Single channel study of the spasmodic mutation α1A52S in recombinant rat glycine receptors

Inherited defects in glycine receptors lead to hyperekplexia, or startle disease. A mutant mouse, spasmodic, that has a startle phenotype, has a point mutation (A52S) in the glycine receptor α1 subunit. This mutation reduces the sensitivity of the receptor to glycine, but the mechanism by which this occurs is not known. We investigated the properties of A52S recombinant receptors by cell-attached patch clamp recording of single-channel currents elicited by 30 – 10000 μM glycine. We used heteromeric receptors, which resemble those found at adult inhibitory synapses. Activation mechanisms were fitted directly to single channel data using the HJCFIT method, which includes an exact correction for missed events. In common with wildtype receptors, only mechanisms with three binding sites and extra shut states could describe the observations. The most physically plausible of these, the ‘flip’ mechanism, suggests that pre-opening isomerisation to the flipped conformation that follows binding is less favoured in mutant than in wild-type receptors, and, especially, that the flipped conformation has a 100-fold lower affinity for glycine than in wildtype receptors. In contrast, the efficacy of the gating reaction was similar to that of wild-type heteromeric receptors. The reduction in affinity for the flipped conformation accounts for the reduction in apparent cooperativity seen in the mutant receptor (without having to postulate interaction between the binding sites) and it accounts for the increased EC50 for responses to glycine that is seen in mutant receptors. This mechanism also predicts accurately the faster decay of synaptic currents that is observed in spasmodic mice

Stem starch reserves studied by on-solid reactions coupled with reflectance detections in water stressed grapevines

Wine grape is usually grown under water deficit conditions that could negatively impact plant reserves, including the organic carbon storage in perennial organs such as woody stems. Assessing the carbohydrate status in woody organs is therefore of interest as it can influence vegetative growth in the successive season. In this study, we aimed to apply an on-solid colour reaction (using Lugol's iodine solution) coupled with reflectance spectroscopy detection to assess the grapevine canes' starch accumulation in response to short drought periods. We used two Vitis vinifera cultivars ('Cabernet Sauvignon' and 'Syrah') that were subjected to three different water conditions (well-watered; early water stress; late water stress) during the growing season as case study. We sampled woody stem tissue during winter rest. The results showed that water stress reduced the starch storage in 'Syrah', especially when imposed late and recovery time was not enough for carbon restoration, while 'Cabernet Sauvignon' was not affected. The results showed that the sensitivity of the method used here is adequate to assess starch accumulation differences due to drought treatments in grapevine canes. Moreover, the analytical approach appears fast, low cost, and promising for future physiological and agronomical research applications

Polyphenolic Characterisation of Vranac, Kratosija and Cabernet Sauvignon (Vitis vinifera L. cv.) Grapes and Wines from Different Vineyard Locations in Montenegro

In Montenegro, red wines are produced predominantly, and Vranac accounts for nearly 80% of thesewines, followed by Kratosija and Cabernet Sauvignon. In order to characterise polyphenols in red varieties,grapes were sampled from representative vineyards at harvest time during 2011 and 2012. The contentand distribution of extractable anthocyanins, low-molecular mass proanthocyanidins (LMP) and highmolecularmass proanthocyanidins (HMP) in the seeds and skins of the grape berries were evaluated byapplying a five-day extraction method using ethanol:water (12:88) as extraction solvent. On average, thehighest content of LMP (2 006 and 1 690 mg/kg of grape fresh mass in years 2011 and 2012 respectively),HMP (2 705 and 2 805 mg/kg in years 2011 and 2012 respectively) and anthocyanins (1 035 mg/kg in theyear 2011) was found in the Cabernet Sauvignon grapes. The highest content of anthocyanins (1 113 mg/kgin the year 2012) and the lowest content of LMP (1 103 and 846 mg/kg in years 2011 and 2012 respectively)was found in Vranac grapes. Kratosija grapes had the lowest anthocyanin content (456 and 517 mg/kg inyears 2011 and 2012 respectively), and levels of LMP were similar to Vranac. The percentage distributionsof LMP between skins and seeds were 34:66, 39:61 and 49:51, whereas the distributions of HMP betweenskins and seeds were 67:33, 62:38 and 64:36 for Vranac, Kratosija and Cabernet Sauvignon respectively.All varieties had more LMP in the seeds and more HMP in the skins of the grapes. The results obtained areimportant to better understand the polyphenolic potential of Montenegrin red grape varieties

Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors

Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking α (α2–α4, α6) and β (β2, β4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (β4_α2, β4_α3, β4_α4, and β2_α4) when coexpressed with the corresponding β monomer subunit. All tandems expressed when injected alone, except for β4_α3, which produced functional channels only together with β4 monomer and was chosen for further characterization. These channels produced from β4_α3 tandem constructs plus β4 monomer were identical with receptors expressed from monomer α3 and β4 constructs in acetylcholine sensitivity and in the number of α and β subunits incorporated in the channel gate. However, separately mutating the β subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of β subunits from the tandem and one from the β monomer construct, whereas the rest incorporated two or three β monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily

Recognition and genotyping of minor germplasm of Friuli Venezia Giulia revealed high diversity

The wealth of vine varieties that used to exist in Friuli Venezia Giulia has been progressively lost. In order to ascertain the current situation regarding vine germplasm in the region, between 2001 and 2008, a wide-ranging study was conducted of recovery, conservation and molecular characterization with microsatellite markers (SSR) of accessions of minor germplasm at risk of erosion or extinction. A total of 178 accessions were analyzed and 93 varieties identified. Of these, 44 are already registered in the Italian Catalogue, 8 have been imported and are well-known foreign varieties even if not registered in the Italian Catalogue, 38 are potentially autochthonous and of these only 15 are described or at least cited in the literature; there are no hypotheses on the remaining three. In order to obtain information on possible genetic similarities, three types of software were used to process the molecular data, but only Structure allowed the existence of two principal groupings to be hypothesized for some of the presumed Friuli autochthons: one that gravitates around ‘Prosecco’ and the other around ‘Refoscone’.

Influence of vineyard inter-row management and clone on 'Sauvignon Blanc' performance in Friuli Venezia Giulia (north-eastern Italy)

The vineyard inter-row management affects grapevine vegetative and bunch health status, as well as yield and grape quality parameters. Several studies assessed that cover-cropped inter-row in place of soil tillage often reduced plant vigour and yield but positively contributed to vineyard ecosystem services and, to a lower extent, to grape quality. In 2013 and 2014, two inter-row management strategies, i.e. soil tillage and mowing of spontaneous cover crops, were compared in an organic vineyard in north-eastern Italy and cultivated with 'Sauvignon Blanc' (Vitis vinifera L.), clones R3 and 297. In particular, the effects of tillage and mowing treatments on grapevine vegetative and bunch health status, yield and grape quality were evaluated. The vegetative parameters were lower in the mowing treatment than in the tillage one and in clone R3 compared to 297. The incidence of Botrytis cinerea was higher in the tillage treatment than in the mowing one and in clone 297 compared to R3. A significant reduction of the yield and bunch weight was ascertained in the mowing treatment, and these parameters were higher for clone 297 compared to clone R3. Titratable acidity was significantly higher in the tillage treatment than in the mowing one and in clone 297 compared to R3. Moreover, hue of berry skin was qualitatively better in the tillage treatment than in the mowing one. In the pedo-climatic conditions of Friuli Venezia Giulia (north-eastern Italy), the management of the vineyard inter-row with spontaneous cover crops proved to be effective to manage grapevine vigour, reducing yield and improving quality of the grapes during maturation

Human alpha 3 beta 4 Neuronal Nicotinic Receptors Show Different Stoichiometry if They Are Expressed in Xenopus Oocytes or Mammalian HEK293 Cells

Background: The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the alpha 3 and beta 4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the alpha 5 and/or beta 2 subunits. We compared the properties of human alpha 3 beta 4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and alpha:beta subunit ratio.Methodology/Principal Findings: Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the alpha subunit, as reported for alpha 4 beta 2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9' of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike alpha 4 beta 2 channels, for alpha 3 beta 4 receptors the putative two-alpha form is the predominant one in oocytes (at 1:1 alpha:beta cRNA ratio). This two-alpha form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-alpha form is the predominant one in HEK cells transfected with a 1:1 alpha:beta DNA ratio or in oocytes at 9:1 alpha:beta RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-alpha form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-alpha form gave rise to short bursts (14 ms) of high conductance (39 pS).Conclusions/Significance: Like other neuronal nicotinic receptors, the alpha 3 beta 4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected

A putative relay circuit providing low-threshold mechanoreceptive input to lamina I projection neurons via vertical cells in lamina II of the rat dorsal horn

Background: Lamina I projection neurons respond to painful stimuli, and some are also activated by touch or hair movement. Neuropathic pain resulting from peripheral nerve damage is often associated with tactile allodynia (touch-evoked pain), and this may result from increased responsiveness of lamina I projection neurons to non-noxious mechanical stimuli. It is thought that polysynaptic pathways involving excitatory interneurons can transmit tactile inputs to lamina I projection neurons, but that these are normally suppressed by inhibitory interneurons. Vertical cells in lamina II provide a potential route through which tactile stimuli can activate lamina I projection neurons, since their dendrites extend into the region where tactile afferents terminate, while their axons can innervate the projection cells. The aim of this study was to determine whether vertical cell dendrites were contacted by the central terminals of low-threshold mechanoreceptive primary afferents. Results: We initially demonstrated contacts between dendritic spines of vertical cells that had been recorded in spinal cord slices and axonal boutons containing the vesicular glutamate transporter 1 (VGLUT1), which is expressed by myelinated low-threshold mechanoreceptive afferents. To confirm that the VGLUT1 boutons included primary afferents, we then examined vertical cells recorded in rats that had received injections of cholera toxin B subunit (CTb) into the sciatic nerve. We found that over half of the VGLUT1 boutons contacting the vertical cells were CTb-immunoreactive, indicating that they were of primary afferent origin. Conclusions: These results show that vertical cell dendritic spines are frequently contacted by the central terminals of myelinated low-threshold mechanoreceptive afferents. Since dendritic spines are associated with excitatory synapses, it is likely that most of these contacts were synaptic. Vertical cells in lamina II are therefore a potential route through which tactile afferents can activate lamina I projection neurons, and this pathway could play a role in tactile allodynia

Scottish and Newcastle antiemetic pre-treatment for paracetamol poisoning study (SNAP)

BACKGROUND: Paracetamol (acetaminophen) poisoning remains the commonest cause of acute liver injury in Europe and North America. The intravenous (IV) N-acetylcysteine (NAC) regimen introduced in the 1970s has continued effectively unchanged. This involves 3 different infusion regimens (dose and time) lasting over 20 hours. The same weight-related dose of NAC is used irrespective of paracetamol dose. Complications include frequent nausea and vomiting, anaphylactoid reactions and dosing errors. We designed a randomised controlled study investigating the efficacy of antiemetic pre-treatment (ondansetron) using standard NAC and a modified, shorter, regimen. METHODS/DESIGN: We designed a double-blind trial using a 2 × 2 factorial design involving four parallel groups. Pre-treatment with ondansetron 4 mg IV was compared against placebo on nausea and vomiting following the standard (20.25 h) regimen, or a novel 12 h NAC regimen in paracetamol poisoning. Each delivered 300 mg/kg bodyweight NAC. Randomisation was stratified on: paracetamol dose, perceived risk factors, and time to presentation. The primary outcome was the incidence of nausea and vomiting following NAC. In addition the frequency of anaphylactoid reactions and end of treatment liver function documented. Where clinically necessary further doses of NAC were administered as per standard UK protocols at the end of the first antidote course. DISCUSSION: This study is primarily designed to test the efficacy of prophylactic anti-emetic therapy with ondansetron, but is the first attempt to formally examine new methods of administering IV NAC in paracetamol overdose. We anticipate, from volunteer studies, that nausea and vomiting will be less frequent with the new NAC regimen. In addition as anaphylactoid response appears related to plasma concentrations of both NAC and paracetamol anaphylactoid reactions should be less likely. This study is not powered to assess the relative efficacy of the two NAC regimens, however it will give useful information to power future studies. As the first formal randomised clinical trial in this patient group in over 30 years this study will also provide information to support further studies in patients in paracetamol overdose, particularly, when linked with modern novel biomarkers of liver damage, patients at different toxicity risk. TRIAL REGISTRATION: EudraCT number 2009-017800-10, ClinicalTrials.gov IdentifierNCT0105027