Stable silencing of SNAP-25 in PC12 cells by RNA interference

BACKGROUND: SNAP-25 is a synaptic protein known to be involved in exocytosis of synaptic vesicles in neurons and of large dense-core vesicles in neuroendocrine cells. Its role in exocytosis has been studied in SNAP-25 knockout mice, in lysed synaptosomes lacking functional SNAP-25 and in cells after treatment with botulinum toxins A or E that specifically cleave SNAP-25. These studies have shown that SNAP-25 appears to be required for most but not all evoked secretion. In order to further study the role of SNAP-25 in catecholamine secretion from PC12 cells we have used the recently developed technique of RNA interference to generate PC12 cell lines with virtually undetectable levels of SNAP-25. RNA interference is the sequence-specific silencing or knockdown of gene expression triggered by the introduction of double-stranded RNA into a cell. RNA interference can be elicited in mammalian cells in a number of ways, one of which is by the expression of small hairpin RNAs from a transfected plasmid. Selection of stably transfected cell lines expressing a small hairpin RNA allows one-time characterization of the degree and specificity of gene silencing and affords a continuing source of well-characterized knockdown cells for experimentation. RESULTS: A PC12 cell line stably transfected with a plasmid expressing an shRNA targeting SNAP-25 has been established. This SNAP-25 knockdown cell line has barely detectable levels of SNAP-25, but normal levels of other synaptic proteins. Catecholamine secretion elicited by depolarization of the SNAP-25 knockdown cells was reduced to 37% of control. CONCLUSION: Knockdown of SNAP-25 in PC12 cells reduces but does not eliminate evoked secretion of catecholamines. Transient expression of human SNAP-25 in the knockdown cells rescues the deficit in catecholamine secretion

A pilot study showing that repeated exposure to stress produces alterations in subsequent responses to anesthetics in rats

The repeated use of a drug frequently leads to alterations in the response to that drug. We undertook this study to determine whether multiple exposures to the general anesthetic produced alterations in subsequent exposures to this anesthetic. For this study, adult male rats were anesthetized with 2.5% isoflurane for one hour. The rats were divided into 4 groups of 8 rats each. Groups 1–3 were transported between their homeroom and the anesthesia testing room and were handled in an identical manner weekly for a period of 12 weeks, but were anesthetized on different schedules. Group 1 was anesthetized weekly for 12 weeks, Group 2 on either a 3 or 4 week schedule and Group 3 was anesthetized a single time, at the end of the 12 week period. To receive anesthesia multiple times, animals were transported from their homeroom to the anesthesia location and handled repeatedly. We took into consideration of the frequency of anesthesia exposure and the stress involved. Rats in groups 2 and 3 were placed in the anesthesia chamber, with O2 but with no anesthetic, every week when they were not scheduled to receive anesthesia. In Group 4, rats were not transported or handled in any way and stayed in the home room for a period of 12 weeks. Rats in this group were anesthetized once, at the very end of the study. Recovery of the rat’s righting reflex was used to assess the acceleration of recovery time from general anesthesia. Group 1 rats showed dramatically faster emergence from anesthesia after several rounds of anesthesia. Surprisingly, Groups 2 and 3 rats, treated in an identical manner as Group 1, but which were anesthetized on different schedules, also exhibited more rapid emergence from anesthesia, when compared to Group 4 rats, which were never handled or transported prior to a single anesthesia. These results suggest that the stress of transportation and handling altered responsiveness to anesthesia. Our results show that responsiveness to anesthetic agents can change over time outside of the normal developmental changes taking place in rats as they age

Illinois Waterfowl Surveys and Investigations W-43-R-62 Annual Progress Report Period: 1 July 2014 – 30 June 2015

This study addresses the following objectives: 1)Inventory abundance and distribution of waterfowl and other waterbirds (a minimum of 10 species and guilds) during autumn migration at a minimum of 40 sites along the Illinois and central Mississippi rivers; 2) Investigate the ecology of canvasback and lesser scaup during spring migration in the central Illinois River valley (IRV) and Pool 19 of the Mississippi River; 3) Estimate waterfowl and other waterbird population sizes (a minimum of 10 species and guilds) during autumn migration using an aerial quadrat survey in the IRV for comparison with aerial inventories (Objective 1); 4) Determine breeding bird use of a minimum of 10 moist-soil wetlands managed for waterfowl during summer in central Illinois; 5) Investigate the breeding ecology of sandhill cranes during spring and summer in northeastern Illinois; 6) Distribute our findings to site managers and biologists, make recommendations for future management, and draw conclusions relevant to regional conservation planning during the project period as appropriate and requested.Illinois Department of Natural Resources, Division of Wildlife & U.S. Fish and Wildlife Service Contract Number: RC09-13FWUIUCunpublishednot peer reviewedOpe

Insulin signaling inhibits the 5-HT(2C )receptor in choroid plexus via MAP kinase

BACKGROUND: G protein-coupled receptors (GPCRs) interact with heterotrimeric GTP-binding proteins (G proteins) to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP) kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways. RESULTS: Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT(2C )receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK) inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1) on the 5-HT(2C )receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT(2C )receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT(2C )receptor abolished the effect of insulin. CONCLUSION: These results show that insulin signaling can inhibit 5-HT(2C )receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR

Simulating the effect of muscle weakness and contracture on neuromuscular control of normal gait in children

Altered neural control of movement and musculoskeletal deficiencies are common in children with spastic cerebral palsy (SCP), with muscle weakness and contracture commonly experienced. Both neural and musculoskeletal deficiencies are likely to contribute to abnormal gait, such as equinus gait (toe-walking), in children with SCP. However, it is not known whether the musculoskeletal deficiencies prevent normal gait or if neural control could be altered to achieve normal gait. This study examined the effect of simulated muscle weakness and contracture of the major plantarflexor/dorsiflexor muscles on the neuromuscular requirements for achieving normal walking gait in children. Initial muscle-driven simulations of walking with normal musculoskeletal properties by typically developing children were undertaken. Additional simulations with altered musculoskeletal properties were then undertaken; with muscle weakness and contracture simulated by reducing the maximum isometric force and tendon slack length, respectively, of selected muscles. Muscle activations and forces required across all simulations were then compared via waveform analysis. Maintenance of normal gait appeared robust to muscle weakness in isolation, with increased activation of weakened muscles the major compensatory strategy. With muscle contracture, reduced activation of the plantarflexors was required across the mid-portion of stance suggesting a greater contribution from passive forces. Increased activation and force during swing was also required from the tibialis anterior to counteract the increased passive forces from the simulated dorsiflexor muscle contracture. Improvements in plantarflexor and dorsiflexor motor function and muscle strength, concomitant with reductions in plantarflexor muscle stiffness may target the deficits associated with SCP that limit normal gait

Hypoxia sensing requires H₂S-dependent persulfidation of olfactory receptor 78

Oxygen (O2) sensing by the carotid body is critical for maintaining cardiorespiratory homeostasis during hypoxia. Hydrogen sulfide (H2S) signaling is implicated in carotid body activation by low O2. Here, we show that persulfidation of olfactory receptor 78 (Olfr78) by H2S is an integral component of carotid body activation by hypoxia. Hypoxia and H2S increased persulfidation in carotid body glomus cells and persulfidated cysteine240 in Olfr78 protein in heterologous system. Olfr78 mutants manifest impaired carotid body sensory nerve, glomus cell, and breathing responses to H2S and hypoxia. Glomus cells are positive for GOlf, adenylate cyclase 3 (Adcy3) and cyclic nucleotide–gated channel alpha 2 (Cnga2), key molecules of odorant receptor signaling. Adcy3 or Cnga2 mutants exhibited impaired carotid body and glomus cell responses to H2S and breathing responses to hypoxia. These results suggest that H2S through redox modification of Olfr78 participates in carotid body activation by hypoxia to regulate breathing

Observation of dose-rate dependence in a Fricke dosimeter irradiated at low dose rates with monoenergetic X-rays

<p>Absolute measurements of the radiolytic yield of Fe3+ in a ferrous sulphate dosimeter formulation (6 mM Fe2+), with a 20 keV x-ray monoenergetic beam, are reported. Dose-rate suppression of the radiolytic yield was observed at dose rates lower than and different in nature to those previously reported with x-rays. We present evidence that this effect is most likely to be due to recombination of free radicals radiolytically produced from water. The method used to make these measurements is also new and it provides radiolytic yields which are directly traceable to the SI standards system. The data presented provides new and exacting tests of radiation chemistry codes.</p

Ecological Consequences of Anomalies in Atmospheric Moisture and Snowpack

Although increased frequency of extreme‐weather events is one of the most secure predictions associated with contemporary climate change, effects of such events on distribution and abundance of climate‐sensitive species remain poorly understood. Montane ecosystems may be especially sensitive to extreme weather because of complex abiotic and biotic interactions that propagate from climate‐driven reductions in snowpack. Snowpack not only protects subnivean biotas from extreme cold, but also influences forage availability through timing of melt‐off and water availability. We related relative abundances of an alpine mammal, the American pika (Ochotona princeps), to measures of weather and snowpack dynamics over an 8‐yr period that included before and after a year of record‐low snowpack in Washington, USA. We sought to (1) quantify any change in pika abundance associated with the snowpack anomaly and (2) identify aspects of weather and snowpack that influenced abundance of pikas. Pikas showed a 1‐yr lag response to the snowpack anomaly and exhibited marked declines in abundance at elevations below 1,400 m simultaneous with increased abundances at higher elevations. Atmospheric moisture, indexed by vapor pressure deficit (VPD), was especially important, evidenced by strong support for the top‐ranked model that included the interaction of VPD with snowpack duration. Notably, our novel application of VPD from gridded climate data for analyses of animal abundances shows strong potential for improving species distribution models because VPD represents an important aspect of weather that influences the physiology and habitat of biota. Pikas were apparently affected by cold stress without snowpack at mid elevations, whereas changes to forage associated with snowpack and VPD were influential at high and low elevations. Our results reveal context dependency in pika responses to weather and illustrate how snow drought can lead to rapid change in the abundance of subnivean animals

Period and chemical evolution of SC stars

The SC and CS stars are thermal-pulsing AGB stars with C/O ratio close to unity. Within this small group, the Mira variable BH Cru recently evolved from spectral type SC (showing ZrO bands) to CS (showing weak C2). Wavelet analysis shows that the spectral evolution was accompanied by a dramatic period increase, from 420 to 540 days, indicating an expanding radius. The pulsation amplitude also increased. Old photographic plates are used to establish that the period before 1940 was around 490 days. Chemical models indicate that the spectral changes were caused by a decrease in stellar temperature, related to the increasing radius. There is no evidence for a change in C/O ratio. The evolution in BH Cru is unlikely to be related to an on-going thermal pulse. Periods of the other SC and CS stars, including nine new periods, are determined. A second SC star, LX Cyg, also shows evidence for a large increase in period, and one further star shows a period inconsistent with a previous determination. Mira periods may be intrinsically unstable for C/O ~ 1; possibly because of a feedback between the molecular opacities, pulsation amplitude, and period. LRS spectra of 6 SC stars suggest a feature at wavelength > 15 micron, which resembles one recently attributed to the iron-sulfide troilite. Chemical models predict a large abundance of FeS in SC stars, in agreement with the proposed association.Comment: 14 pages, 20 figures. MNRAS, 2004, accepted for publication. Janet Mattei, one of the authors, died on 22 March, 2004. This paper is dedicated to her memor

Reverberation Mapping of the Kepler-Field AGN KA1858+4850

KA1858+4850 is a narrow-line Seyfert 1 galaxy at redshift 0.078 and is among the brightest active galaxies monitored by the Kepler mission. We have carried out a reverberation mapping campaign designed to measure the broad-line region size and estimate the mass of the black hole in this galaxy. We obtained 74 epochs of spectroscopic data using the Kast Spectrograph at the Lick 3-m telescope from February to November of 2012, and obtained complementary V-band images from five other ground-based telescopes. We measured the H-beta light curve lag with respect to the V-band continuum light curve using both cross-correlation techniques (CCF) and continuum light curve variability modeling with the JAVELIN method, and found rest-frame lags of lag_CCF = 13.53 (+2.03, -2.32) days and lag_JAVELIN = 13.15 (+1.08, -1.00) days. The H-beta root-mean-square line profile has a width of sigma_line = 770 +/- 49 km/s. Combining these two results and assuming a virial scale factor of f = 5.13, we obtained a virial estimate of M_BH = 8.06 (+1.59, -1.72) x 10^6 M_sun for the mass of the central black hole and an Eddington ratio of L/L_Edd ~ 0.2. We also obtained consistent but slightly shorter emission-line lags with respect to the Kepler light curve. Thanks to the Kepler mission, the light curve of KA1858+4850 has among the highest cadences and signal-to-noise ratios ever measured for an active galactic nucleus; thus, our black hole mass measurement will serve as a reference point for relations between black hole mass and continuum variability characteristics in active galactic nuclei