Overall Lack of Regulated Secretion in a PC12 Variant Cell Clone

Abstract A stable clone of PC12 neuroendocrine cells, named 27, known from previous studies to exhibit a defect of regulated secretion (lack of regulated secretory proteins, of synaptophysin, of dense granules and of catecholamine uptake and release; Clementi, E., Racchetti, G., Zacchetti, D., Panzeri, M. C., and Meldolesi, J. (1992) Eur. J. Neurosci. 4, 944-953) was characterized in detail to clarify the nature of its phenotype and the mechanisms of its establishment. The neuroendocrine nature of the PC12-27 phenotype was documented by specific markers: synapsins, neurofilament subunit H, neuronal kinesin, and α-latrotoxin receptor. Moreover, various intracellular membrane systems of PC12-27, including the endoplasmic reticulum and the Golgi complex, appeared similar to control PC12 in both morphology and marker expression. In contrast, all the investigated markers located either in dense granules (dopamine-β-hydroxylase), in synaptic-like microvesicles (the acetylcholine transporter) or in both these regulated secretory organelles (VAMP2/synaptobrevin-2, synaptotagmin) were missing in PC12-27 cells, and the same was true also for the cytosolic and plasmalemma proteins involved in regulated exocytosis (Rab3, SNAP25, syntaxin). Pulse labeling and in vitro translation experiments revealed the defect to consist in a protein synthesis blockade that mRNA studies (reverse transcription-polymerase chain reaction, Northern blotting, and actinomycin D experiments) revealed to take place primarily at the transcriptional level. The secretion defect of PC12-27 cells was modified neither by various types of long term stimulation nor by nerve growth factor treatment. Moreover, when one of the missing regulated secretory proteins, chromogranin B, was expressed by cDNA transfection, it was secreted, however via the constitutive pathway. Our results demonstrate that PC12-27 cells are fully incompetent for both branches of regulated secretion, those of dense granules and synaptic-like microvesicles, possibly because of the impairment of a general expression control system that appears to operate independently of neuroendocrine cell differentiation

Sensitivity of projected long-term CO 2 emissions across the Shared Socioeconomic Pathways

Scenarios showing future greenhouse gas emissions are needed to estimate climate impacts and the mitigation efforts required for climate stabilization. Recently, the Shared Socioeconomic Pathways (SSPs) have been introduced to describe alternative social, economic and technical narratives, spanning a wide range of plausible futures in terms of challenges to mitigation and adaptation. Thus far the key drivers of the uncertainty in emissions projections have not been robustly disentangled. Here we assess the sensitivities of future CO 2 emissions to key drivers characterizing the SSPs. We use six state-of-the-art integrated assessment models with different structural characteristics, and study the impact of five families of parameters, related to population, income, energy efficiency, fossil fuel availability, and low-carbon energy technology development. A recently developed sensitivity analysis algorithm allows us to parsimoniously compute both the direct and interaction effects of each of these drivers on cumulative emissions. The study reveals that the SSP assumptions about energy intensity and economic growth are the most important determinants of future CO 2 emissions from energy combustion, both with and without a climate policy. Interaction terms between parameters are shown to be important determinants of the total sensitivities

Sympathetic Innervation of the Mammalian Pineal Gland: Its Involvement in Ontogeny and Physiology, and in Pineal Dysfunction

In mammals, the melatonin-producing pineal gland (PG) receives sympathetic innervation from the superior cervical ganglia (SCG). This chapter describes the role of this innervation on the PG’s ontogeny and rhythmic function, along with consequences to physiology when this regulation is disrupted. The PG and the SCG are components of the circadian timing system (CTS). Therefore, the overall CTS is described, including its oscillatory basis, its synchronization to the light: dark (L:D) cycles, and the dissemination of timing cues to all cells throughout the body. Pineal cellular composition and heterogeneity, cell-cell interactions, and the molecular mechanisms involved in the circadian rhythm of melatonin (MEL), are discussed. The SCG’s bilateral placement among surrounding anatomical landmarks, as well as their afferent and efferent connections, are described and illustrated. In addition, the SCG-related surgical models and the state-of-the art technology used to investigate the connection between SCG and PG are presented. Perspectives and gaps in our understanding are also discussed. We hope this chapter inspires readers to delve deeper into the field of the pineal gland and its main messenger, melatonin, as well as MEL’s impact in health and disease, including as a remedial therapy

Temporal properties of gamma-ray bursts as signatures of jets from the central engine

A comprehensive temporal analysis has been performed on the 319 brightest GRBs with T90>2s from the BATSE current catalog. The rise times, fall times, full-widths at half maximum (FWHM), pulse amplitudes and pulse areas were measured and the frequency distributions are presented here. The distribution of time intervals between pulses is not random but compatible with a lognormal distribution when allowance was made for the 64 ms time resolution and a small excess (5%) of long duration intervals that is often referred to as a Pareto-Levy tail. A range of correlations are presented on pulse and burst properties. The rise and fall times, FWHM and area of the pulses are highly correlated with each other. The time intervals between pulses and pulse amplitudes of neighbouring pulses are correlated with each other. It was also found that the number of pulses, N, in GRBs is strongly correlated with the fluence and the duration and that can explain the well known correlation between duration and fluence. The GRBs were sorted into three catagories based on N i.e. 3=25. The properties of pulses before and after the stongest pulse were compared for the three catagories of bursts. This analysis revealed that the GRBs with large numbers of pulses have narrower and faster pulses and also larger fluences, longer durations and higher hardness ratios than the GRBs with smaller numbers of pulses.Comment: 19 pages, 22 figures. Submitted to A&A July 200

Cloning and characterization of mouse UBPy, a deubiquitinating enzyme that interacts with the Ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1

We used yeast "two-hybrid" screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25(Mm). Three independent overlapping clones were isolated from a mouse embryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction. It encodes a large protein (1080 amino acids) highly homologous to the human deubiquitinating enzyme hUBPy and contains a well conserved domain typical of ubiquitin isopeptidases. Therefore we called this new protein mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA present in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25(Mm) is exclusively expressed. Using a glutathione S-transferase fusion protein, we demonstrated an "in vitro" interaction between mUBPy and the N-terminal half (amino acids 1-625) of CDC25(Mm). In addition "in vivo" interaction was demonstrated after cotransfection in mammalian cells. We also showed that CDC25Mm, expressed in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decreases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of the human homolog hUBPy is not defined, although its expression was correlated with cell proliferation. Our results suggest that mUBPy may play a role in controlling degradation of CDC25(Mm), thus regulating the level of this Ras-guanine nucleotide exchange factor

Challenging GRB models through the broadband dataset of GRB060908

Context: Multiwavelength observations of gamma-ray burst prompt and afterglow emission are a key tool to disentangle the various possible emission processes and scenarios proposed to interpret the complex gamma-ray burst phenomenology. Aims: We collected a large dataset on GRB060908 in order to carry out a comprehensive analysis of the prompt emission as well as the early and late afterglow. Methods: Data from Swift-BAT, -XRT and -UVOT together with data from a number of different ground-based optical/NIR and millimeter telescopes allowed us to follow the afterglow evolution from about a minute from the high-energy event down to the host galaxy limit. We discuss the physical parameters required to model these emissions. Results: The prompt emission of GRB060908 was characterized by two main periods of activity, spaced by a few seconds of low intensity, with a tight correlation between activity and spectral hardness. Observations of the afterglow began less than one minute after the high-energy event, when it was already in a decaying phase, and it was characterized by a rather flat optical/NIR spectrum which can be interpreted as due to a hard energy-distribution of the emitting electrons. On the other hand, the X-ray spectrum of the afterglow could be fit by a rather soft electron distribution. Conclusions: GRB060908 is a good example of a gamma-ray burst with a rich multi-wavelength set of observations. The availability of this dataset, built thanks to the joint efforts of many different teams, allowed us to carry out stringent tests for various interpretative scenarios showing that a satisfactorily modeling of this event is challenging. In the future, similar efforts will enable us to obtain optical/NIR coverage comparable in quality and quantity to the X-ray data for more events, therefore opening new avenues to progress gamma-ray burst research.Comment: A&A, in press. 11 pages, 5 figure

Multi-wavelength observations of the energetic GRB 080810: detailed mapping of the broadband spectral evolution

GRB 080810 was one of the first bursts to trigger both Swift and the Fermi Gamma-ray Space Telescope. It was subsequently monitored over the X-ray and UV/optical bands by Swift, in the optical by ROTSE and a host of other telescopes and was detected in the radio by the VLA. The redshift of z= 3.355 +/- 0.005 was determined by Keck/HIRES and confirmed by RTT150 and NOT. The prompt gamma/X-ray emission, detected over 0.3-10^3 keV, systematically softens over time, with E_peak moving from ~600 keV at the start to ~40 keV around 100 s after the trigger; alternatively, this spectral evolution could be identified with the blackbody temperature of a quasithermal model shifting from ~60 keV to ~3 keV over the same time interval. The first optical detection was made at 38 s, but the smooth, featureless profile of the full optical coverage implies that this originated from the afterglow component, not the pulsed/flaring prompt emission. Broadband optical and X-ray coverage of the afterglow at the start of the final X-ray decay (~8 ks) reveals a spectral break between the optical and X-ray bands in the range 10^15 - 2x10^16 Hz. The decay profiles of the X-ray and optical bands show that this break initially migrates blueward to this frequency and then subsequently drifts redward to below the optical band by ~3x10^5 s. GRB 080810 was very energetic, with an isotropic energy output for the prompt component of 3x10^53 erg and 1.6x10^52 erg for the afterglow; there is no evidence for a jet break in the afterglow up to six days following the burst.Comment: 15 pages, 9 figures, 4 in colour. Accepted for publication in MNRA

A survey of clinical features of allergic rhinitis in adults

Background: Allergic rhinitis (AR) has high prevalence and substantial socio-economic burden. Material/Methods: The study included 35 Italian Centers recruiting an overall number of 3383 adult patients with rhinitis (48% males, 52% females, mean age 29.1, range 18–45 years). For each patient, the attending physician had to fill in a standardized questionnaire, covering, in particular, some issues such as the ARIA classification of allergic rhinitis (AR), the results of skin prick test (SPT), the kind of treatment, the response to treatment, and the satisfaction with treatment. Results: Out of the 3383 patients with rhinitis, 2788 (82.4%) had AR: 311 (11.5%) had a mild intermittent, 229 (8.8%) a mild persistent, 636 (23.5%) a moderate-severe intermittent, and 1518 (56.1%) a moderate-severe persistent form. The most frequently used drugs were oral antihistamines (77.1%) and topical corticosteroids (60.8%). The response to treatment was judged as excellent in 12.2%, good in 41.3%, fair in 31.2%, poor in 14.5%, and very bad in 0.8% of subjects. The rate of treatment dissatisfaction was significantly higher in patients with moderate-to-severe AR than in patients with mild AR (p<0.0001). Indication to allergen immunotherapy (AIT) was significantly more frequent (p<0.01) in patients with severe AR than with mild AR. . Conclusions: These fndings confirm the appropriateness of ARIA guidelines in classifying the AR patients and the association of severe symptoms with unsuccessful drug treatment. The optimal targeting of patients to be treated with AIT needs to be reassessed

R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.Fil: Mesquita, Fernanda C. P.. Universidade Federal do Rio de Janeiro; BrasilFil: Arantes, Paulo C.. Universidade Federal do Rio de Janeiro; BrasilFil: Kasai Brunswick, Tais H.. Universidade Federal do Rio de Janeiro; BrasilFil: Araujo, Dayana S.. Universidade Federal do Rio de Janeiro; BrasilFil: Gubert, Fernanda. Universidade Federal do Rio de Janeiro; BrasilFil: Monnerat, Gustavo. Universidade Federal do Rio de Janeiro; BrasilFil: Silva dos Santos, Danúbia. Universidade Federal do Rio de Janeiro; BrasilFil: Neiman, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Leitão, Isabela C.. Universidade Federal do Rio de Janeiro; BrasilFil: Barbosa, Raiana A. Q.. Universidade Federal do Rio de Janeiro; BrasilFil: Coutinho, Jorge L.. National Institute Of Cardiology; BrasilFil: Vaz, Isadora M.. Pontificia Universidad Catolica de Parana; BrasilFil: dos Santos, Marcus N.. Universidade Federal do Rio de Janeiro; BrasilFil: Borgonovo, Tamara. Pontificia Universidad Catolica de Parana; BrasilFil: Cruz, Fernando E. S.. National Institute of Cardiology; BrasilFil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Medei, Emiliano H.. Universidade Federal do Rio de Janeiro; BrasilFil: Campos de Carvalho, Antonio C.. Universidade Federal do Rio de Janeiro; Brasil. National Institute of Cardiology; Brasil. National Institute for Science and Technology in Regenerative Medicine; BrasilFil: Carvalho, Adriana B.. Universidade Federal do Rio de Janeiro; Brasil. National Institute for Science and Technology in Regenerative Medicine; Brasi