Prolyl hydroxylase PHD3 enhances the hypoxic survival and G1 to S transition of carcinoma cells

Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells.     

AS03 adjuvanted AH1N1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in Finland

Background

Narcolepsy is a chronic sleep disorder with strong genetic predisposition causing excessive daytime sleepiness and cataplexy. A sudden increase in childhood narcolepsy was observed in Finland soon after pandemic influenza epidemic and vaccination with ASO3-adjuvanted Pandemrix. No increase was observed in other age groups.

Methods

Retrospective cohort study. From January 1, 2009 to December 31, 2010 we retrospectively followed the cohort of all children living in Finland and born from January 1991 through December 2005. Vaccination data of the whole population was obtained from primary health care databases. All new cases with assigned ICD-10 code of narcolepsy were identified and the medical records reviewed by two experts to classify the diagnosis of narcolepsy according to the Brighton collaboration criteria. Onset of narcolepsy was defined as the first documented contact to health care because of excessive daytime sleepiness. The primary follow-up period was restricted to August 15, 2010, the day before media attention on post-vaccination narcolepsy started.

Findings

Vaccination coverage in the cohort was 75%. Of the 67 confirmed cases of narcolepsy, 46 vaccinated and 7 unvaccinated were included in the primary analysis. The incidence of narcolepsy was 9.0 in the vaccinated as compared to 0.7/100,000 person years in the unvaccinated individuals, the rate ratio being 12.7 (95% confidence interval 6.1–30.8). The vaccine-attributable risk of developing narcolepsy was 1∶16,000 vaccinated 4 to 19-year-olds (95% confidence interval 1∶13,000–1∶21,000).

Conclusions

Pandemrix vaccine contributed to the onset of narcolepsy among those 4 to 19 years old during the pandemic influenza in 2009–2010 in Finland. Further studies are needed to determine whether this observation exists in other populations and to elucidate potential underlying immunological mechanism. The role of the adjuvant in particular warrants further research before drawing conclusions about the use of adjuvanted pandemic vaccines in the future.     

Foraging habitats of bats in southern Finland

We determined the foraging habitats of the northern batEptesicus nilssonii (Keyserling et Blasius, 1839), Brandt’s batMyotis brandtii (Eversmann, 1845), whiskered batMyotis mystacinus (Kuhl, 1819), Daubenton’s batMyotis daubentonii (Kuhl, 1819) and brown long-eared batPlecotus auritus (Linnaeus, 1758) in southern Finland. Among these species, we compared the diversities of foraging habitats, linear feature preference and the bats’ tendencies to forage simultaneously.Eptesicus nilssonii was the most opportunistic, foraging in a wide range of habitats.Myotis daubentonii (94%) foraged mainly on water habitats, whileM. brandtii/mystacinus (89%) andP. auritus (66%) foraged mainly in forest habitats. The diversities of foraging habitats used byE. nilssonii andP. auritus were higher than those ofM. brandtii/mystacinus andM. daubentonii. Both E.nilssonii andP. auritus foraged mostly alone or in small numbers, whileM. brandtii/mystacinus tended to gather in large numbers to forage in the same habitat. Half ofE. nilssonii and 46% ofM. daubentonii foraged over linear features, while other species did not use linear features to such an extent. Management and conservation of foraging habitats are needed especially forM. brandtii/mystacinus andM. daubentonii, which are more specialized thanE. nilssonii and P. auritus.     

Modelling the occurrence of threatened plant species in taiga landscapes: methodological and ecological perspectives

Aim Understanding the spatial patterns of species distribution and predicting the occurrence of high biological diversity and rare species are central themes in biogeography and environmental conservation. The aim of this study was to model and scrutinize the relative contributions of climate, topography, geology and land‐cover factors to the distributions of threatened vascular plant species in taiga landscapes in northern Finland. Location North‐east Finland, northern Europe. Methods The study was performed using a data set of 28 plant species and environmental variables at a 25‐ha resolution. Four different stepwise selection algorithms [Akaike information criterion (AIC), Bayesian information criterion (BIC), adaptive backfitting, cross selection] with generalized additive models (GAMs) were fitted to identify the main environmental correlates for species occurrences. The accuracies of the distribution models were evaluated using fourfold cross‐validation based on the area under the curve (AUC) derived from receiver operating characteristic plots. The GAMs were tentatively extrapolated to the whole study area and species occurrence probability maps were produced using GIS techniques. The effect of spatial autocorrelation on the modelling results was also tested by including autocovariate terms in the GAMs. Results According to the AUC values, the model performance varied from fair to excellent. The AIC algorithm provided the highest mean performance (mean AUC = 0.889), whereas the lowest mean AUC (0.851) was obtained from BIC. Most of the variation in the distribution of threatened plant species was related to growing degree days, temperature of the coldest month, water balance, cover of mire and mean elevation. In general, climate was the most powerful explanatory variable group, followed by land cover, topography and geology. Inclusion of the autocovariate only slightly improved the performance of the models and had a minor effect on the importance of the environmental variables. Main conclusions The results confirm that the landscape‐scale distribution patterns of plant species can be modelled well on the basis of environmental parameters. A spatial grid system with several environmental variables derived from remote sensing and GIS data was found to produce useful data sets, which can be employed when predicting species distribution patterns over extensive areas. Landscape‐scale maps showing the predicted occurrences of individual or multiple threatened plant species may provide a useful basis for focusing field surveys and allocating conservation efforts.     

In vivo effect of mutations at the PRPP binding site of the Bacillus subtilis purine repressor

The Bacillus subtilis PurR mediates adenine repression and guanosine induction of purA. PRPP inhibits binding of PurR to DNA in vitro. Mutations in the PRPP binding motif of PurR caused strong repression regardless of purine exclusions or additions, establishing the role of PRPP as regulator of PurR.     

Targeted disruption of spermidine/spermine N1-acetyltransferase gene in mouse embryonic stem cells. Effects on polyamine homeostasis and sensitivity to polyamine analogues

We have generated mouse embryonic stem cells with targeted disruption of spermidine/spermine N(1)-acetyltransferase (SSAT) gene. The targeted cells did not contain any inducible SSAT activity, and the SSAT protein was not present. The SSAT-deficient cells proliferated normally and appeared to maintain otherwise similar polyamine pools as did the wild-type cells, with the possible exception of constantly elevated (about 30%) cellular spermidine. As expected, the mutated cells were significantly more resistant toward the growth-inhibitory action of polyamine analogues, such as N(1),N(11)-diethylnorspermine. However, this resistance was not directly attributable to cellular depletion of the higher polyamines spermidine and spermine, as the analogue depleted the polyamine pools almost equally effectively in both wild-type and SSAT-deficient cells. Tracer experiments with [C(14)]-labeled spermidine revealed that SSAT activity is essential for the back-conversion of spermidine to putrescine as radioactive N(1)-acetylspermidine and putrescine were readily detectable in N(1),N(11)-diethylnorspermine-exposed wild-type cells but not in SSAT-deficient cells. Similar experiments with [C(14)]spermine indicated that the latter polyamine was converted to spermidine in both cell lines and, unexpectedly, more effectively in the targeted cells than in the parental cells. This back-conversion was only partly inhibited by MDL72527, an inhibitor of polyamine oxidase. These results indicated that SSAT does not play a major role in the maintenance of polyamine homeostasis, and the toxicity exerted by polyamine analogues is largely not based on SSAT-induced depletion of the natural polyamines. Moreover, embryonic stem cells appear to operate an SSAT-independent system for the back-conversion of spermine to spermidine.     

Contributions of galectin-3 and -9 to epithelial cell adhesion analyzed by single cell force spectroscopy

Galectins are widely expressed in epithelial tissues and have been implicated in a variety of cellular processes, including adhesion and polarization. Here we studied the contributions of galectins in cell adhesion and cyst formation of Madin-Darby canine kidney cells. Quantitative single cell force spectroscopy and standard adhesion assays were employed to study both early (<2 min) and long term (90 min) adhesion of cells to different extracellular matrix components. Inhibitors were used to examine the contribution of integrins and galectins in general and RNA interference to specifically address the role of two abundantly expressed galectins, galectin-3 and -9. We found that both galectin-3 and -9 were required for optimal long term cell adhesion to both collagen I and laminin-111. Early adhesion to laminin was found to be integrin-independent and was instead mediated by carbohydrate interactions and galectin-3 and -9. The opposite was observed for early adhesion to collagen. Although similar, the contributions of galectin-3 and -9 to adhesion appeared to be by distinct processes. These defects in adhesion of the two galectin knockdown cell lines may underlie the epithelial phenotypes observed in the cyst assays. Our findings emphasize the complex regulation of epithelial cell functions by galectins.