A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization

We describe a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction by FRET measure. As a proof of concept we analyzed two alternative splicing events originating from lymphocyte antigen 6 (LY6) complex, locus G5B (LY6G5B) pre-mRNA. These are characterized by the removal of the first intron (Fully Spliced Isoform, FSI) or by retention of such intron (Intron-Retained Isoform, IRI). The use of PNA probe pairs labeled with donor (Cy3) and acceptor (Cy5) fluorophores, suitable to FRET, flanking FSI and IRI specific splice junctions specifically detected both mRNA isoforms in HeLa cells. We have observed that the method works efficiently with probes 5–11 nt apart. The data supports that this FRET-based PNA fluorescence in situ hybridization (FP–FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location and dynamics but also potentially a wide variety of close range RNA–RNA interactions.     

Cytokine Release Assays as Tests for Exposure to Leishmania,and for Confirming Cure from Leishmaniasis,in Solid Organ Transplant Recipients

Spain has one of the world’s largest pools of organ donors and is a global leader in terms of the number of transplants it performs. The current outbreak of leishmaniasis in Fuenlabrada (in the southwest of the region of Madrid, Spain) has involved 600 clinical cases since late 2009 (prevalence 0.2%). It may therefore be wise to monitor the town’s transplanted population for Leishmania infantum; its members are immunosuppressed and at greater risk of infection and relapse following treatment. The present work examines the use of cytokine release assays to determine the prevalence of Leishmania infection in this population, and to confirm recovery following treatment for visceral leishmaniasis (VL). The humoral and cellular immune responses to L. infantum were characterized in 63 solid organ transplant (SOT) recipients from Fuenlabrada, 57 of whom reported no previous episode of VL (NVL subjects), and six of whom had been cured of VL (CVL subjects). Seventeen subjects (12 NVL and 5 CVL) showed a patent lymphoproliferative response to soluble Leishmania antigen (SLA). Stimulation of peripheral blood mononuclear cell cultures and of whole blood with SLA led to the production of different combinations of cytokines that might serve to confirm Leishmania infection or recovery from VL and help prevent cured patients from relapsing into this serious condition.     

Effect of the relation between neural cholinergic action and nitric oxide on ovarian steroidogenesis in prepubertal rats

The coeliac ganglion and the ovary are related by the superior ovarian nerve, which penetrates into the ovary by the hilium and innervates mainly the ovarian stroma. On the other hand, it is known that the gaseous neurotransmitter nitric oxide (NO) and the two isoforms of its synthesis enzyme, the nitric oxide synthetase (NOS), are present in the ovary. Both innervation and NO participate in ovarian steroidogenesis. Therefore, the purposes of this work were (a) to standardize an in vitro coeliac ganglion-superior ovarian nerve-ovary integrated system in prepubertal rats; (b) to determine the presence of NO in the ovary and analyze the ganglionic cholinergic effect on the ovarian release of androstenedione, progesterone and NO; and (c) to assess the steroids/NO relationship. The system was incubated in buffer solution for 120 min, with the ganglion and ovary located in different compartments and linked by the superior ovarian nerve. From the results obtained, it is concluded that the system is viable and functional. The presence of basal NO is stimulated by the cholinergic action, while the release of the steroids is inhibited, which might indicate that the ganglionic cholinergic effect is probably mediated by NO. To our knowledge, this work constitutes the first study of the relationship between the neural cholinergic action and NO on the ovarian steroidogenesis of prepubertal rats.     

Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.     

PGE(2) decreases muscle cell proliferation in patients with non-asthmatic eosinophilic bronchitis

Non-asthmatic eosinophilic bronchitis (NAEB) is characterized by chronic cough and sputum eosinophilia without bronchial hyperresponsiveness. The aim of the present study is to determine whether increased levels of PGE(2) from NAEB sputum supernatants play a protective role in airway inflammation and muscular hyperplasia. Twenty-one patients with NAEB, 15 asthmatic patients, and 12 healthy subjects were studied. An up-regulated PGE(2) enzymatic pathway was observed in bronchial biopsies from patients with NAEB as compared with samples from asthmatic patients. Also, EP2 and EP4 receptor expression was increased in these samples. BSMC proliferation was inhibited to a greater extent in NAEB sputum supernatants than in those taken from asthmatic subjects and healthy controls. This inhibition was mostly due to PGE(2) levels, a fact which was confirmed by employing synthetic EP2 and EP4 agonist and antagonist receptors.These findings suggest that PGE(2) inhibits BSMC proliferation entailing a reduction of smooth muscle hyperplasia and thus protecting against the onset of airflow obstruction.     

The type 1 cysteinyl leukotriene receptor triggers calcium influx and chemotaxis in mouse alpha beta- and gamma delta effector T cells

Linker for activation of T cells (LAT) is essential for T cell activation. Mice with mutations of distinct LAT tyrosine residues (LatY136F and Lat3YF) develop lymphoproliferative disorders involving TCR alphabeta or gammadelta T cells that trigger symptoms resembling allergic inflammation. We analyzed whether these T cells share a pattern of gene expression that may account for their pathogenic properties. Both LatY136F alphabeta and Lat3YF gammadelta T cells expressed high levels of the type 1 cysteinyl leukotriene receptor (CysLT(1)). Upon binding to the 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTD(4)) cysteinyl leukotriene, CysLT(1) induced Ca(2+) flux and caused chemotaxis in both LatY136F alphabeta and Lat3YF gammadelta T cells. Wild-type in vitro-activated T cells, but not resting T cells, also migrated toward LTD(4) however with a lower magnitude than T cells freshly isolated from LatY136F and Lat3YF mice. These results suggest that CysLT(1) is likely involved in the recruitment of activated alphabeta and gammadelta T cells to inflamed tissues.     

Cysteine string proteins are associated with cortical granules of Xenopus laevis oocytes

Cysteine string proteins (csps) are associated with secretory organelles in a wide range of eukaryotic cells. Functional studies of these proteins indicate that they subserve one or more vital steps in the pathway of regulated exocytosis. Here, we document the presence of csps in fully grown (stage VI) oocytes of the frog, Xenopus laevis. Both Northern and immunoblot data support the conclusion that csps are expressed in these cells. In addition, immunoreactive csp is seen even at the earliest stage of oocyte development, namely, in stage I oocytes. Finally, immunoblot and immunocytochemical results indicate that csps are associated with cortical granules of stage II-VI oocytes. These observations suggest that csps participate in the cortical reaction that underlies the sustained block to polyspermy in Xenopus eggs. Moreover, because of the relative ease of manipulating cells as large as Xenopus oocytes, this system harbors considerable promise as a model for studying the role of csps and other proteins in exocytotic events.