1H, 13C and 15N resonances of the AlgE62 subunit from Azotobacter vinelandii mannuronan C5-epimerase

The 17.7 kDa R2 module from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been isotopically labeled (¹³C,¹⁵N) and recombinantly expressed. Here we report the ¹H, ¹³C, ¹⁵N resonance assignment of AlgE6R2.     

NMR assignments of 1H, 13C and 15N resonances of the C-terminal subunit from Azotobacter vinelandii mannuronan C5-epimerase 6 (AlgE6R3)

The 19.9 kDa C-terminal module (R3) from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been ¹³C, ¹⁵N isotopically labelled and recombinantly expressed. We report here the ¹H, ¹³C, ¹⁵N resonance assignment of AlgE6R3.     

Floating Ice-Algal Aggregates below Melting Arctic Sea Ice

During two consecutive cruises to the Eastern Central Arctic in late summer 2012, we observed floating algal aggregates in the melt-water layer below and between melting ice floes of first-year pack ice. The macroscopic (1-15 cm in diameter) aggregates had a mucous consistency and were dominated by typical ice-associated pennate diatoms embedded within the mucous matrix. Aggregates maintained buoyancy and accumulated just above a strong pycnocline that separated meltwater and seawater layers. We were able, for the first time, to obtain quantitative abundance and biomass estimates of these aggregates. Although their biomass and production on a square metre basis was small compared to ice-algal blooms, the floating ice-algal aggregates supported high levels of biological activity on the scale of the individual aggregate. In addition they constituted a food source for the ice-associated fauna as revealed by pigments indicative of zooplankton grazing, high abundance of naked ciliates, and ice amphipods associated with them. During the Arctic melt season, these floating aggregates likely play an important ecological role in an otherwise impoverished near-surface sea ice environment. Our findings provide important observations and measurements of a unique aggregate-based habitat during the 2012 record sea ice minimum year.     

The rise and fall of local populations of ortolan buntings Emberiza hortulana: importance of movements of adult males

Changes in population size of local populations of birds have usually been interpreted in relation to adult return rate and recruitment of young individuals after natal dispersal. Little is known about the importance of redistribution of adult individuals through breeding dispersal. The small Norwegian population of ortolan buntings Emberiza hortulana has a patchy distribution with about 30 long‐term local populations. During a period of general population decline (29% decrease over 7 years), the population trends of local populations (measured as number of males recorded) were highly variable, with some even increasing four‐fold. Comparisons of demographic parameters showed that adult immigration rate (i.e. dispersal of adult males) explained both yearly changes in male population size and population trends over the whole study period better than adult return rate or adult emigration rate, or a measure of recruitment of young males. Adult immigration rates and recruitment rates were correlated, suggesting that both young and adult males find the same places attractive. In the study area, adult sex ratio was strongly male‐biased, and immigration rate was higher when local sex ratio was less skewed. In addition, less skewed sex ratio was related to higher adult return rate and lower emigration rate. We found no relationships between measures of breeding success and population change. We suggest that conspecific attraction may explain the observed patterns. Some local populations may act as hot‐spots attracting adult males from other populations. Thus, local population changes need not reflect overall population growth rate, but may be a consequence of redistribution of adult birds.     

Serglycin: a structural and functional chameleon with wide impact on immune cells

Among the different proteoglycans expressed by mammals, serglycin is in most immune cells the dominating species. A unique property of serglycin is its ability to adopt highly divergent structures, because of glycosylation with variable types of glycosaminoglycans when expressed by different cell types. Recent studies of serglycin-deficient animals have revealed crucial functions for serglycin in a diverse array of immunological processes. However, its exact function varies to a large extent depending on the cellular context of serglycin expression. Based on these findings, serglycin is emerging as a structural and functional chameleon, with radically different properties depending on its exact cellular and immunological context.     

Metabolism of gibberellin A19 is under photoperiodic control in Populus, Salix and Betula, but not in daylength-insensitive Populus overexpressing phytochrome A

Application experiments have suggested that short‐day‐induced cessation of elongation growth in trees is caused by photoperiodic regulation of the conversion of gibberellin GA₁₉ to GA₂₀. In the present study we examined further the photoperiodic control of GA metabolism in trees with focus on the conversion of GA₁₉ in Salix pentandra, hybrid aspen (Populus tremula × tremuloides) and silver birch (Betula pendula) using [17,17‐²H₂]‐GA₁₉ or unlabelled GAs in application studies. GA₂₀ and GA₁ were able to restore growth also in hybrid aspen and silver birch under short days (SD), whereas GA₁₉ had no or only a very small activity. Contrary to hybrid aspen and S. pentandra, the activity of GA₂₀ in silver birch was significantly lower than that of GA₁. Gas chromatography‐mass spectrometry (GC‐MS) analysis revealed a smaller turnover of [²H₂]‐GA₁₉ in SD than in long days (LD) in hybrid aspen. No such difference in turnover of [²H₂]‐GA₁₉ was observed in photoperiod‐insensitive hybrid aspen overexpressing PHYA. Application of unlabelled GAs to seedlings of S. pentandra, hybrid aspen and silver birch under SD followed by quantification of metabolites by GC‐MS analysis, showed that applied GA₁₉ was not readily converted to GA₂₀ and GA₁. Although the sensitivity to GAs is also known to decrease under SD, the present data are in favour of a photoperiodic regulation of the metabolism of GA₁₉in vivo in the woody species S. pentandra, hybrid aspen and silver birch. The data might also suggest that silver birch differs from S. pentandra and hybrid aspen by exhibiting a possible photoperiodic control also of the conversion of GA₂₀ to GA₁.     

Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2

Tryptophan hydroxylase (TPH) catalyses the rate-limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (V(max)) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild-type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.     

T cell responses in melanoma patients after vaccination with tumor-mRNA transfected dendritic cells

We have developed an individualized melanoma vaccine based on autologous dendritic cells (DCs) transfected with autologous tumor-mRNA. The vaccine targets the unique spectrum of tumor antigens in each patient and may recruit multiple T cell clones. In a recent phase I/II trial, we demonstrated T cell responses against vaccine antigens in 9/19 patients evaluable by T cell assays. Here, we report a follow-up study that was conducted to characterize interesting T cell responses and to investigate the effects of long-term booster vaccination. Two patients were selected for continued vaccine therapy. The clinical follow-up suggested a favorable clinical development in both patients. The immunological data (T cell proliferation/IFNgamma ELISPOT/Bioplex cytokine assays) indicated sustained T cell responses and suggested an enhancing effect of booster vaccinations. Both CD4(+) and CD8(+) T cell responses were demonstrated. From post-vaccination samples, we generated 39 T cell clones that responded specifically to stimulation by mRNA-transfected DCs and 12 clones that responded to mock-transfected DCs. These data clearly indicate a two-component vaccine response, against transfected and non-transfected antigens. T cell receptor (TCR) clonotype mapping, performed on 11 tDC-specific clones, demonstrated that 10/11 clones had different TCRs. The results thus indicate a broad spectrum T cell response against antigens encoded by the transfected tumor-mRNA. We generally observed mixed Th1/Th2 cytokine profiles, even in T cell clones that were confirmed to be derived from a single cell. This finding suggests that cytokine patterns after cancer vaccination may be more complex than indicated by the classic Th1/Th2 dichotomy.     

Macrogeographic variation in the call of the corncrake Crex crex

This study was conducted to characterise macrogeographic variation in the vocalisation of the corncrake Crex crex, a bird species with a non‐learned and highly stereotyped call. We also examined: 1) whether call characteristics remained stable across successive breeding seasons within two of the study populations and 2) whether call similarity was related to distance between populations. Recordings of 352 males from eight populations were analysed. The analyses focused on variation in 1) temporal characteristics (duration of syllables and intervals, duration of the intervals between consecutive maximal amplitude peaks within syllables, called pulse‐to‐pulse duration (PPD)), and 2) spectral characteristics (minimal and maximal frequency, frequencies below which 25%, 50% and 75% acoustic energy of signal is distributed). We found significant differences in most of the temporal and all of the spectral characteristics between populations. No differences were found in PPD. Significant interannual differences in spectral characteristics were found in both of the populations examined, whereas differences in temporal characteristics were only observed in one population. In general, geographic variation in calls showed clinal distance‐dependence, where distant populations showed larger differences in call than neighbouring populations. Our results show that geographic variation in corncrake calls may be very dynamic in the short term and that within‐population variation may occur on the same scale as between‐population variation. This finding is surprising because call characteristics in non‐learners are essentially inherited, and genetic transmission should be very slow. We suggest that the social interactions between males and/or the specific dispersal patterns of this species and the low site fidelity of adult and young birds may be responsible for such pattern.     

Tumor Phosphatidylinositol-3-Kinase Signaling and Development of Metastatic Disease in Locally Advanced Rectal Cancer

Background

Recognizing EGFR as key orchestrator of the metastatic process in colorectal cancer, but also the substantial heterogeneity of responses to anti-EGFR therapy, we examined the pattern of composite tumor kinase activities governed by EGFR-mediated signaling that might be implicated in development of metastatic disease.

Patients and Methods

Point mutations in KRAS, BRAF, and PIK3CA and ERBB2 amplification were determined in primary tumors from 63 patients with locally advanced rectal cancer scheduled for radical treatment. Using peptide arrays with tyrosine kinase substrates, ex vivo phosphopeptide profiles were generated from the same baseline tumor samples and correlated to metastasis-free survival.

Results

Unsupervised clustering analysis of the resulting phosphorylation of 102 array substrates defined two tumor classes, both consisting of cases with and without KRAS/BRAF mutations. The smaller cluster group of patients, with tumors generating high ex vivo phosphorylation of phosphatidylinositol-3-kinase-related substrates, had a particularly aggressive disease course, with almost a half of patients developing metastatic disease within one year of follow-up.

Conclusion

High phosphatidylinositol-3-kinase-mediated signaling activity of the primary tumor, rather than KRAS/BRAF mutation status, was identified as a hallmark of poor metastasis-free survival in patients with locally advanced rectal cancer undergoing radical treatment of the pelvic cavity.