Ana I. Jiménez

Potentiation of ATP calcium responses by A2B receptor stimulation and other signals coupled to Gs proteins in type‐1 cerebellar astrocytes

We have studied the interaction between P1 and P2 purinoceptors in purified type‐1 astrocyte cultures from postnatal days 7–8 rat cerebella using single cell microfluorimetry with fura‐2. The stimulation of astrocytes with ATP elicits rapid [Ca2+]i transients showing an EC50 value of 7.9 ± 0.3 μM. Costimulation of type‐1 astrocytes with adenosine and ineffective ATP concentrations (0.1 or 1 μM) evoked [Ca2+]i transients that correspond to 60% of the maximal ATP response. NECA (5′‐N‐ethylcarboxamidoadenosine) was the only agonist that mimicked the adenosine effect and showed an EC50 value of 0.17 ± 0.01 μM. This value was identical to that obtained for the cAMP production stimulation, indicating that A2B receptors coupled to adenylate cyclase activation were involved. The presence of A2B adenosine receptors was also confirmed by immocytochemistry experiments. When astrocytes were costimulated with isoproterenol and ineffective ATP concentrations similar [Ca2+]i transients were observed. The treatment of astrocytes with cholera toxin potentiated ATP calcium signals, lowering the EC50 value for ATP to 1.5 ± 0.2 μM. However, the pretreatment of cells with forskolin or a permeable cAMP analogue had no effect on ATP calcium responses. These results indicated that the potentiation mechanism was elicited before the adenylate cyclase activation. We could conclude that in type‐1 astrocytes, the activation of A2B adenosine receptors or other signals positively coupled to adenylate cyclase stimulation strongly potentiate metabotropic calcium responses to ATP. The potentiation was parallel but independent on cAMP accumulation suggesting the involvement of βγ subunits released after Gs stimulation. GLIA26:119–128, 1999.

Coexpression of Several Types of Metabotropic Nucleotide Receptors in Single Cerebellar Astrocytes

Abstract: We have examined the expression of mRNA for several P2Y nucleotide receptors by northern blot analysis in purified type 1 cerebellar astrocyte cultures. These results suggest that different P2Y subtypes could be responsible for ATP metabotropic calcium responses in single type 1 astrocytes. To identify these subtypes we have studied the pharmacological profile of ATP calcium responses using fura‐2 microfluorimetry. All tested astrocytes responded to ATP and UTP stimulations evoking similar calcium transients. Most astrocytes also responded to 2‐methylthioATP and ADP challenges. The agonist potency order was 2‐methylthioATP > ADP > ATP = UTP. Cross‐desensitization experiments carried out with ATP, UTP, and 2‐methylthioATP showed that 2‐methylthioATP and UTP interact with different receptors, P2Y1 and P2Y2 or P2Y4. In a subpopulation of type 1 astrocytes, ATP prestimulation did not block UTP responses, and UDP elicited clear intracellular Ca2+ concentration responses at very low concentrations. 2‐MethylthioATP and UTP calcium responses exhibited different sensitivity to pertussis toxin and different inhibition patterns in response to P2 antagonists. The P2Y1‐specific antagonist N6‐methyl‐2′‐deoxyadenosine 3′,5′‐bisphosphate (MRS 2179) specifically blocked the 2‐methylthio‐ATP responses. We can conclude that all single astrocytes coexpressed at least two types of P2Y metabotropic receptors: P2Y1 and either P2Y2 or P2Y4 receptors. Moreover, 30‐40% of astrocytes also coexpressed specific pyrimidine receptors of the P2Y6 subtype, highly selective for UDP coupled to pertussis‐toxin insensitive G protein.

Epigenomic profiling in polycythaemia vera and essential thrombocythaemia shows low levels of aberrant DNA methylation

Aims The purpose of this study was to compare the DNA-methylation signature in classic chronic Philadelphia negative myeloproliferative neoplasms (MPN), polycythaemia vera (PV) and essential thrombocythaemia (ET), in order to obtain a global insight into DNA-methylation changes associated with these malignancies. Methods Thirty-five MPN samples from 11 ET JAK2 V617F, 12 ET JAK2 wild type (WT) and 12 PV JAK2 V617F patients as well as 12 from healthy donors were analysed. DNA samples extracted from whole peripheral blood were hybridised to the ‘HumanMethylation27 DNA Analysis BeadChip.’ Results All groups showed a very homogeneous methylation pattern. Only the ZNF577 gene showed a differential methylation profile between PV JAK2 V617F positive and controls. This aberrant methylation was correlated with a differential gene expression of ZNF577. No aberrant hypermethylation was found in the SOCS-1 and SOCS-3 genes. Conclusions According to our results, an aberrant methylation pattern does not seem to play a crucial role in MPN pathogenesis; nor does it justify phenotypical differences between PV and ET.

Real Time PCR to detect hazelnut allergen coding sequences in processed foods

A quantitative RT-PCR method, employing novel primer sets designed on Cor a 9, Cor a 11 and Cor a 13 allergen-coding sequences has been setup and validated. Its specificity, sensitivity and applicability have been compared. The effect of processing on detectability of these hazelnut targets in complex food matrices was also studied. The DNA extraction method based on CTAB-phenol-chloroform was the best for hazelnut. RT-PCR using primers for Cor a 9, 11 and 13 allowed a specific and accurate amplification of these sequences. The limit of detection was 1 ppm of raw hazelnut. The method sensitivity and robustness were confirmed with spiked samples. Thermal treatments (roasting and autoclaving) reduced yield and amplificability of hazelnut DNA, however, high-hydrostatic pressure did not affect. Compared with an ELISA assay, this RT-PCR showed higher sensitivity to detected hazelnut traces in commercial foodstuffs. The RT-PCR method described is the most sensitive of those reported for the detection of hazelnut traces in processed foods.

Inhibition of related JAK/STAT pathways with molecular targeted drugs shows strong synergy with ruxolitinib in chronic myeloproliferative neoplasm

This study aimed to assess the antitumour effects, molecular mechanisms of action, and potential synergy of ruxolitinib with sorafenib, KNK437, dasatinib, and perifosine, in Philadelphia‐negative chronic myeloproliferative neoplasms (MPN). Cytotoxic and cytostatic effects of the different compounds were determined in the JAK2 V617F‐positive cell lines, HEL and Ba/F3 JAK2V617F EPOR, and in primary mononuclear and bone marrow CD34‐positive cells from 19 MPN patients. Ruxolitinib [50% inhibitory concentration (IC50)PV = 15 nmol/l], as well as sorafenib ( IC50 PV=8μmol/l ), KNK437 ( IC50 PV=100μmol/l ), and perifosine ( IC50 PV=15μmol/l ), were able to inhibit proliferation in cell line models and in primary cells from MPN patients. Dasatinib, KNK437, and sorafenib showed a strong synergistic effect in combination with ruxolitinib [combination index (CI)PV < 0·3]. Western blot confirmed that ruxolitinib blocked ERK, and consequently STAT5 activation, sorafenib inhibited ERK, P38 and STAT5, dasatinib blocked SRC and STAT5, and KNK437 decreased the stability of the JAK2 protein, reducing its expression. Inhibiting JAK2‐related proliferative pathways has the potential to inhibit cell proliferation in MPNs. Furthermore, the combination of ruxolitinib with inhibitors that target these pathways has a strong synergistic effect, which may be due to decreased activation of the common effector, STAT5.

Specific diadenosine pentaphosphate receptor coupled to extracellular regulated kinases in cerebellar astrocytes

In this study, we show specific intracellular responses evoked by the stimulation of astrocytes with the P1,P5‐di(adenosine‐5′)pentaphosphate, Ap5A. The stimulation of astrocytes with micromolar concentrations of the dinucleotide elicited rapid increases in intracellular calcium concentration ([Ca2+]i), showing an EC50 value of 15.27 ± 0.61 µm. Moreover, the stimulation of cells with nanomolar concentrations of Ap5A, unable to induce calcium responses, increased the phosphorylated forms of extracellular‐signal regulated kinase 1/2 (ERK) with an EC50 value of 9.8 ± 2.4 nm. The maximal activation was observed at 100 nm Ap5A, which was similar to that produced by epidermal growth factor (EGF) under the same experimental conditions. The present data reported here indicate that Ap5A mediated these effects by interacting with a specific receptor, not yet identified, which was different from the P2Y1 and P2Y2/P2Y4 receptors present in all individual astrocytes.

Cross-talk among epidermal growth factor, Ap5A, and nucleotide receptors causing enhanced ATP Ca2+ signaling involves extracellular kinase activation in cerebellar astrocytes

In previous papers, we reported that ATP calcium responses in cerebellar astrocytes were strongly potentiated by preincubation with nanomolar concentrations of the diadenosine pentaphosphate Ap5A. However, the intracellular signaling pathway mediating this effect was not defined. We also showed that stimulation of astrocytes with the dinucleotide led to the activation of extracellular regulated kinases (ERKs). Here, we examined whether ERKs are involved in the potentiating mechanism and intracellular mechanism leading to their activation. Epidermal growth factor (EGF) exactly reproduced the potentiation displayed by the dinucleotide. Moreover, the potentiation of ATP responses by Ap5A and EGF was completely abolished by the MAP kinase (MEK) inhibitor U‐0126, indicating that ERK activation is a required step for the potentiation event. Our data also indicated that ERK activation and the potentiation of ATP calcium responses were sensitive to the src‐like kinase inhibitor herbimycin A, p21ras farnesyltransferase inhibitor peptide, and some PKC inhibitors. Taken together, our findings reveal that Ap5A triggers the potentiation of ATP calcium responses through an intracellular mechanism that is insensitive to pertussis toxin and that this potentiation requires src protein‐mediated ERK activation and the participation of an atypical protein kinase C isoform activated downstream from ERK.

Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera

JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

Use of combined transmucosal fentanyl, nitrous oxide, and hematoma block for fracture reduction in a pediatric emergency department.

Objective The objective of this study was to evaluate the use of combined inhaled nitrous oxide (NO), hematoma block (HB), and transmucosal fentanyl (TMF) as sedoanalgesia in the reduction of radioulnar fractures in children in a pediatric emergency department (PED). Methods A retrospective, analytical observational study examining the cases of radioulnar fracture reduction in PED from 2007 to 2009 in children from 4 to 15 years old. The cases were divided into 2 groups: those in which only NO + HB was used and those in which TMF was combined with NO + HB. The pain perceived by the child, the doctor, and the nurse was studied during the procedure with 0- to 10-point scales (10 being severe pain). Satisfaction of the medical professionals, duration of the procedure, and the adverse effects that appeared were also studied. Results Eighty-one children were included. Sixty-four children (79%) received NO + TMF + HB, and 17 children (21%) received NO + HB only. The pain perceived by the child during the procedure in the group receiving NO + TMF + HB was 2.5 (95% confidence interval [CI], 1.8–3.1) compared with 3.9 (95% CI, 2.3–5.5) in the NO + HB group (P = 0.035), the pain perceived by the doctor was 2.6 (95% CI, 2–3.2) compared with 4 (95% CI, 1.6–4), and by the nurse was 2.7 (95% CI, 2–3.3) compared with 3.9 (95% CI, 2.3–5.5), respectively. Adverse events appeared in 15.3% of the NO + TMF + HB group and in 40% of the NO + HB group. Conclusions The association of NO + TMF + HB in the reduction of radioulnar fractures in PED improves pain control compared with the NO + HB combination. New studies are required to confirm the benefit and safety of this drug combination.

Bilateral tuberculous otomastoiditis in an immmunocompetent 5-year-old child: CT and MRI findings (2009: 3b)

Bilateral tuberculous mastoiditis (TOM) in an immunocompetent child is a very uncommon form of tuberculous infection presentation. This report shows the CT and MR imaging of bilateral tuberculous otomastoiditis consisting of aggressive signs of middle ear and mastoid involvement with bony destruction and periauricular collections with no signs of brain involvement. Differential diagnosis at pediatric age of destructive lesions such as mainly aggressive forms of histiocytosis is underscored. This form of bilateral TOM at this early age has not been described from a radiological perspecitve.