Human neuronal stargazin-like proteins, γ_2, γ_3 and γ_4; an investigation of their specific localization in human brain and their influence on Ca_V2.1 voltage-dependent calcium channels expressed in Xenopus oocytes

Background: Stargazin (γ2) and the closely related γ3, and γ4 transmembrane proteins are part of a family of proteins that may act as both neuronal voltage-dependent calcium channel (VDCC) γ subunits and transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazoleproponinc (AMPA) receptor regulatory proteins (TARPs). In this investigation, we examined the distribution patterns of the stargazin-like proteins γ2, γ3, and γ4 in the human central nervous system (CNS). In addition, we investigated whether human γ2 or γ4 could modulate the electrophysiological properties of a neuronal VDCC complex transiently expressed in Xenopus oocytes. Results: The mRNA encoding human γ2 is highly expressed in cerebellum, cerebral cortex, hippocampus and thalamus, whereas γ3 is abundant in cerebral cortex and amygdala and γ4 in the basal ganglia. Immunohistochemical analysis of the cerebellum determined that both γ2 and γ4 are present in the molecular layer, particularly in Purkinje cell bodies and dendrites, but have an inverse expression pattern to one another in the dentate cerebellar nucleus. They are also detected in the interneurons of the granule cell layer though only γ2 is clearly detected in granule cells. The hippocampus stains for γ2 and γ4 throughout the layers of the every CA region and the dentate gyrus, whilst γ3 appears to be localized particularly to the pyramidal and granule cell bodies. When co-expressed in Xenopus oocytes with a CaV2.1/β4 VDCC complex, either in the absence or presence of an α2δ2 subunit, neither γ2 nor γ4 significantly modulated the VDCC peak current amplitude, voltage-dependence of activation or voltage-dependence of steady-state inactivation. Conclusion: The human γ2, γ3 and γ4 stargazin-like proteins are detected only in the CNS and display differential distributions among brain regions and several cell types in found in the cerebellum and hippocampus. These distribution patterns closely resemble those reported by other laboratories for the rodent orthologues of each protein. Whilst the fact that neither γ2 nor γ4 modulated the properties of a VDCC complex with which they could associate in vivo in Purkinje cells adds weight to the hypothesis that the principal role of these proteins is not as auxiliary subunits of VDCCs, it does not exclude the possibility that they play another role in VDCC function

Human neuronal stargazin-like proteins, γ(2), γ(3 )and γ(4); an investigation of their specific localization in human brain and their influence on Ca(V)2.1 voltage-dependent calcium channels expressed in Xenopus oocytes.

BACKGROUND: Stargazin (γ(2)) and the closely related γ(3), and γ(4 )transmembrane proteins are part of a family of proteins that may act as both neuronal voltage-dependent calcium channel (VDCC) γ subunits and transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazoleproponinc (AMPA) receptor regulatory proteins (TARPs). In this investigation, we examined the distribution patterns of the stargazin-like proteins γ(2), γ(3), and γ(4 )in the human central nervous system (CNS). In addition, we investigated whether human γ(2 )or γ(4 )could modulate the electrophysiological properties of a neuronal VDCC complex transiently expressed in Xenopus oocytes. RESULTS: The mRNA encoding human γ(2 )is highly expressed in cerebellum, cerebral cortex, hippocampus and thalamus, whereas γ(3 )is abundant in cerebral cortex and amygdala and γ(4 )in the basal ganglia. Immunohistochemical analysis of the cerebellum determined that both γ(2 )and γ(4 )are present in the molecular layer, particularly in Purkinje cell bodies and dendrites, but have an inverse expression pattern to one another in the dentate cerebellar nucleus. They are also detected in the interneurons of the granule cell layer though only γ(2 )is clearly detected in granule cells. The hippocampus stains for γ(2 )and γ(4 )throughout the layers of the every CA region and the dentate gyrus, whilst γ(3 )appears to be localized particularly to the pyramidal and granule cell bodies. When co-expressed in Xenopus oocytes with a Ca(V)2.1/β(4 )VDCC complex, either in the absence or presence of an α2δ(2 )subunit, neither γ(2 )nor γ(4 )significantly modulated the VDCC peak current amplitude, voltage-dependence of activation or voltage-dependence of steady-state inactivation. CONCLUSION: The human γ(2), γ(3 )and γ(4 )stargazin-like proteins are detected only in the CNS and display differential distributions among brain regions and several cell types in found in the cerebellum and hippocampus. These distribution patterns closely resemble those reported by other laboratories for the rodent orthologues of each protein. Whilst the fact that neither γ(2 )nor γ(4 )modulated the properties of a VDCC complex with which they could associate in vivo in Purkinje cells adds weight to the hypothesis that the principal role of these proteins is not as auxiliary subunits of VDCCs, it does not exclude the possibility that they play another role in VDCC function

Effect of pasture species on internal parasites of lambs

Paper presented at the 58th New Zealand Grassland Association Conference, 21-24 October 1996, Oamaru.Increasing resistance of gastro-intestinal nematode parasites to anthelmintics and consumer resistance to the possibility of residues in animal products have prompted research on the effect of pasture species on nematodes and animal performance. Lambs (either infected with high rates of gastrointestinal nematodes or maintained nematode-free) were grazed on pure swards of chicory, high- or low-endophyte ryegrass, cocksfoot, tall fescue, lucerne, lotus, white clover or plantain. Infected lambs that grazed chicory had lower faecal egg counts and adult nematode populations, and higher carcass weights, than lambs grazed on plantain or the grass species; lambs that grazed legumes generally had intermediate counts, populations and weights. When kept parasite-free, carcass weights were up to 48% greater than in the nematode infected treatments. On farmlets run over 3 years, substituting 30% of the ryegrass area with lucerne or replacing the ryegrass with a multi-species mix consisting predominantly of bromes, tall fescue, phalaris, timothy and red and white clover, had no effect on gastro-intestinal nematode larvae, lamb faecal worm egg or adult nematode numbers. It is concluded that a diet of pure chicory affects internal parasite populations but the small proportion included in the farmlet studies had no effect.We wish to thank MRDC for funding Project 1 and AGMARDT for funding Project 2

The Stargazin-Related Protein {gamma}7 Interacts with the mRNA-Binding Protein Heterogeneous Nuclear Ribonucleoprotein A2 and Regulates the Stability of Specific mRNAs, Including CaV2.2

The role(s) of the novel stargazin-like {gamma}-subunit proteins remain controversial. We have shown previously that the neuron-specific {gamma}7 suppresses the expression of certain calcium channels, particularly CaV2.2, and is therefore unlikely to operate as a calcium channel subunit. We now show that the effect of {gamma}7 on CaV2.2 expression is via an increase in the degradation rate of CaV2.2 mRNA and hence a reduction of CaV2.2 protein level. Furthermore, exogenous expression of {gamma}7 in PC12 cells also decreased the endogenous CaV2.2 mRNA level. Conversely, knockdown of endogenous {gamma}7 with short-hairpin RNAs produced a reciprocal enhancement of CaV2.2 mRNA stability and an increase in endogenous calcium currents in PC12 cells. Moreover, both endogenous and expressed {gamma}7 are present on intracellular membranes, rather than the plasma membrane. The cytoplasmic C terminus of {gamma}7 is essential for all its effects, and we show that {gamma}7 binds directly via its C terminus to a heterogeneous nuclear ribonucleoprotein (hnRNP A2), which also binds to a motif in CaV2.2 mRNA, and is associated with native CaV2.2 mRNA in PC12 cells. The expression of hnRNP A2 enhances CaV2.2 IBa, and this enhancement is prevented by a concentration of {gamma}7 that alone has no effect on IBa. The effect of {gamma}7 is selective for certain mRNAs because it had no effect on {alpha}2{delta}-2 mRNA stability, but it decreased the mRNA stability for the potassium-chloride cotransporter, KCC1, which contains a similar hnRNP A2 binding motif to that in CaV2.2 mRNA. Our results indicate that {gamma}7 plays a role in stabilizing CaV2.2 mRNA

The impact of population-based faecal occult blood test screening on colorectal cancer mortality:a matched cohort study

BACKGROUND: Randomised trials show reduced colorectal cancer (CRC) mortality with faecal occult blood testing (FOBT). This outcome is now examined in a routine, population-based, screening programme. METHODS: Three biennial rounds of the UK CRC screening pilot were completed in Scotland (2000–2007) before the roll out of a national programme. All residents (50–69 years) in the three pilot Health Boards were invited for screening. They received a FOBT test by post to complete at home and return for analysis. Positive tests were followed up with colonoscopy. Controls, selected from non-pilot Health Boards, were matched by age, gender, and deprivation and assigned the invitation date of matched invitee. Follow-up was from invitation date to 31 December 2009 or date of death if earlier. RESULTS: There were 379 655 people in each group (median age 55.6 years, 51.6% male). Participation was 60.6%. There were 961 (0.25%) CRC deaths in invitees, 1056 (0.28%) in controls, rate ratio (RR) 0.90 (95% confidence interval (CI) 0.83–0.99) overall and 0.73 (95% CI 0.65–0.82) for participants. Non-participants had increased CRC mortality compared with controls, RR 1.21 (95% CI 1.06–1.38). CONCLUSION: There was a 10% relative reduction in CRC mortality in a routine screening programme, rising to 27% in participants

Nicotine Normalizes Intracellular Subunit Stoichiometry of Nicotinic Receptors Carrying Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is linked with high penetrance to several distinct nicotinic receptor (nAChR) mutations. We studied (α4)_3(2β)_2 versus (α4)_2(β2)_3 subunit stoichiometry for five channel-lining M2 domain mutations: S247F, S252L, 776ins3 in α4, V287L, and V287M in β2. α4 and β2 subunits were constructed with all possible combinations of mutant and wild-type (WT) M2 regions, of cyan and yellow fluorescent protein, and of fluorescent and nonfluorescent M3-M4 loops. Sixteen fluorescent subunit combinations were expressed in N2a cells. Forster resonance energy transfer (FRET) was analyzed by donor recovery after acceptor photobleaching and by pixel-by-pixel sensitized emission, with confirmation by fluorescence intensity ratios. Because FRET efficiency is much greater for adjacent than for nonadjacent subunits and the α4 and β2 subunits occupy specific positions in nAChR pentamers, observed FRET efficiencies from (α4)_3(β2)_2 carrying fluorescent α4 subunits were significantly higher than for (α4)_2(β2)_3; the converse was found for fluorescent 2 subunits. All tested ADNFLE mutants produced 10 to 20% increments in the percentage of intracellular (α4)_3(β2)_2 receptors compared with WT subunits. In contrast, 24- to 48-h nicotine (1 µM) exposure increased the proportion of (α4)_2(β2)_3 in WT receptors and also returned subunit stoichiometry to WT levels for α4S248F and β2V287L nAChRs. These observations may be relevant to the decreased seizure frequency in patients with ADNFLE who use tobacco products or nicotine patches. Fluorescence-based investigations of nAChR subunit stoichiometry may provide efficient drug discovery methods for nicotine addiction or for other disorders that result from dysregulated nAChRs

Förster Resonance Energy Transfer (FRET) Correlates of Altered Subunit Stoichiometry in Cys-Loop Receptors, Exemplified by Nicotinic α4β2

We provide a theory for employing Förster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose α4 and β2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (α4)3(β2)2 vs. (α4)2(β2)3. Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (α4)3(β2)2 exceeds that of (α4)2(β2)3. The simulations account for published data on transfected Neuro2a cells in which α4β2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (α4)3(β2)2 stoichiometry to mostly (α4)2(β2)3. The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (α4)2(β2)3 and (α4)3(β2)2 populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors

Successful Establishment of Oversown Chicory and Plantain on Uncultivatable Hill Country

All-year grazing of livestock on steep, non-arable hill country (\u3e 20o slope angle, \u3c 1,000 m elevation) is a significant feature of New Zealand agriculture. Hill country pastures are in various states of improvement depending on factors such as extent of subdivision, fertiliser inputs, plant species introduction, and grazing management. Numerous introduced grass, legume and herb species are available to match the many micro-sites in steep hill country (Kemp et al. 1999). There has been increasing use of the perennial herbs chicory (Chicorium intybus L.) and plantain (Plantago lanceolata L.) in seed mixtures used on a range of topographies, mostly flat to undulating terrain. Advantages of these species include tolerance of drought and high summer temperatures, highly palatable foliage, enhanced mineral content, and high animal growth rates (Stewart 1996; Li and Kemp 2005). Farmers have sown these species on hill country but there is negligible information on their establishment in such landscapes. As part of a large, New Zealand-wide programme to increase pasture productivity on non-arable hill country through new germplasm introduction, chicory and plantain were included in a seed mixture broadcast-sown at a range of sites. This paper reports on the seedling establishment of these two species

Intrinsic and extrinsic drivers of activity budgets in sympatric grey and harbour seals

D. J. F. Russell was funded by the UK Department of Energy and Climate Change (DECC) as part of their Offshore Energy Strategic Environmental Assessment programme and by Scottish Government as part of their Marine Mammal Scientific Support Research Programme (MMSS/001/11). The telemetry tags and their deployment were funded by DECC, the Natural Environment Research Council, Scottish Government, Marine Scotland Science and The European Commission.Investigation of activity budgets in relation to seasonal, intrinsic (age, sex) and extrinsic (time of day, spatial) covariates enables an understanding of how such covariates shape behavioural strategies. However, conducting such investigations in the wild is challenging, because of the required large sample size of individuals across the annual cycle, and difficulties in categorising behavioural states and analysing the resulting individual-referenced and serially correlated data. In this study, from telemetry tags deployed on 63 grey seals (Halichoerus grypus) and 126 harbour seals (Phoca vitulina) we used behavioural data, and movement data within a Bayesian state-space model (SSM), to define population-level activity budgets around Britain. Using generalised estimating equations (GEEs) we then examined how time spent in four states (resting on land (hauled out), resting at sea, foraging and travelling) was influenced by seasonal, intrinsic and extrinsic covariates. We present and discuss the following key findings. (1) We found no evidence that regional variation in foraging effort was linked to regional population trajectories in harbour seals. (2) Grey seals demonstrated sex-specific seasonal differences in their activity budgets, independent from those related to reproductive costs. (3) In these sympatric species there was evidence of temporal separation in time hauled out, but not in time foraging. (4) In both species, time spent resting at sea was separated into inshore (associated with tidal haul out availability) and offshore areas. Time spent resting at sea and on land was interchangeable to some extent, suggesting a degree of overlap in their functionality. This may result in a relaxation of the constraints associated with a central place foraging strategy. More generally, we demonstrate how a large dataset, incorporating differing tag parameters, can be analysed to define activity budgets and subsequently address important ecological questions.PostprintPeer reviewe

Novel RPTPγ and RPTPζ splice variants from mixed neuron–astrocyte hippocampal cultures as well as from the hippocampi of newborn and adult mice

Receptor protein tyrosine phosphatases γ and ζ (RPTPγ and RPTPζ) are transmembrane signaling proteins with extracellular carbonic anhydrase–like domains that play vital roles in the development and functioning of the central nervous system (CNS) and are implicated in tumor suppression, neurodegeneration, and sensing of extracellular [CO2] and [HCO3−]. RPTPγ expresses throughout the body, whereas RPTPζ preferentially expresses in the CNS. Here, we investigate differential RPTPγ-RPTPζ expression in three sources derived from a wild-type laboratory strain of C57BL/6 mice: (a) mixed neuron–astrocyte hippocampal (HC) cultures 14 days post isolation from P0–P2 pups; (b) P0–P2 pup hippocampi; and (c) 9- to 12-week-old adult hippocampi. Regarding RPTPγ, we detect the Ptprg variant-1 (V1) transcript, representing canonical exons 1–30. Moreover, we newly validate the hypothetical assembly [XM_006517956] (propose name, Ptprg-V3), which lacks exon 14. Both transcripts are in all three HC sources. Regarding RPTPζ, we confirm the expression of Ptprz1-V1, detecting it in pups and adults but not in cultures, and Ptprz1-V3 through Ptprz1-V7 in all three preparations. We newly validate hypothetical assemblies Ptprz1-X1 (in cultures and pups), Ptprz1-X2 (in all three), and Ptprz1-X5 (in pups and adults) and propose to re-designate them as Ptprz1-V0, Ptprz1-V2, and Ptprz1-V8, respectively. The diversity of RPTPγ and RPTPζ splice variants likely corresponds to distinct signaling functions, in different cellular compartments, during development vs later life. In contrast to previous studies that report divergent RPTPγ and RPTPζ protein expressions in neurons and sometimes in the glia, we observe that RPTPγ and RPTPζ co-express in the somata and processes of almost all HC neurons but not in astrocytes, in all three HC preparations