Gender Differences in S-Nitrosoglutathione Reductase Activity in the Lung

S-nitrosothiols have been implicated in the etiology of various pulmonary diseases. Many of these diseases display gender preferences in presentation or altered severity that occurs with puberty, the mechanism by which is unknown. Estrogen has been shown to influence the expression and activity of endothelial nitric oxide synthase (eNOS) which is associated with increased S-nitrosothiol production. The effects of gender hormones on the expression and activity of the de-nitrosylating enzyme S-nitrosoglutathione reductase (GSNO-R) are undefined. This report evaluates the effects of gender hormones on the activity and expression of GSNO-R and its relationship to N-acetyl cysteine (NAC)-induced pulmonary hypertension (PH). GSNO-R activity was elevated in lung homogenates from female compared to male mice. Increased activity was not due to changes in GSNO-R expression, but correlated with GSNO-R S-nitrosylation: females were greater than males. The ability of GSNO-R to be activated by S-nitrosylation was confirmed by: 1) the ability of S-nitrosoglutathione (GSNO) to increase the activity of GSNO-R in murine pulmonary endothelial cells and 2) reduced activity of GSNO-R in lung homogenates from eNOS−/− mice. Gender differences in GSNO-R activity appear to explain the difference in the ability of NAC to induce PH: female and castrated male animals are protected from NAC-induced PH. Castration results in elevated GSNO-R activity that is similar to that seen in female animals. The data suggest that GSNO-R activity is modulated by both estrogens and androgens in conjunction with hormonal regulation of eNOS to maintain S-nitrosothiol homeostasis. Moreover, disruption of this eNOS-GSNO-R axis contributes to the development of PH

First observations of separated atmospheric nu_mu and bar{nu-mu} events in the MINOS detector

The complete 5.4 kton MINOS far detector has been taking data since the beginning of August 2003 at a depth of 2070 meters water-equivalent in the Soudan mine, Minnesota. This paper presents the first MINOS observations of nuµ and [overline nu ]µ charged-current atmospheric neutrino interactions based on an exposure of 418 days. The ratio of upward- to downward-going events in the data is compared to the Monte Carlo expectation in the absence of neutrino oscillations, giving Rup/downdata/Rup/downMC=0.62-0.14+0.19(stat.)±0.02(sys.). An extended maximum likelihood analysis of the observed L/E distributions excludes the null hypothesis of no neutrino oscillations at the 98% confidence level. Using the curvature of the observed muons in the 1.3 T MINOS magnetic field nuµ and [overline nu ]µ interactions are separated. The ratio of [overline nu ]µ to nuµ events in the data is compared to the Monte Carlo expectation assuming neutrinos and antineutrinos oscillate in the same manner, giving R[overline nu ][sub mu]/nu[sub mu]data/R[overline nu ][sub mu]/nu[sub mu]MC=0.96-0.27+0.38(stat.)±0.15(sys.), where the errors are the statistical and systematic uncertainties. Although the statistics are limited, this is the first direct observation of atmospheric neutrino interactions separately for nuµ and [overline nu ]µ

The N-Terminal Domain of the Drosophila Retinoblastoma Protein Rbf1 Interacts with ORC and Associates with Chromatin in an E2F Independent Manner

The retinoblastoma (Rb) tumor suppressor protein can function as a DNA replication inhibitor as well as a transcription factor. Regulation of DNA replication may occur through interaction of Rb with the origin recognition complex (ORC).We characterized the interaction of Drosophila Rb, Rbf1, with ORC. Using expression of proteins in Drosophila S2 cells, we found that an N-terminal Rbf1 fragment (amino acids 1-345) is sufficient for Rbf1 association with ORC but does not bind to dE2F1. We also found that the C-terminal half of Rbf1 (amino acids 345-845) interacts with ORC. We observed that the amino-terminal domain of Rbf1 localizes to chromatin in vivo and associates with chromosomal regions implicated in replication initiation, including colocalization with Orc2 and acetylated histone H4.Our results suggest that Rbf1 can associate with ORC and chromatin through domains independent of the E2F binding site. We infer that Rbf1 may play a role in regulating replication directly through its association with ORC and/or chromatin factors other than E2F. Our data suggest an important role for retinoblastoma family proteins in cell proliferation and tumor suppression through interaction with the replication initiation machinery

Charge-separated atmospheric neutrino-induced muons in the MINOS far detector

14 pages, 15 figuresWe found 140 neutrino-induced muons in 854.24 live days in the MINOS far detector. We looked for evidence of neutrino disappearance in this data set by computing the ratio of the number of low momentum muons to the sum of the number of high momentum and unknown momentum muons for both data and Monte Carlo expectation in the absence of neutrino oscillations. The ratio of data and Monte Carlo ratios is consistent with an oscillation signal. A fit to the data for the oscillation parameters excludes the null oscillation hypothesis at the 94% confidence level. We separated the muons by charge sign in both the data and Monte Carlo events and found the ratio of the total number of negative to positive muons in both samples. The ratio of those ratios is a test of CPT conservation. The result is consistent with CPT conservation

Observation of Muon Neutrino Disappearance with the MINOS Detectors in the NuMI Neutrino Beam

This Letter reports results from the MINOS experiment based on its initial exposure to neutrinos from the Fermilab NuMI beam. The rates and energy spectra of charged current νμ interactions are compared in two detectors located along the beam axis at distances of 1 and 735 km. With 1.27×1020 120 GeV protons incident on the NuMI target, 215 events with energies below 30 GeV are observed at the Far Detector, compared to an expectation of 336±14 events. The data are consistent with νμ disappearance via oscillations with |Δm322|=2.74-0.26+0.44×10-3  eV2 and sin⁡2(2θ23)>0.87 (68% C.L.)

Hypoglycaemic stimulation of macrophage cytokine release is suppressed by AMP-activated protein kinase activation

This is the author accepted manuscript.Data Availability: All data generated or analysed during this study are included in the published article (and its ESM). The file is available from the corresponding author upon reasonable request.Aims/hypothesis: Acute hypoglycaemia promotes pro-inflammatory cytokine production, increasing risk for cardiovascular events in diabetes. AMP-activated protein kinase (AMPK) is regulated by and influences production of pro-inflammatory cytokines. We sought to examine the mechanistic role of AMPK in low glucose-induced changes in the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), which is elevated in people with diabetes. Methods: Macrophage cell line Raw264.7 cells, primary macrophage bone marrow derived macrophages obtained from wild type mice or AMPK γ1 gain-of-function mice were used, as were AMPK1/2 knockout mouse embryonic fibroblasts (MEF). Allosteric AMPK activators PF-06409577 and BI-9774 were used, in conjunction with inhibitor SBI-0206965. We examined changes in protein phosphorylation/expression using western blotting, and protein localisation using immunofluorescence. Metabolic function was assessed using extracellular flux analyses and luciferase-based ATP assay. Cytokine release was quantified by ELISA. Oxidative stress was detected using a fluorescence-based ROS assay, and cell viability was examined using flow cytometry. Results: Macrophages exposed to low glucose showed a transient and modest activation of AMPK and a metabolic shift towards increased oxidative phosphorylation. Moreover, low glucose increased oxidative stress and augmented release of macrophage migration inhibitory factor (MIF). However, pharmacological activation of AMPK by PF-06409577 and BI-9774 attenuated low glucose-induced MIF release, with a similar trend noted with genetic activation using AMPKγ1 gain-of-function (D316A) mice, which produced a mild effect on low glucose-induced MIF release. Inhibition of NFĸB signalling diminished MIF release and AMPK activation modestly but significantly reduced low glucose-induced nuclear translocation of NFĸB. Conclusions/interpretation Taken together, these data indicate that pharmacological AMPK activation suppresses release of MIF from macrophages caused by energy stress, suggesting that AMPK activation could be a useful strategy for mitigating hypoglycaemia-induced inflammation.Medical Research Council (MRC)Diabetes UKAstraZenecaBiotechnology and Biological Sciences Research Council (BBSRC