Structural characterization and agonist binding to human α4β2 nicotinic receptors

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition – particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences.     

Blood pressure changes alter tracheobronchial cough: computational model of the respiratory-cough network and in vivo experiments in anesthetized cats

We tested the hypothesis, motivated in part by a coordinated computational cough network model, that alterations of mean systemic arterial blood pressure (BP) influence the excitability and motor pattern of cough. Model simulations predicted suppression of coughing by stimulation of arterial baroreceptors. In vivo experiments were conducted on anesthetized spontaneously breathing cats. Cough was elicited by mechanical stimulation of the intrathoracic airways. Electromyograms (EMG) of inspiratory parasternal, expiratory abdominal, laryngeal posterior cricoarytenoid (PCA), and thyroarytenoid muscles along with esophageal pressure (EP) and BP were recorded. Transiently elevated BP significantly reduced cough number, cough-related inspiratory, and expiratory amplitudes of EP, peak parasternal and abdominal EMG, and maximum of PCA EMG during the expulsive phase of cough, and prolonged the cough inspiratory and expiratory phases as well as cough cycle duration compared with control coughs. Latencies from the beginning of stimulation to the onset of cough-related diaphragm and abdominal activities were increased. Increases in BP also elicited bradycardia and isocapnic bradypnea. Reductions in BP increased cough number; elevated inspiratory EP amplitude and parasternal, abdominal, and inspiratory PCA EMG amplitudes; decreased total cough cycle duration; shortened the durations of the cough expiratory phase and cough-related abdominal discharge; and shortened cough latency compared with control coughs. Reduced BP also produced tachycardia, tachypnea, and hypocapnic hyperventilation. These effects of BP on coughing likely originate from interactions between barosensitive and respiratory brainstem neuronal networks, particularly by modulation of respiratory neurons within multiple respiration/cough-related brainstem areas by baroreceptor input.     

Development of a near-isogenic line population of Arabidopsis thaliana and comparison of mapping power with a recombinant inbred line population

In Arabidopsis recombinant inbred line (RIL) populations are widely used for quantitative trait locus (QTL) analyses. However, mapping analyses with this type of population can be limited because of the masking effects of major QTL and epistatic interactions of multiple QTL. An alternative type of immortal experimental population commonly used in plant species are sets of introgression lines. Here we introduce the development of a genomewide coverage near-isogenic line (NIL) population of Arabidopsis thaliana, by introgressing genomic regions from the Cape Verde Islands (Cvi) accession into the Landsberg erecta (Ler) genetic background. We have empirically compared the QTL mapping power of this new population with an already existing RIL population derived from the same parents. For that, we analyzed and mapped QTL affecting six developmental traits with different heritability. Overall, in the NIL population smaller-effect QTL than in the RIL population could be detected although the localization resolution was lower. Furthermore, we estimated the effect of population size and of the number of replicates on the detection power of QTL affecting the developmental traits. In general, population size is more important than the number of replicates to increase the mapping power of RILs, whereas for NILs several replicates are absolutely required. These analyses are expected to facilitate experimental design for QTL mapping using these two common types of segregating populations.     

Pre-steady-state currents in neutral amino acid transporters induced by photolysis of a new caged alanine derivative

Na+-Dependent transmembrane transport of small neutral amino acids, such as glutamine and alanine, is mediated, among others, by the neutral amino acid transporters of the solute carrier 1 [SLC1, alanine serine cysteine transporter 1 (ASCT1), and ASCT2] and SLC38 families [sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4]. Many mechanistic aspects of amino acid transport by these systems are not well-understood. Here, we describe a new photolabile alanine derivative based on protection of alanine with the 4-methoxy-7-nitroindolinyl (MNI) caging group, which we use for pre-steady-state kinetic analysis of alanine transport by ASCT2, SNAT1, and SNAT2. MNI-alanine has favorable photochemical properties and is stable in aqueous solution. It is also inert with respect to the transport systems studied. Photolytic release of free alanine results in the generation of significant transient current components in HEK293 cells expressing the ASCT2, SNAT1, and SNAT2 proteins. In ASCT2, these currents show biphasic decay with time constants, tau, in the 1-30 ms time range. They are fully inhibited in the absence of extracellular Na+, demonstrating that Na+ binding to the transporter is necessary for induction of the alanine-mediated current. For SNAT1, these transient currents differ in their time course (tau = 1.6 ms) from previously described pre-steady-state currents generated by applying steps in the membrane potential (tau approximately 4-5 ms), indicating that they are associated with a fast, previously undetected, electrogenic partial reaction in the SNAT1 transport cycle. The implications of these results for the mechanisms of transmembrane transport of alanine are discussed. The new caged alanine derivative will provide a useful tool for future, more detailed studies of neutral amino acid transport.     

An integrative approach to characterize Malagasy bats of the subfamily Vespertilioninae Gray, 1821, with the description of a new species of Hypsugo

Although important advances have been made in recent years in the taxonomy of different families and subfamilies of Malagasy bats, those belonging to the Vespertilioninae remain partially unresolved. Herein using a mitochondrial marker (cytochrome b) as the point of departure for 76 specimens of Malagasy vespers and appropriate African taxa, we diagnose the six taxa of this subfamily on the island by overlaying different morphological and bioacoustic characters on the clade structure of sequenced animals. The species include: endemic Neoromicia matroka, which is sister to African N. capensis; endemics N. malagasyensis and N. robertsi, which form sister species; a member of the genus Hypsugo, which is sister to African H. anchietae and described herein as new to science; Pipistrellus hesperidus for which Madagascar animals are genetically close but distinct from African populations of the same species; and endemic P. raceyi, which shows segregation of eastern (mesic) and western (dry) populations and its sister species relationships are unresolved. While the external and craniodental measurements, as well as bioacoustic variables, allow only partial differentiation of these six species of Vespertilioninae, molecular characters provide definitive separation of the taxa, as do male bacular morphology. © 2015 The Linnean Society of London     

Quantitative trait loci for glucosinolate accumulation in Brassica rapa leaves

Glucosinolates and their breakdown products have been recognized for their effects on plant defense, human health, flavor and taste of cruciferous vegetables. Despite this importance, little is known about the regulation of the biosynthesis and degradation in Brassica rapa. Here, the identification of quantitative trait loci (QTL) for glucosinolate accumulation in B. rapa leaves in two novel segregating double haploid (DH) populations is reported: DH38, derived from a cross between yellow sarson R500 and pak choi variety HK Naibaicai; and DH30, from a cross between yellow sarson R500 and Kairyou Hakata, a Japanese vegetable turnip variety. An integrated map of 1068 cM with 10 linkage groups, assigned to the international agreed nomenclature, is developed based on the two individual DH maps with the common parent using amplified fragment length polymorphism (AFLP) and single sequence repeat (SSR) markers. Eight different glucosinolate compounds were detected in parents and F(1)s of the DH populations and found to segregate quantitatively in the DH populations. QTL analysis identified 16 loci controlling aliphatic glucosinolate accumulation, three loci controlling total indolic glucosinolate concentration and three loci regulating aromatic glucosinolate concentrations. Both comparative genomic analyses based on Arabidopsis-Brassica rapa synteny and mapping of candidate orthologous genes in B. rapa allowed the selection of genes involved in the glucosinolate biosynthesis pathway that may account for the identified QTL.     

Characterization and mapping of a gene controlling shoot regeneration in tomato

The superior regeneration capacity of Lycopersicon peruvianum was introduced into the cultivated tomato Lycopersicon esculentum by backcrossing hybrid material with the tomato genotype VF11. In segregating material derived from these backcrosses, the ability to regenerate shoots on root explants cultured on a zeatin-containing medium, was highly correlated with the ability to regenerate shoots on established callus cultures. The efficient shoot-regenerating root explant system permitted us to study the genetics of this trait and to locate the genes involved, using a set of morphological markers defining all 12 tomato chromosomes. Depending on the tomato genotype, mono, -di- or trigenic ratios were observed. It is concluded that a dominant L. peruvianum allele at a locus (Rg-1) near the middle of chromosome 3 determines efficient shoot regeneration on root explants in tomato in combination with dominant alleles at one or two other loci of either L. peruvianum or L. esculentum origin. The map location of the Rg-1 locus was refined further using a number of chromosome-3-specific RFLPs. The addition of new classical and RFLP linkage data to existing literature data and subsequent processing resulted in a revised and integrated map of tomato chromosome 3. From a morphological and physiological analysis of genotypes differing in Rg phenotype, it is concluded that the genetic component associated with regeneration determines the maintenance of morphogenetic competence and not the sensitivity to hormones.     

Outcomes and indications of corpus callosum section for intractable seizure control

Callosotomy (CCS) was performed in 9 cases of rigorously classified drug-refractory epilepsy not eligible for focal cortical resection. Complete CCS was carried out in two stages in 8 patients. 1 case had an anterior CCS only. None of the 9 patients has become seizure free. In 7, seizure frequency has diminished by a factor of 3-60 for at least one seizure type. Drop attacks in 2 cases have ceased. Clinical seizure patterns appeared more helpful in predicting outcome than preoperative EEG and stereoelectroencephalographic studies. Complete CCS may result in prolonged behavioral disturbances in the areas of language, hemisphere competition and attention-memory sequencing with impact on the sphere of daily living.