Cloning, expression, and localization of a rat brain high-affinity glycine transporter

A cDNA clone encoding a glycine transporter has been isolated from rat brain by a combined PCR and plaque-hybridization strategy. mRNA synthesized from this clone (designated GLYT1) directs the expression of sodium-and chloride-dependent, high-affinity uptake of [3H]glycine by Xenopus oocytes. [3H]Glycine transport mediated by clone GLYT1 is blocked by sarcosine but is not blocked by methylaminoisobutyric acid or L-alanine, a substrate specificity similar to that described for a previously identified glycine-uptake system called system Gly. In situ hybridization reveals that GLYT1 is prominently expressed in the cervical spinal cord and brainstem, two regions of the central nervous system where glycine is a putative neurotransmitter. GLYT1 is also strongly expressed in the cerebellum and olfactory bulb and is expressed at lower levels in other brain regions. The open reading frame of the GLYT1 cDNA predicts a protein containing 633 amino acids with a molecular mass of ≈70 kDa. The primary structure and hydropathicity profile of GLYT1 protein reveal that this protein is a member of the sodium- and chloride-dependent superfamily of transporters that utilize neurotransmitters and related substances as substrates

Protein kinase C modulates the activity of a cloned gamma-aminobutyric acid transporter expressed in Xenopus oocytes via regulated subcellular redistribution of the transporter

We report that activators and inhibitors of protein kinase C (PKC) and protein phosphatases regulate the activity of a cloned rat brain gamma- aminobutyric acid (GABA) transporter (GAT1) expressed in Xenopus oocytes. Four compounds known to activate PKC increased GABA uptake 2- 3.5-fold over basal control levels. Inhibition of PKC by bisindolylmaleimide reduced basal GABA uptake 80% and blocked the phorbol 12-myristate 13-acetate (PMA)-induced stimulation of transport. Okadaic acid, a protein phosphatase inhibitor, stimulated transport 2.5- fold; a 4-fold increase in GABA uptake occurred when oocytes were treated with cyclosporin A, a specific inhibitor of protein phosphatase 2B. Modulation resulted in changes to Vmax but not to Km and was influenced by the functional expression level of the transporter protein; as expression level increased, the ability to up-regulate transporter activity decreased. Down-regulation of transporter activity was independent of expression level. Modulation did not occur through phosphorylation of the three consensus PKC sites predicted by the primary protein sequence since their removal had no effect on the susceptibility of the transporter to modulation by PMA or bisindolylmaleimide. Subcellular fractionation of oocyte membranes demonstrated that under basal level conditions, the majority of GAT1 was targeted to a cytoplasmic compartment corresponding to the trans- Golgi or low density vesicles. Stimulation of PKC with PMA resulted in a translocation of transporters from this compartment to the plasma membrane. At higher expression levels of GAT1 protein, a larger portion of GAT1 was found on the plasma membrane during basal level conditions and treatment with bisindolylmaleimide resulted in removal of these transporters from the plasma membrane. At expression levels demonstrated to be resistant to modulation by PMA, PMA-treatment still resulted in translocation of transporters from the cytoplasm to the plasma membrane. Thus, the inability of PMA to increase uptake at high expression of the GAT1 protein is due to saturation at a step subsequent to translocation. These findings 1) demonstrate the presence of a novel regulated secretory pathway in oocytes and 2) suggest a modulatory mechanism for neurotransmitter transporters that could have significant effects upon synaptic function

Alternative Complementation in Partially Schematic Constructions: a Quantitative Corpus-based Examination of COME to V2 and GET to V2

This paper examines two English polyverbal constructions, COME to V2 and GET to V2, as exemplified in Examples 1 and 2, respectively. (1) the senator came to know thousands of his constituents (2) Little Johnny got to eat ice cream after every little league game. Previous studies considered these types of constructions (though come and get as used here have not been sufficiently studied) as belonging to a special class of complement constructions, in which the infinitive is regarded as instantiating a separate, subordinate predication from that of the “matrix” or leftward finite verb. These constructions, however, exhibit systematic deviation from the various criteria proposed in previous research. This study uses the American National Corpus to investigate the statistical propensities of the target phenomena via lexico-syntactic (collostructional analysis) and morpho-syntactic (binary logistic regression) features, as captured through the lens of construction grammar

Gut mucosal colonisation with extended-spectrum beta-lactamase producing Enterobacteriaceae in sub-Saharan Africa: a systematic review and meta-analysis.

Background: Extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) threaten human health; and, in areas of sub-Saharan Africa (sSA) where carbapenems are not available, may render ESBL-E infections untreatable. Gut mucosal colonisation probably occurs before infection, making prevention of colonisation an attractive target for intervention, but the epidemiology of ESBL-E in sSA is poorly described. Objectives: Describe ESBL-E colonisation prevalence in sSA and risk factors associated with colonisation. Methods: Studies included were prospective cross-sectional or cohort studies reporting gut mucosal ESBL-E colonisation in any population in sSA. We searched PubMed and Scopus on 18 December 2018. We summarise the range of prevalence across sites and tabulated risk factors for colonisation. The protocol was registered (Prospero ID CRD42019123559). Results: From 2975 abstracts we identified 32 studies including a total of 8619 participants from a range of countries and settings. Six studies were longitudinal; no longitudinal studies followed patients beyond hospital discharge.  Prevalence varied between 5 and 84% with a median of 31%, with a relationship to setting: pooled ESBL-E colonisation in community studies was 18% (95% CI 12 to 28, 12 studies); in studies recruiting people at admission to hospital colonisation was 32% (95% CI 24 to 41% 8 studies); and for inpatients, colonisation was 55% (95% CI 49 to 60%, 7 studies). Antimicrobial use was associated with increased risk of ESBL-E colonisation, and protected water sources or water treatment by boiling may reduce risk. Conclusions: ESBL-E colonisation is common in sSA, but how people become carriers and why is not well understood. To inform the design of interventions to interrupt transmission in this setting requires longitudinal, community studies

Chronic nicotine improves cognitive and social impairment in mice overexpressing wild type α-synuclein

In addition to dopaminergic and motor deficits, patients with Parkinson's disease (PD) suffer from non-motor symptoms, including early cognitive and social impairment, that do not respond well to dopaminergic therapy. Cholinergic deficits may contribute to these problems, but cholinesterase inhibitors have limited efficacy. Mice over-expressing α-synuclein, a protein critically associated with PD, show deficits in cognitive and social interaction tests, as well as a decrease in cortical acetylcholine. We have evaluated the effects of chronic administration of nicotine in mice over-expressing wild type human α-synuclein under the Thy1-promoter (Thy1-aSyn mice). Nicotine was administered subcutaneously by osmotic minipump for 6 months from 2 to 8 months of age at 0.4 mg/kg/h and 2.0 mg/kg/h. The higher dose was toxic in the Thy1-aSyn mice, but the low dose was well tolerated and both doses ameliorated cognitive impairment in Y-maze performance after 5 months of treatment. In a separate cohort of Thy1-aSyn mice, nicotine was administered at the lower dose for one month beginning at 5 months of age. This treatment partially eliminated the cognitive deficit in novel object recognition and social impairment. In contrast, chronic nicotine did not improve motor deficits after 2, 4 or 6 months of treatment, nor modified α-synuclein aggregation, tyrosine hydroxylase immunostaining, synaptic and dendritic markers, or microglial activation in Thy1-aSyn mice. These results suggest that cognitive and social impairment in synucleinopathies like PD may result from deficits in cholinergic neurotransmission and may benefit from chronic administration of nicotinic agonists

Control of the diffracted response of a metallic wire array with double period: experimental demonstration

In recent papers, it has been theoretically shown that by using dual-period wire gratings, it is possible to control the relative efficiencies of the diffracted orders, regardless of the wires’ material, incident polarization and wavelength. In this Letter, we experimentally demonstrate, for the first time, that by appropriately choosing the geometrical parameters of a nanometric periodic structure, it is possible to control the optical response in the visible range. We show examples of nanostructures designed to cancel out or to intensify a particular diffraction order. Such nanostructures allow a broad control over the directionality and the intensity of the diffracted light, which makes them useful for applications such as highly directional optical nanoantennas and photonic multiplexers.Centro de Investigaciones Óptica

Uropathic Observations in Mice Expressing a Constitutively Active Point Mutation in the 5-HT_(3A) Receptor Subunit

Mutant mice with a hypersensitive serotonin (5-HT)_(3A) receptor were generated through targeted exon replacement. A valine to serine mutation (V13′S) in the channel-lining M2 domain of the 5-HT_(3A) receptor subunit rendered the 5-HT₃ receptor ∼70-fold more sensitive to serotonin and produced constitutive activity when combined with the 5-HT_(3B) subunit. Mice homozygous for the mutant allele (5-HT_(3A)^(vs/vs)) had decreased levels of 5-HT_(3A) mRNA. Measurements on sympathetic ganglion cells in these mice showed that whole-cell serotonin responses were reduced, and that the remaining 5-HT₃ receptors were hypersensitive. Male 5-HT_(3A)^(vs/vs) mice died at 2-3 months of age, and heterozygous (5-HT_(3A)^(vs/+)) males and homozygous mutant females died at 4-6 months of age from an obstructive uropathy. Both male and female 5-HT_(3A) mutant mice had urinary bladder mucosal and smooth muscle hyperplasia and hypertrophy, whereas male mutant mice had additional prostatic smooth muscle and urethral hyperplasia. 5-HT_(3A) mutant mice had marked voiding dysfunction characterized by a loss of micturition contractions with overflow incontinence. Detrusor strips from 5-HT_(3A)^(vs/vs) mice failed to contract to neurogenic stimulation, despite overall normal responses to a cholinergic agonist, suggestive of altered neuronal signaling in mutant mouse bladders. Consistent with this hypothesis, decreased nerve fiber immunoreactivity was observed in the urinary bladders of 5-HT_(3A)^(vs/vs) compared with 5-HT_(3A) wild-type (5-HT_(3A)^(+/+)) mice. These data suggest that persistent activation of the hypersensitive and constitutively active 5-HT_(3A) receptor in vivo may lead to excitotoxic neuronal cell death and functional changes in the urinary bladder, resulting in bladder hyperdistension, urinary retention, and overflow incontinence

Pathogenic forms of tau inhibit kinesin-dependent axonal transport through a mechanism involving activation of axonal phosphotransferases

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Neuroscience 31 (2011): 9858-9868, doi:10.1523/JNEUROSCI.0560-11.2011.Aggregated filamentous forms of hyperphosphorylated tau (a microtubule-associated protein) represent pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. While axonal transport dysfunction is thought to represent a primary pathogenic factor in AD and other neurodegenerative diseases, the direct molecular link between pathogenic forms of tau and deficits in axonal transport remain unclear. Recently, we demonstrated that filamentous, but not soluble, forms of wild-type tau inhibit anterograde, kinesin-based fast axonal transport (FAT) by activating axonal protein phosphatase 1 (PP1) and glycogen synthase kinase 3 (GSK3), independent of microtubule binding. Here, we demonstrate that amino acids 2–18 of tau, comprising a phosphatase-activating domain (PAD), are necessary and sufficient for activation of this pathway in axoplasms isolated from squid giant axons. Various pathogenic forms of tau displaying increased exposure of PAD inhibited anterograde FAT in squid axoplasm. Importantly, immunohistochemical studies using a novel PAD-specific monoclonal antibody in human postmortem tissue indicated that increased PAD exposure represents an early pathogenic event in AD that closely associates in time with AT8 immunoreactivity, an early marker of pathological tau. We propose a model of pathogenesis in which disease-associated changes in tau conformation lead to increased exposure of PAD, activation of PP1-GSK3, and inhibition of FAT. Results from these studies reveal a novel role for tau in modulating axonal phosphotransferases and provide a molecular basis for a toxic gain-of-function associated with pathogenic forms of tau.This work was supported by NIH Grants T32 AG020506-07 (N.M.K.); AG09466 (L.I.B.); and NS23868, NS23320, and NS41170 (S.T.B.); as well as 2007/2008 MBL Summer Research Fellowships and an ALS/CVS Therapy Alliance grant (G.M.)

From collocations to call-ocations : using linguistic methods to quantify animal call combinations

Emerging data in a range of non-human animal species have highlighted a latent ability to combine certain pre-existing calls together into larger structures. Currently, however, the quantification of context-specific call combinations has received less attention. This is problematic because animal calls can co-occur with one another simply through chance alone. One common approach applied in language sciences to identify recurrent word combinations is collocation analysis. Through comparing the co-occurrence of two words with how each word combines with other words within a corpus, collocation analysis can highlight above chance, two-word combinations. Here, we demonstrate how this approach can also be applied to non-human animal signal sequences by implementing it on artificially generated data sets of call combinations. We argue collocation analysis represents a promising tool for identifying non-random, communicatively relevant call combinations and, more generally, signal sequences, in animals