Differential signalling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes

Stimulation by both adrenergic and non-adrenergic pathways can induce proliferation of brown pre-adipocytes. To understand the signalling pathways involved in non-adrenergic stimulation of cell proliferation, we examined Erk1/2 activation. In primary cultures of mouse brown pre-adipocytes, both EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) induced Erk1/2 activation. EGF-stimulated Erk1/2 activation involved Src tyrosine kinases, but not PKC or PI3K, whereas in PDGF-induced Erk1/2 activation, PI3K, PKC (probably the atypical ζ isoform) and Src were involved sequentially. Both EGF and PDGF induced PI3K-dependent Akt activation that was not involved in Erk1/2 activation. By comparing effects of signalling inhibitors (wortmannin, SH-6, TPA, Gö6983, PP2, PD98059) on EGF- and PDGF-induced Erk1/2 activation and cell proliferation (³H-thymidine incorporation), we conclude that while the signal transduction pathways initiated by these growth factors are clearly markedly different, their effects on cell proliferation can be fully explained through their stimulation of Erk1/2 activation; thus Erk1/2 is a common, essential step for stimulation of proliferation in these cells.     

Quantitative analysis of RU38486 (mifepristone) by HPLC triple quadrupole mass spectrometry

A sensitive liquid chromatography–mass spectrometric method was validated for the quantification of RU38486 (mifepristone) in human and murine plasma. The analyte and internal standard (alfaxolone) were extracted by liquid–liquid extraction with diethyl ether, resolved on a C18 column using gradient elution with methanol and ammonium acetate and detected after positive electrospray ionization (m/z 430 → 372; m/z 333 → 297, respectively). Quantification was linear over the range 0.5–500 ng (r² > 0.997), precise and accurate (intra-assay RSD ≤ 7.2%, RME ≤ 8.2%; inter-assay RSD ≤ 15.7% RME ≤ 10.2%). The limit of quantification (LOQ) was 50 pg injected on column, permitting reproducible analysis of RU38486 in small volumes of plasma.     

Phenotyping of <Emphasis Type="Italic">E. coli</Emphasis> serotypes associated to oedema disease

Background  

Oedema disease is a severe disease, mainly affecting recently weaned pigs. It is caused by E. coli strains that express fimbriae F18 and produce verotoxin 2e, mainly belonging to serotype O138, O139 or O141. The aim of this study was to compare E. coli isolates within these serotypes with respect to diversity.     

Biosphere-atmosphere interactions of ammonia with grasslands: Experimental strategy and results from a new European initiative

A new study to address the biosphere-atmosphere exchange of ammonia (NH₃) with grasslands is applying a European transect to interpret NH₃ fluxes in relation to atmospheric conditions, grassland management and soil chemistry. Micrometeorological measurements using the aerodynamic gradient method (AGM) with continuous NH₃ detectors are supported by bioassays of the NH₃ `stomatal compensation point' (<sub>s</sub>). Relaxed eddy accumulation (REA) is also applied to enable flux measurements at one height; this is relevant to help address flux divergence due to gas-particle inter-conversion or the presence of local sources in a landscape.Continuous measurements that contrast intensively managed grasslands with semi-natural grasslands allow a scaling up from 15 min values to seasonal means. The measurements demonstrate the bi-directional nature of NH<sub>3</sub> fluxes, with typically daytime emission and small nocturnal deposition. They confirm the existence of enhanced NH<sub>3</sub> emissions (e.g. 30 g N ha<sup>–1</sup> d<sup>–1</sup>) following cutting of intensively managed swards. Further increased emissions follow fertilization with NH<sub>4</sub>NO<sub>3</sub> (typically 70 g N ha<sup>–1</sup> d<sup>–1</sup>). Measurements using REA support these patterns, but require a greater analytical precision than with the AGM.The results are being used to develop models of NH<sub>3</sub> exchange. `Canopy compensation point' resistance models reproduce bi-directional diurnal patterns, but currently lack a mechanistic basis to predict changes in relation to grassland phenology. An advance proposal here is the coupling of _s to dynamic models of grassland C–N cycling, and a relationship with modelled plant substrate-N is shown. Applications of the work include incorporation of the resistance models in NH₃ dispersion modelling and assessment of global change scenarios.     

Mortality after long-term exposure to radioactive Thorotrast: a forty-year follow-up survey in Sweden

To evaluate temporal patterns of cause-specific mortality after long-term exposure to the alpha-particle-emitting contrast medium Thorotrast, we investigated a cohort consisting of 693 Swedish patients with neurological disorders who received Thorotrast during cerebral angiography, with follow-up ending in 1993. Standardized mortality ratios (SMRs) were calculated as the ratio of observed cases in the cohort to expected cases in the general population. Persons exposed to Thorotrast had significant excesses of all causes of death (SMR = 2.8; 95% CI 2.5-3.0), with similar increases noted for men and women. The largest risks were observed for deaths from hematological causes (SMR = 16.4; n = 8), cerebrovascular diseases (SMR = 10.1; n = 18), gastrointestinal disorders including liver cirrhosis (SMR = 5.2; n = 36), and tumors (SMR = 4.7; n = 187). There was a significant decrease in SMR with time since injection for cerebrovascular and circulatory diseases, indicative of the impact of underlying neurological disorders. In contrast, the SMR increased significantly with time for tumors and gastrointestinal diseases, suggestive of a detrimental effect of cumulative radiation dose. A significant dose-response relationship was found for all causes of death and malignant tumors among all age groups, and since SMR increased with time for the latter category, this is consistent with an effect of cumulative radiation exposure on cancer development. However, the findings should be treated with caution, since selection bias may have influenced some of the results.     

Pyrosequencing analysis identifies discrete populations of Haemonchus contortus from small ruminants

The genus Haemonchus consists of blood-sucking parasitic nematodes in the abomasum of ruminants. Members of this genus are responsible for extensive production losses, particularly of small ruminants in the tropics but are also found in temperate regions. In this study, we examined the internal transcribed spacers-1 and -2 of rRNA in Haemonchus spp. The rRNA region spanning the internal transcribed spacers-1, -2 and the 5.8S rRNA gene was amplified by PCR from each of 10 worms from Swedish sheep, a Swedish goat and Kenyan sheep. The fragments were sequenced and examined with respect to genetic differences fixed among the three isolates. These and additional worms were further analysed with Pyrosequencing technology. This technique allowed us to rapidly analyse 110 individuals in three putative polymorphic nucleotide positions that were initially identified with dideoxy sequencing. The geographical isolates could to a large extent be genetically distinguished, but none of the polymorphic positions were consistent among all individuals within each isolate. Furthermore an alignment of our sequences and a consensus sequence published for Haemonchus contortus revealed two differences in positions 123 and 196 in internal transcribed spacers-2. Although these positions were previously reported as heterogenic, no polymorphism was detected among the 30 worms sequenced in the present study. Modelling of the internal transcribed spacers-2 secondary structure based on our data also identified a new putative long-range interaction. The isolates are best described as populations. In conclusion, consistent differences were not identified and the studied isolates are therefore best described as discrete populations. This study also reveals for the first time the potential of Pyrosequencing technology as a tool in the analysis of nematode population genetics.     

Inability of 50 Hz magnetic fields to regulate PKC- and Ca(2+)-dependent gene expression in Jurkat cells

We have previously reported that the T cell line Jurkat registers the exposure of a sinusoidal extremely low frequency magnetic field at the level of the plasma membrane, resulting in activation of the tyrosine kinase p56(lck), increase in inositol-3-phosphate levels and increase in intracellular calcium concentration within minutes. To elucidate if these events associated with changes in intracellular calcium ion levels were biologically significant, transient transfections of Jurkat cells were performed with calcium-ion dependent reporter constructs. Three different enhancer/promoter constructs were studied coupled to the luciferase reporter gene. The luciferase activity of each construct was measured after treatment of transfected cells to EMF exposure alone, or in combination with ionomycin, phorbol ester or cross-linking anti-CD3 antibodies. There was no indication that the used EMFs could influence any of these reporter constructs.     

Immunoglobulin-secreting cells of maternal origin can be detected in B cell-deficient mice

It is well known that the transfer of immunoglobulins (Igs) from mother to young via milk contributes to the offspring's immune defense. The present study suggests that not only is IgG transmitted to progeny, but that functional maternal Ig-secreting cells (or B cells) can also be transferred to the neonate. We have used B cell-deficient (micro(-/-)) mice and found that a high proportion of them obtain long-lasting, partial reconstitution of their serum Ig levels if born to micro(+/-) mothers. In some of these serum IgG-positive micro(-/-) mice, Ig-secreting cells were detected in spleen and bone marrow. To ensure that cells of maternal origin were present in the progeny, micro(-/-) offspring born to micro(+/-) dams transgenic for green fluorescent protein (GFP) were used. In spleens and bone marrow from some of these micro(-/-)GFP(-/-) offspring, GFP-positive cells were detected, which demonstrated that cells of maternal origin could infiltrate the progeny. In addition, splenic Ig-secreting cells were detected in micro(-/-) mice that were born to micro(-/-) dams and transferred to a lactating micro(+/+) foster dam at birth. This indicates that maternal Ig-secreting cells can be transferred postnatally via milk.