A mouse model for Li-Fraumeni-Like Syndrome with cardiac angiosarcomas associated to POT1 mutations

The shelterin protein POT1 has been found mutated in many different familial and sporadic cancers, however, no mouse models to understand the pathobiology of these mutations have been developed so far. To address the molecular mechanisms underlying the tumorigenic effects of POT1 mutant proteins in humans, we have generated a mouse model for the human POT1^R117C mutation found in Li-Fraumeni-Like families with cases of cardiac angiosarcoma by introducing this mutation in the Pot1a endogenous locus, knock-in for Pot1a^R117C. We find here that both mouse embryonic fibroblasts (MEFs) and tissues from Pot1a^+/ki mice show longer telomeres than wild-type controls. Longer telomeres in Pot1a^+/ki MEFs are dependent on telomerase activity as they are not found in double mutant Pot1a^+/ki Tert^-/- telomerase-deficient MEFs. By using complementation assays we further show that POT1a pR117C exerts dominant-negative effects at telomeres. As in human Li-Fraumeni patients, heterozygous Pot1a^+/ki mice spontaneously develop a high incidence of angiosarcomas, including cardiac angiosarcomas, and this is associated to the presence of abnormally long telomeres in endothelial cells as well as in the tumors. The Pot1a^+/R117C mouse model constitutes a useful tool to understand human cancers initiated by POT1 mutations.     

Cholecystokinin Outcome on Markers of Intestinal IgA Antibody Response

Cholecystokinin 8 (CCK8) is an entero-octapeptide that participates in crosstalk with components of intestinal immunity via the CCK receptor (CCKR), but its role in modulation of the IgA response is not fully known under physiological conditions. Male eight-week-old BALB/c mice each were intraperitoneally injected once during 7 days with CCK8, devazapide (CCKR1 antagonist), L365,260 (CCKR2 antagonist) or vehicle (sham group). In intestinal lavages, total and secretory IgA (SIgA) were determined by ELISA; in lamina propria, IgA⁺ B lymphocytes and IgA⁺ plasma cells were analyzed by flow cytometry; mRNA levels of polymeric immunoglobulin receptor (pIgR) in epithelial cells and α chain, interleukins (ILs) in lamina propria cells were assessed by qRTPCR. Regarding the sham conditions, IgA⁺ plasma-cell percentage and IL-2, IL-5, IL-10 and transforming growth factor-β (TGF-β) mRNA levels were either increased by CCK8 or decreased by both CCKR antagonists. For IgA/SIgA responses, IL-4/IL-6 mRNA levels were decreased by all drugs and pIgR mRNA was increased by CCK8 and reduced by L365,260. IgA⁺ B cell percentage and α chain mRNA levels were elicited by CCK8 and L365,260. Data suggested a presumable differential role of CCK/CCKR on the IgA-response; outcome of L365,260 on the elicitation of IgA⁺ B cells and α chain mRNA needs further examination.     

Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver

MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5′ end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3′ UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context.     

A physiological and molecular study of the effects of nickel deficiency and phenylphosphorodiamidate (PPD) application on urea metabolism in oilseed rape (Brassica napus L.)

Background and aims

Urea is the major nitrogen (N) form supplied as fertilizer in agriculture. However, urease, a nickel-dependent enzyme, allows plants to use external or internally generated urea as a nitrogen source. Since a urease inhibitor is frequently applied in conjunction with urea fertilizer, the N-metabolism of plants may be affected. The aim of this study was to determine physiological and molecular effects of nickel deficiency and a urease inhibitor on urea uptake and assimilation in oilseed rape.

Methods

Plants were grown on hydroponic solution with urea as the sole N source under three treatments: plants treated with nickel (+Ni) as a control, without nickel (?Ni) and with nickel and phenylphosphorodiamidate (+Ni+PPD). Urea transport and assimilation were investigated.

Results

The results show that Ni-deficiency or PPD supply led to reduced growth and reduced ¹⁵N-uptake from urea. This effect was more pronounced in PPD-treated plants, which accumulated high amounts of urea and ammonium. Thus, Ni-deficiency or addition of PPD, limit the availability of N and decreased shoot and root amino acid content. The up-regulation of BnDUR3 in roots indicated that this gene is a component of the stress response to nitrogen-deficiency. A general decline of glutamine synthetase (GS) activity and activation of glutamate dehydrogenase (GDH) and increases in its expression level were observed in control plants. At the same time, in (?N) or (+Ni+PPD) treated plants, no increases in GS or GDH activities and expression level were found.

Conclusions

Overall results showed that plants require Ni as a nutrient (while most widely used nutrient solutions are devoid of Ni), whether they are grown with or without a urea supply, and that urease inhibitors may have deleterious effects at least in hydroponic grown oilseed rape.     

Relationships between leaf morphological traits, nutrient concentrations and isotopic signatures for Mediterranean woody plant species and communities

Background and aims

Soil factors are driving forces that influence spatial distribution and functional traits of plant species. We test whether two anchor morphological traits—leaf mass per area (LMA) and leaf dry matter content (LDMC)—are significantly related to a broad range of leaf nutrient concentrations in Mediterranean woody plant species. We also explore the main environmental filters (light availability, soil moisture and soil nutrients) that determine the patterns of these functional traits in a forest stand.

Methods

Four morphological and 19 chemical leaf traits (macronutrients and trace elements and δ¹³C and δ¹⁵N signatures) were analysed in 17 woody plant species. Community-weighted leaf traits were calculated for 57 plots within the forest. Links between LMA, LDMC and other leaf traits were analysed at the species and the community level using standardised major axis (SMA) regressions

Results

LMA and LDMC were significantly related to many leaf nutrient concentrations, but only when using abundance-weighted values at community level. Among-traits links were much weaker for the cross-species analysis. Nitrogen isotopic signatures were useful to understand different resource-use strategies. Community-weighted LMA and LDMC were negatively related to light availability, contrary to what was expected.

Conclusion

Community leaf traits have parallel shifts along the environmental factors that determine the community assembly, even though they are weakly related across individual taxa. Light availability is the main environmental factor determining this convergence of the community leaf traits.     

Ecology,morphology and physiology of Chroothece richteriana (Rhodophyta,Stylonematophyceae) in the highly calcareous Río Chícamo,south-east Spain

The ecology of Chroothece was studied in the highly calcareous Río Chícamo, south-east Spain, in order to explain its success there, but rarity elsewhere. The river, which originates mainly from an underground aquifer, has water with high conductivity, sulphate and nitrate but low phosphate concentrations, the latter mainly organic. Chroothece occurs in mats and in lobed colonies reaching 4 cm in the broadest dimension. The colony surface consists of one layer of cells, each of which is attached to a stalk, which dichotomizes when the cell divides; stalks often extend to the colony base. The central region of many mat cells and almost all colony cells has a yellow to orange-brown colour, associated with the numerous lipid droplets densely covering the surface of the pyrenoid and arms of the star-shaped chloroplast. Field material and laboratory isolates indicate that stalk formation occurs under moderate P limitation and both stalks and cell sheath show high phosphatase activities. This also occurred in a culture collection strain maintained for 30 years in a very P-rich medium, but then transferred to a moderately P-limiting medium (c. 0.9 mg l^?1). We suggest that colony formation is initiated by aggregation of motile cells following P pulses in the water. Comparisons are made with Rivularia, a competitor in this nitrate-rich river, in spite of being a N₂-fixer. One difference is that Chroothece cells lie at the periphery of the colonies and are therefore exposed to maximum sunlight, whereas Rivularia trichomes grow inside colonies with photoprotection by scytonemin. However, the ability to withstand heavy grazing pressure may be an especially important factor favouring Rivularia here.     

Nuclear localization of TRK-A in liver cells

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.