Store-operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells.

KEY POINTS: Depletion of Ca(2+) stores activates store-operated channels (SOCs), which mediate Ca(2+) entry pathways that regulate cellular processes such as contraction, proliferation and gene expression. In vascular smooth muscle cells (VSMCs), stimulation of SOCs composed of canonical transient receptor potential channel 1 (TRPC1) proteins requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1/protein kinase C (PKC) activity. We studied the role of stromal interaction molecule 1 (STIM1) in coupling store depletion to this activation pathway using patch clamp recording, GFP-PLCδ1-PH imaging and co-localization techniques. Store-operated TRPC1 channel and PLCβ1 activities were inhibited by STIM1 short hairpin RNA (shRNA) and absent in TRPC1(-/-) cells, and store-operated PKC phosphorylation of TRPC1 was inhibited by STIM1 shRNA. Store depletion induced interactions between STIM1 and TRPC1, Gαq and PLCβ1, which required STIM1 and TRPC1. Similar effects were produced with noradrenaline. These findings identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, where store-operated STIM1-TRPC1 interactions stimulate Gαq/PLCβ1/PKC activity to induce channel gating. ABSTRACT: In vascular smooth muscle cells (VSMCs), stimulation of canonical transient receptor potential channel 1 (TRPC1) protein-based store-operated channels (SOCs) mediates Ca(2+) entry pathways that regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1-based SOCs requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1 activities and protein kinase C (PKC) phosphorylation, although it is unclear how store depletion stimulates this gating pathway. The present study examines this issue by focusing on the role of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum Ca(2+) sensor. Store-operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 short hairpin RNA (shRNA) in wild-type VSMCs, and was absent in TRPC1(-/-) VSMCs. Store-operated PKC phosphorylation of TRPC1 was reduced by knockdown of STIM1. Moreover, store-operated PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH was reduced by STIM1 shRNA and absent in TRPC1(-/-) cells. Immunocytochemistry, co-immunoprecipitation and proximity ligation assays revealed that store depletion activated STIM1 translocation from within the cell to the plasma membrane (PM) where it formed STIM1-TRPC1 complexes, which then associated with Gαq and PLCβ1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCβ1, which were inhibited by STIM1 knockdown. Effects of N-terminal and C-terminal STIM1 antibodies on TRPC1-based SOCs and STIM1 staining suggest that channel activation may involve insertion of STIM1 into the PM. The findings of the present study identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, in which store-operated STIM1-TRPC1 interactions stimulate PLCβ1 activity to induce PKC phosphorylation of TRPC1 and channel gating

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells.

Ca(2+)-permeable store-operated channels (SOCs) mediate Ca(2+) entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca(2+) sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCβ1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1(-/-) mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1(-/-) VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1(-/-) VSMCs, store depletion induced PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1(-/-) VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCβ1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype

The Distribution of H2O Maser Emission in the Nucleus of NGC 4945

We present the first interferometer map of the water maser emission in the active nucleus of NGC 4945. Although the declination of the galaxy is about -49 degrees, we were able to make the observations with the southernmost antennas of the Very Long Baseline Array. Strong maser emission is present in three velocity ranges, one near the systemic velocity and two shifted roughly symmetrically by +/-(100-150) km/s. This is the first detection of highly blue-shifted water emission in NGC 4945. We determined the position of the maser to be RA(B1950)= 13 02 32.28 +/- 0.02 ; Dec(B1950)= -49 12 01.9 +/- 0.1. The uncertainties in earlier estimates are at least several arcseconds. The maser lies within 2'' (36 pc at a distance of 3.7 Mpc) of the peaks in 1.4 GHz continuum and 1.6 micron emission from the nucleus. The mappable maser emission is distributed roughly linearly over about 40 milliarcseconds (0.7 pc) at a position angle of about 45 degrees, which is close to the 43 +/- 2 degree position angle of the galactic disk. The red and blue-shifted emission symmetrically stradle the systemic emission on the sky, which suggests material in edge-on circular motion around a central object. The position-velocity structure indicates a binding mass of about one million Suns, within a volume of radius about 0.3 pc. This implies that the central engine radiates on the order of 10% of its Eddington luminosity.Comment: 18 pages, including 5 Postscript figures. Accepted for publication in ApJ Letter

The Molonglo Galactic Plane Survey: I. Overview and Images

The first epoch Molonglo Galactic Plane Survey (MGPS1) is a radio continuum survey made using the Molonglo Observatory Synthesis Telescope (MOST) at 843 MHz with a resolution of 43" X 43" cosec |delta|. The region surveyed is 245 deg < l < 355 deg, |b| < 1.5 deg. The thirteen 9 deg X 3 deg mosaic images presented here are the superposition of over 450 complete synthesis observations, each taking 12 h and covering 70' X 70' cosec |delta|. The root-mean-square sensitivity over much of the mosaiced survey is 1-2 mJy/beam (1 sigma), and the positional accuracy is approximately 1" X 1" cosec |delta| for sources brighter than 20 mJy. The dynamic range is no better than 250:1, and this also constrains the sensitivity in some parts of the images. The survey area of 330 sq deg contains well over 12,000 unresolved or barely resolved objects, almost all of which are extra-galactic sources lying in the Zone of Avoidance. In addition a significant fraction of this area is covered by extended, diffuse emission associated with thermal complexes, discrete H II regions, supernova remnants, and other structures in the Galactic interstellar medium.Comment: Paper with 3 figures and 1 table + Table 2 + 7 jpg grayscales for Fig 4. Astrophysical Journal Supplement (in press) see also http://www.astrop.physics.usyd.edu.au/MGP

Mixing hetero- and homogeneous models in weighted ensembles

The effectiveness of ensembling for improving classification performance is well documented. Broadly speaking, ensemble design can be expressed as a spectrum where at one end a set of heterogeneous classifiers model the same data, and at the other homogeneous models derived from the same classification algorithm are diversified through data manipulation. The cross-validation accuracy weighted probabilistic ensemble is a heterogeneous weighted ensemble scheme that needs reliable estimates of error from its base classifiers. It estimates error through a cross-validation process, and raises the estimates to a power to accentuate differences. We study the effects of maintaining all models trained during cross-validation on the final ensemble's predictive performance, and the base model's and resulting ensembles' variance and robustness across datasets and resamples. We find that augmenting the ensemble through the retention of all models trained provides a consistent and significant improvement, despite reductions in the reliability of the base models' performance estimates

PKS 1018-42: A Powerful Kinetically Dominated Quasar

We have identified PKS 1018-42 as a radio galaxy with extraordinarily powerful jets, over twice as powerful as any 3CR source of equal or lesser redshift except for one (3C196). It is perhaps the most intrinsically powerful extragalactic radio source in the, still poorly explored, Southern Hemisphere. PKS 1018-42 belongs to the class of FR II objects that are kinetically dominated, the jet kinetic luminosity, $Q \sim 6.5 \times 10^{46}\mathrm{ergs/s}$ (calculated at 151 MHz), is 3.4 times larger than the total thermal luminosity (IR to X-ray) of the accretion flow, $L_{bol} \sim 1.9 \times 10^{46}\mathrm{ergs/s}$. It is the fourth most kinetically dominated quasar that we could verify from existing radio data. From a review of the literature, we find that kinetically dominated sources such as PKS 1018-42 are rare, and list the 5 most kinetically dominated sources found from our review. Our results for PKS 1018-42 are based on new observations from the Australia Telescope Compact Array.Comment: To appear in ApJ Letter

The relationship between web enjoyment and student perceptions and learning using a web-based tutorial

Web enjoyment has been regarded as a component of system experience. However, there has been little targeted research considering the role of web enjoyment alone in student learning using web-based systems. To address this gap, this study aims to examine the influence of web enjoyment on learning performance and perceptions by controlling system experience as a variable in the study. 74 students participated in the study, using a web-based tutorial covering subject matter in the area of 'Computation and algorithms'. Their learning performance was assessed with a pre-test and a post-test and their learning perceptions were evaluated with a questionnaire. The results indicated that there are positive relationships between the levels of web enjoyment and perceived usefulness and non-linear navigation for users with similar, significant levels of system experience. The implications of these findings in relation to web-based learning are explored and ways in which the needs of students who report different levels of web enjoyment might be met are discussed

Stringent neutron-star limits on large extra dimensions

Supernovae (SNe) are copious sources for Kaluza-Klein gravitons which are generic for theories with large extra dimensions. These massive particles are produced with average velocities ~0.5 c so that many of them are gravitationally retained by the SN core. Every neutron star thus has a halo of KK gravitons which decay into nu bar-nu, e^+e^- and gamma gamma on time scales \~10^9 years. The EGRET gamma-flux limits (E_gamma ~ 100 MeV) for nearby neutron stars constrain the fundamental scale for n=2 extra dimensions to M >500 TeV, and M>30 TeV for n=3. The upcoming GLAST satellite is a factor ~30 more sensitive and thus may detect KK decays, for example at the nearby neutron star RX J185635--3754. The requirement that neutron stars are not excessively heated by KK decays implies M>1700 TeV for n=2, and M>60 TeV for n=3.Comment: Minor changes, matches version to appear in PR

The optical-ultraviolet continuum of Seyfert 2 galaxies

This paper aims to understand the continuum of Seyfert 2 galaxies. By fitting the single galaxies in the sample of Heckman et al. (1995) with composite models (shock+ photoionization from the active center), we show that five main components characterize the SED of the continuum. Shocks play an important role since they produce a high temperature zone where soft X-rays are emitted. We show that in the optical-UV range, the slope of the NLR emission reproduces the observed values, and may be the main component of the featureless continuum. The presence of star forming regions cannot be excluded in the circumnuclear region of various Seyfert galaxies. An attempt is made to find their fingerprints in the observed AGN spectra. Finally, it is demonstrated that multi-cloud models are necessary to interpret the spectra of single objects, even in the global investigation of a sample of galaxies.Comment: 26 pages, LaTeX (including 5 Tables) + 17 PostScript figures. To appear in "The Astrophysical Journal