cloning assay was made in parallel. Finally the content of B/T-lymphocytes and monocytes was measured in phytohemaglutinin (PHA)-stimulated cultures at different time intervals. The results showed a higher DNA repair activity in cryopreserved samples compared with fresh samples. We also found differences in mutant frequencies with higher values in fresh samples. A significant correlation of frequencies was seen when comparing fresh with cryopreserved samples. Furthermore we recommend fresh human plasma used in UDS incubation media.     

Hydrophobic thickness of fluid planar monooleylglycerol membranes maximally thinned by inversed micellisation

A procedure of making membranes of amphiphilic materials at the bottom of a U-shaped flexible plastic tube within an aqueous medium is described. The membranes were made sufficiently large in order for the annulus area to be neglected. Consequently the hydrophobic thickness of the membrane could be measured by a capacitance technique assuming the relative permittivity of the hydrophobic part of the bilayer. Introduction of an AC microvolt technique allowed manufacture of stable thick membranes by quenching the electroconstriction observed when DC electrical potentials in the millivolt range are used. By continuously monitoring the hydrophobic thickness and by use of the AC microvolt technique the membrane-thinning process by chemical means could be studied in isolation because the electroconstriction was quenched. The maximally thinned hydrophobic thickness of a monooleylglycerol membrane measured at 38 degrees C was found to be 25+/-1.2 A. Criteria and argumentation for maximal thinning of the membrane are put forward. A distinction between genuine and modified cholesterol was demonstrated to be possible by the described method.     

RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (γH2AX) foci, which indicate DNA double-strand breaks. The formation of γH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak γH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these γH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce γH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.     

Moisture Effects on Temperature Sensitivity of CO<Subscript>2</Subscript> Exchange in a Subarctic Heath Ecosystem

Carbon fluxes between natural ecosystems and the atmosphere have received increased attention in recent years due to the impact they have on climate. In order to investigate independently how soil moisture and temperature control carbon fluxes into and out of a dry subarctic dwarf shrub dominated heath, monoliths containing soil and plants were incubated at three different moisture levels and subjected to four different temperature levels between 7 and 20 °C. Ecosystem CO₂ exchange was monitored continuously day and night during the 16 to 18 days that each of three experiments lasted. Additionally, the carbon allocation pattern of the plants was investigated by labelling monoliths with ¹⁴CO₂ followed by harvest of above and below ground plant parts. The results revealed that the three different soil moisture levels caused distinctly differing levels of CO₂ fluxes. Also, both carbon fixation calculated as gross ecosystem production (GEP) and carbon release measured as ecosystem respiration (ER) increased with increasing temperatures, with ER increasing faster than GEP. Hence, short term carbon loss from the ecosystem accelerated with raised temperatures. Temperature sensitivity of the ecosystem was dependent on the soil moisture level, shown by differing Q10 values of both GEP and ER at different soil moisture levels. It is therefore highly important to take soil moisture levels into consideration when evaluating responses of ecosystem carbon balance to changes in temperature. The greatest C fixation took place via the two most dominant species of the ecosystem, Vaccinium uliginosum and Empetrum hermaphroditum, with the former being responsible for the different size of C fixation at the three moisture levels.     

Response characteristics of the mitochondrial DNA genome in developmental health and disease

This review focuses on mitochondrial biology in mammalian development; specifically, the dynamics of information transfer from nucleus to mitochondrion in the regulation of mitochondrial DNA genomic expression, and the reverse signaling of mitochondrion to nucleus as an adaptive response to the environment. Data from recent studies suggest that the capacity of embryonic cells to react to oxygenation involves a tradeoff between factors that influence prenatal growth/development and postnatal growth/function. For example, mitochondrial DNA replication and metabolic set points in nematodes may be determined by mitochondrial activity early in life. The mitochondrial drug PK11195, a ligand of the peripheral benzodiazepine receptor, has antiteratogenic and antidisease action in several developmental contexts in mice. Protein malnutrition during early life in rats can program mitochondrial DNA levels in adult tissues and, in humans, epidemiological data suggest an association between impaired fetal growth and insulin resistance. Taken together, these findings raise the provocative hypothesis that environmental programming of mitochondrial status during early life may be linked with diseases that manifest during adulthood. Genetic defects that affect mitochondrial function may involve the mitochondrial DNA genome directly (maternal inheritance) or indirectly (Mendelian inheritance) through nuclear-coded mitochondrial proteins. In a growing number of cases, the depletion of, or deletion in, mitochondrial DNA is seen to be secondary to mutation of key nuclear-coded mitochondrial proteins that affect mitochondrial DNA replication, expression, or stability. These defects of intergenomic regulation may disrupt the normal cross-talk or structural compartmentation of signals that ultimately regulate mitochondrial DNA integrity and copy number, leading to depletion of mitochondrial DNA.