Muscarinic activation of mitogen-activated protein kinase in rat thyroid epithelial cells

Carbachol (Cch), a muscarinic acetylcholine receptors (mAChR) agonist, produces time- and dose-dependent increases in mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in nondifferentiated Fischer rat thyroid (FRT) epithelial cells. Cells pretreatment with the selective phospholipase C inhibitor U73122 resulted in a decrease of Cch-stimulated ERK1/2 phosphorylation. These data indicated that the effect of mAChR on ERK activation could be mediated through agonist-induced Ca(2+) mobilization or PKC activation. Phosphorylation of ERK1/2 was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate acetate (PMA), but was not altered either by PKC inhibitor GF109203X or by down-regulation of PKC. Phosphorylation of ERK1/2 was elevated by a direct [Ca(2+)](i) increase caused by thapsigargin or ionophore. Additionally, Cch-induced ERK1/2 phosphorylation was reduced after either inhibition of Ca(2+) influx or intracellular Ca(2+) release. Nevertheless, Cch-mediated ERK1/2 activation was genistein sensitive, indicating the involvement of protein tyrosine kinases on the downstream signalling of mAChR. Pretreatment of the cells with PP2 markedly decreased Cch-induced ERK1/2 phosphorylation, suggesting a role of Src family of tyrosine kinases in the signal transduction pathway involved in ERK1/2 activation by mAChR. To test the biological consequences of ERK activation, we examined the effect of mAChR on cell functions. Cch stimulation of FRT cells did not affect cell proliferation, but increased protein synthesis. This effect was significantly attenuated by PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK). This study demonstrated that muscarinic receptor-mediated increase in the ERK1/2 phosphorylation was dependent on [Ca(2+)](i) but independent of PKC and was mediated by the Src family of tyrosine kinases. Our results also supported the idea that the protein synthesis stimulated by mAChR in polarized FRT epithelial cells was regulated by the ERK1/2 phosphorylation pathway.     

Proteomics as a Tool to Identify New Targets Against Aspergillus and Scedosporium in the Context of Cystic Fibrosis

Mycopathologia - Cystic fibrosis (CF) is a genetic disorder that increases the risk of suffering microbial, including fungal, infections. In this paper, proteomics-based information was collated...     

High-resolution chemo- and biostratigraphic records of the Early Aptian oceanic anoxic event in Cantabria (N Spain): Palaeoceanographic and palaeoclimatic implications

During the Early Aptian, major palaeoenvironmental changes occurred leading to an oceanic anoxic event (OAE 1a) and a perturbation of the global carbon cycle. New detailed litho-, bio-, and chemostratigraphic (TOC, δ¹³C_carb, δ¹³C_org) records of two superbly exposed and expanded Lower Aptian sections in Cantabria (La Florida and Cuchía) allow to recognize the expression of the OAE 1a in shallow shelf environments of northern Spain. The succession consists of shallow platform limestones that include a marly unit (Patrocinio Formation), the deposition of which occurred mostly at the onset of the OAE 1a (~ 120.5 Ma). This study presents a new integrated biostratigraphy based on ammonites, planktonic foraminifera, calcareous nannofossils and palynomorphs that allows an accurate age resolution of the succession. The marly unit records an abrupt negative δ¹³C excursion in both bulk organic matter (up to 5‰) and carbonate (up to 6‰, mean 3‰), as has been already observed at the onset of the OAE 1a in other Lower Aptian deposits worldwide. In detail, however, the negative excursion presents two minima in the studied sections. This negative spike is confidently attributed to the upper half of the Hayesites irregularis nannofossil Zone, to the upper part of the Blowiella blowi foraminiferal Zone, and to the middle–upper part of the Deshayesites weissi ammonite Zone. A third negative excursion occurs at the base of the Rhagodiscus angustus nannofossil Zone, which may be correlatable with the Dufrenoyia furcata ammonite Zone. This data set refines the age of the OAE 1a and reveals the existence of a stratigraphic gap in the westernmost margin of the Basque Cantabrian Basin that covers at least a portion of the upper part of the Early Aptian. Sedimentary facies and quantitative analysis of palynomorphs and nannofossils document significant environmental changes associated with the OAE 1a: compositional changes of neritic carbonates and calcareous nannofossils data indicate the occurrence of a biocalcification crisis inferred to have been related to CO₂-induced changes in seawater chemistry, and palynomorphs identify a thermal maximum followed by a cooling phase. The latter show a Classopollis maximum during the OAE 1a, which is followed by a decrease in Classopollis and an increase of bisaccate pollen after the event.     

DNA Hypomethylation Affects Cancer-Related Biological Functions and Genes Relevant in Neuroblastoma Pathogenesis

Neuroblastoma (NB) pathogenesis has been reported to be closely associated with numerous genetic alterations. However, underlying DNA methylation patterns have not been extensively studied in this developmental malignancy. Here, we generated microarray-based DNA methylation profiles of primary neuroblastic tumors. Stringent supervised differential methylation analyses allowed us to identify epigenetic changes characteristic for NB tumors as well as for clinical and biological subtypes of NB. We observed that gene-specific loss of DNA methylation is more prevalent than promoter hypermethylation. Remarkably, such hypomethylation affected cancer-related biological functions and genes relevant to NB pathogenesis such as CCND1, SPRR3, BTC, EGF and FGF6. In particular, differential methylation in CCND1 affected mostly an evolutionary conserved functionally relevant 3′ untranslated region, suggesting that hypomethylation outside promoter regions may play a role in NB pathogenesis. Hypermethylation targeted genes involved in cell development and proliferation such as RASSF1A, POU2F2 or HOXD3, among others. The results derived from this study provide new candidate epigenetic biomarkers associated with NB as well as insights into the molecular pathogenesis of this tumor, which involves a marked gene-specific hypomethylation.     

Radiopotentiation of enzalutamide over human prostate cancer cells as assessed by real-time cell monitoring

AimTo evaluate the radiopotentiation of enzalutamide in human prostate cancer cells.BackgroundWhile radiotherapy is the first line of treatment for prostate cancer, androgen blockade therapies are demonstrating significant survival benefit as monotherapies. As androgen blockade can cause cell death by apoptosis, it is likely that androgen blockade will potentiate the cytotoxic activities of radiotherapy.Materials and methodsHere, we tested the potential synergistic effects of these two treatments over two human metastatic prostate cancer cells by real-time cell analysis (RTCA), androgen-sensitive LNCaP cells (Lymph Node Carcinoma of the Prostate) and androgen-independent PC-3. Both cell lines were highly resistant to high doses of radiotherapy.ResultsA pre-treatment of LNCaP cells with IC50 concentrations of enzalutamide significantly sensitized them to radiotherapy through enhanced apoptosis. In contrast, enzalutamide resistant PC-3 cells were not sensitized to radiotherapy by androgen blockade.ConclusionsThese results provide evidence that the enzalutamide/radiotherapy combination could maximize therapeutic responses in patients with enzalutamide-sensitive prostate cancer.     

Responsiveness of Durum Wheat to Mycorrhizal Inoculation Under Different Environmental Scenarios

A greater understanding of how climate change will affect crop photosynthetic performance has been described as a target goal to improve yield potential. Other concomitant stressors can reduce the positive effect of elevated atmospheric CO₂ on wheat yield. Arbuscular mycorrhizal fungi (AMF) are symbiotic fungi predicted to be important in defining plant responses to rising atmospheric CO₂, but their role in response to global climatic change is still poorly understood. This study aimed to assess if increased atmospheric CO₂ interacting with drought can modify the effects of mycorrhizal symbiosis on flag leaf physiology in winter wheat. The study was performed in climate-controlled greenhouses with ambient (400 ppm, ACO₂) or elevated (700 ppm, ECO₂) CO₂ concentrations in the air. Within each greenhouse half of the plants were inoculated with Rhizophagus intraradices. When ear emergence began, half of the plants from each mycorrhizal and CO₂ treatment were subjected to terminal drought. At ACO₂ AMF improved the photochemistry efficiency of PSII compared with non-mycorrhizal plants, irrespective of irrigation regime. Mycorrhizal wheat accumulated more fructan than non-mycorrhizal plants under optimal irrigation. The level of proline in the flag leaf increased only in mycorrhizal wheat after applying drought. Mycorrhizal association avoided photosynthetic acclimation under ECO₂. However, nitrogen availability to flag leaves in mycorrhizal plants was lower under ECO₂ than at ACO₂. Results suggest that the mechanisms underlying the interactions between mycorrhizal association and atmospheric CO₂ concentration can be crucial for the benefits that this symbiosis can provide to wheat plants undergoing water deficit.