Differentiation of postmitotic neuroblasts into substance P-immunoreactive sensory neurons in dissociated cultures of chick dorsal root ganglion.

Counts performed on dissociated cell cultures of E10 chick embryo dorsal root ganglia (DRG) showed after 4-6 days of culture a pronounced decline of the neuronal population in neuron-enriched cultures and a net gain in the number of ganglion cells in mixed DRG cell cultures (containing both neurons and nonneuronal cells). In the latter case, the increase in the number of neurons was found to depend on NGF and to average 119% in defined medium or 129% in horse serum-supplemented medium after 6 days of culture. The lack of [3H]thymidine incorporation into the neuronal population indicated that the newly formed ganglion cells were not generated by proliferation. On the contrary, the differentiation of postmitotic neuroblasts present in the nonneuronal cell compartment was supported by sequential microphotographs of selected fields taken every hour for 48-55 hr after 3 days of culture. Apparently nonneuronal flat dark cells exhibited morphological changes and gradually evolved into neuronal ovoid and refringent cell bodies with expanding neurites. The ultrastructural organization of these evolving cells corresponded to that of primitive or intermediate neuroblasts. The neuronal nature of these rounding up cell bodies was indeed confirmed by the progressive expression of various neuronal cell markers (150 and 200-kDa neurofilament triplets, neuron specific enolase, and D2/N-CAM). Besides a constant lack of immunoreactivity for tyrosine hydroxylase, somatostatin, parvalbumin, and calbindin-D 28K and a lack of cytoenzymatic activity for carbonic anhydrase, all the newly produced neurons expressed three main phenotypic characteristics: a small cell body, a strong immunoreactivity to MAG, and substance P. Hence, ganglion cells newly differentiated in culture would meet characteristics ascribed to small B sensory neurons and more specifically to a subpopulation of ganglion cells containing substance P-immunoreactive material.

Quantification of abscisic Acid in a single maize root.

Quantitative analyses of abscisic acid in the elongating zone of a single maize root (Zea mays L. cv LG 11) were performed by gas chromatography-mass spectrometry using negative chemical ion ionization. Data showed that the more abscisic acid, the slower the growth, but a large dispersion of individual values was observed. We assume that abscisic acid is perhaps not correlated only to the growth rate.

Functional and molecular evolutionary analysis of the "Brevix Radix" gene family in mono-and dicotyledonous plants

ABSTRACTIn contrast to animals, plants cannot move from their place of birth and, therefore, need to adapt to their particular habitat in order to survive. Thus, plant development is remarkably plastic, making plants an ideal system for the isolation of genes that account for intraspecific natural variation and possibly environmental adaptation. However, to date, this approach mostly identified null alleles and missed mutations with subtle effects. For instance, BREVIS RADIX (BRX) has been isolated as a key regulator of root growth through a naturally occurring loss-of-function allele in the Arabidopsis thaliana accession Uk-1 and is the founding member of a highly-conserved plant-specific gene family.In this work, we show that a strong selective pressure is acting on the BRX gene family and dates back before the monocot-dicot divergence. However, functional diversification is observed mainly in dicotyledon BRX family genes and is correlated with acceleration in the evolutionary rates in the N-terminal regions. Population genetic data revealed that BRX is highly conserved across Arabidopsis accessions and presents signatures of adaptation. Interestingly, a seven amino acid deletion polymorphism in BRX sequence was found in a few accessions, which seems to be responsible for their enhanced primary root growth. Nevertheless, BRX might not only be active in the root, as suggested by its expression in the shoot. Indeed, leaves and cotyledons of brx mutants are significantly smaller than wild- type. This phenotype is a direct consequence of the absence of BRX function in the shoot rather than an indirect effect of an altered root system growth. Interestingly, cotyledons of brx plants reflect the same physiological defects as the root. Moreover, phenotypes in BRX gain-of-function plants, such as epinastic leaves and increased epidermal cell size, could be associated with an increase in leaf brassinosteroid content.Collectively, these results indicate that BRX contributes to local adaptation by ubiquitously regulating plant growth, probably through the modulation of brassinosteroid biosynthesis.RÉSUMÉContrairement à la plupart des animaux, les plantes ne peuvent se mouvoir et doivent ainsi s'adapter à leur environnement pour survivre. Pour cette raison, elles représentent un système idéal pour l'identification de gènes contribuant à la variation naturelle intra- spécifique, ainsi qu'à l'adaptation. Cependant, cette approche a, jusqu'à présent, surtout permis d'isoler des allèles nuls et non des mutations conférant des effets plus subtiles. C'est le cas du gène Β REVIS RADIX (BRX), un régulateur clé de la croissance racinaire, qui a été identifié grâce à un allèle non-fonctionnel présent dans l'accession naturelle d'Arabidopsis thaliana Uk-1. BRX et ses homologues des plantes mono- et dicotylédones forment une famille très conservée et spécifique aux plantes.Dans ce travail, nous démontrons que la famille de gènes BRX est soumise à une forte pression de sélection qui remonte avant la divergence entre mono- et dicotylédones. Cependant, une diversification fonctionnelle a été observée chez les gènes des dicotylédones et corrèle avec une accélération de la vitesse d'évolution dans leur région N- terminale. Une analyse génétique de différentes accessions naturelles d'Arabidopsis a révélé que BRX est hautement conservé et présente des signatures d'adaptation. Remarquablement, un polymorphisme de délétion de sept acides aminés a été détecté dans quelques accessions et a pour conséquence une plus forte croissance de la racine primaire. Néanmoins, il semble que le rôle de BRX ne se limite pas qu'à la racine, comme indiqué par son expression dans les parties aériennes de la plante. En effet, les mutants brx présentent des cotylédons et des feuilles significativement plus petits que le type sauvage, une conséquence directe de l'absence d'activité de BRX dans ces organes. Nous avons aussi noté que les cotylédons des mutants brx, à l'instar des racines, ont une perception altérée de l'auxine et peuvent être complémentés par l'application exogène de brassinostéroïdes. De plus, dans des plantes présentant un gain de fonction BRX, les feuilles sont épinastiques et les cellules de leur épiderme plus grandes. Ces phénotypes sont accompagnés d'une augmentation de la concentration de brassinostéroïdes dans les feuilles. Conjointement, ces résultats démontrent que BRX contribue à une adaptation locale de la plante par la régulation générale de sa croissance, probablement en modulant la biosynthèse des brassinostéroïdes.

The effect of host plant and isolation on the genetic structure of phytophagous insects: A preliminary study on a bruchid beetle

Genetic differentiation is a consequence of the combination of drift and restriction in gene flow between populations due to barriers to dispersal, or selection against individuals resulting from inter-population matings In phytophagous insects, local adaptation to different kinds of host plants can sometimes lead to reproductive isolation and thus to genetic structuring, or even to speciation Acanthoscelides. obtectus Say is a bean bruchid specialized on beans of the Phaseolus vulgaris group, attacking both wild and domesticated forms of P vulgaris., and P coccineus This study reveals that the genetic structure of populations of this bruchid is explained mainly by their geographical location and is not related to a particular kind (wild or domesticated) of bean In contrast, the species of bean might have led, to some extent, to genetic structuring in these bruchids, although our sampling is too limited to address such process unambiguously. If confirmed, it would corroborate preliminary results found for the parasitoid species that attack Acanthoscelides species, which might show a genetic structure depending on the species of host plant

Brain spectrin isoforms during development of chick dorsal root ganglion cells in vivo and in vitro

Brain spectrin is one of the major cytoskeletal proteins associated with the plasma membrane. In many tissues this protein occurs in a variety of isoforms, for which at least three have been described in the brain: i) brain spectrin 240/235 is localized in neurons most prominently in axons and is present early during brain development. ii) Brain spectrin 240/235E is immunologicaly related to erythrocyte spectrin and restricted to somato-dendritic regions in neurons and to glia. It appears late in brain development. iii) A third form, brain spectrin 240/ 235A, is found exclusively in astrocytes. In this study we have investigated the appearance and distribution of brain spectrins 240/235 and 240/235E during embryonic chick dorsal root ganglia development in vivo and in vitro. This system provides a unique model due to the lack of dendrites on developing sensory neurons. Both isoforms first appeared at embryonic day 6. Brain spectrin 240/235 increased transiently around embryonic day 10 and 14, and was first expressed in ventrolateral neurons. It was localized abundantly in perikarya and their axons. This somato-axonal distribution pattern found in situ was also observed in vitro. In contrast, brain spectrin 240/235E only slightly increased between E6 and E15 and remained unchanged thereafter. It was localized mainly in small neurons of the mediodorsal area, where it was found as punctate staining in the cytoplasm, forming first a nuclear cap and in subsequent stages becoming distributed evenly throughout cytoplasm. This brain spectrin isoform was absent from axons, both in situ and in vitro. In conclusion, this study suggests i) that brain spectrin 240/235 may contribute towards the outgrowth, elongation and possibly maintenance of axonal processes, ii) that brain spcctrin 240/235E could be involved in the stablization of the cytoarchilecture of cell bodies in a sclected population of ganglion cells, and iii) that isoform expression of brain spectrin 240/235E in DRG cells may depend on environmental factors.

Ross procedure: is the root replacement technique superior to the sub-coronary implantation technique? Long-term results.

There is controversy over the use of the Ross procedure with regard to the sub-coronary and root replacement technique and its long-term durability. A systematic review of the literature may provide insight into the outcomes of these two surgical subvariants. A systematic review of reports between 1967 and February 2013 on sub-coronary and root replacement Ross procedures was undertaken. Twenty-four articles were included and divided into (i) sub-coronary technique and (ii) root replacement technique. The 10-year survival rate for a mixed-patient population in the sub-coronary procedure was 87.3% with a 95% confidence interval (CI) of 79.7-93.4 and 89.1% (95% CI, 85.3-92.1) in the root replacement technique category. For adults, it was 94 vs 95.3% (CI, 88.9-98.1) and in the paediatric series it was 90 vs 92.7% (CI, 86.9-96.0), respectively. Freedom from reoperation at 10 years was, in the mixed population, 83.3% (95% CI, 69.9-93.4) and 93.3% (95% CI, 89.4-95.9) for sub-coronary versus root replacement technique, respectively. In adults, it was 98 vs 91.2% (95% CI, 82.4-295.8), and in the paediatric series 93.3 vs 92.0% (95% CI, 86.1-96.5) for sub-coronary versus root replacement technique, respectively. The Ross procedure arguably has satisfactory results over 5 and 10 years for both adults and children. The results do not support the advantages of the sub-coronary technique over the root replacement technique. Root replacement was of benefit to patients undergoing reoperations on neoaorta and for long-term survival in mixed series.

Dialogues of root-colonizing biocontrol pseudomonads

Among biocontrol agents that are able to suppress root diseases caused by fungal pathogens, root-colonizing fluorescent pseudomonads have received particular attention because many strains of these bacteria trigger systemic resistance in host plants and produce antifungal compounds and exoenzymes. In general, the expression of these plant-beneficial traits is regulated by autoinduction mechanisms and may occur on roots when the pseudomonads form microcolonies. Three major classes of antibiotic compounds reviewed here in detail (2,4-diacetylphloroglucinol, pyoluteorin and various phenazine compounds) are all produced under cell population density-dependent autoinduction control acting at transcriptional and post-transcriptional levels. This regulation can either be reinforced or attenuated by a variety of chemical signals emanating from the pseudomonads themselves, other microorganisms or root exudates. Signals stimulating biocontrol factor expression via the Gac/Rsm signal transduction pathway in the biocontrol strain Pseudomonas fluorescens CHA0 are synthesized by many different plant-associated bacteria, warranting a more detailed investigation in the future.

Dos cuestiones pendientes de la sintaxis de los pronombres átonos: enclisis como fenómeno asociado a las oraciones matrices y doble clítico en las construcciones perifrásticas = The Remainders of the Syntax of Clitics: Enclisis as a Root Phenomenon, and Clitic Doubling in Periphrastic Constructions

Conferència centrada en dos aspectes concrets de la sintaxi dels pronoms àtons: l' enclisis que es dona en algunes oracions subordinades de les variants iberromàniques modernes de l'oest peninsular i les configuracions de doble cíclic en les construccions perifràstiques

Seasonal changes of whole root system conductance by a drought-tolerant grape root system

The role of root systems in drought tolerance is a subject of very limited information compared with above-ground responses. Adjustments to the ability of roots to supply water relative to shoot transpiration demand is proposed as a major means for woody perennial plants to tolerate drought, and is often expressed as changes in the ratios of leaf to root area (AL:AR). Seasonal root proliferation in a directed manner could increase the water supply function of roots independent of total root area (AR) and represents a mechanism whereby water supply to demand could be increased. To address this issue, seasonal root proliferation, stomatal conductance (gs) and whole root system hydraulic conductance (kr) were investigated for a drought-tolerant grape root system (Vitis berlandieri×V. rupestris cv. 1103P) and a non-drought-tolerant root system (Vitis riparia×V. rupestris cv. 101-14Mgt), upon which had been grafted the same drought-sensitive clone of Vitis vinifera cv. Merlot. Leaf water potentials (ψL) for Merlot grafted onto the 1103P root system (–0.91±0.02 MPa) were +0.15 MPa higher than Merlot on 101-14Mgt (–1.06±0.03 MPa) during spring, but dropped by approximately –0.4 MPa from spring to autumn, and were significantly lower by –0.15 MPa (–1.43±0.02 MPa) than for Merlot on 101-14Mgt (at –1.28±0.02 MPa). Surprisingly, gs of Merlot on the drought-tolerant root system (1103P) was less down-regulated and canopies maintained evaporative fluxes ranging from 35–20 mmol vine−1 s−1 during the diurnal peak from spring to autumn, respectively, three times greater than those measured for Merlot on the drought-sensitive rootstock 101-14Mgt. The drought-tolerant root system grew more roots at depth during the warm summer dry period, and the whole root system conductance (kr) increased from 0.004 to 0.009 kg MPa−1 s−1 during that same time period. The changes in kr could not be explained by xylem anatomy or conductivity changes of individual root segments. Thus, the manner in which drought tolerance was conveyed to the drought-sensitive clone appeared to arise from deep root proliferation during the hottest and driest part of the season, rather than through changes in xylem structure, xylem density or stomatal regulation. This information can be useful to growers on a site-specific basis in selecting rootstocks for grape clonal material (scions) grafted to them.

Radial transport of nutrients: the plant root as a polarized epithelium.

In higher plants, roots acquire water and soil nutrients and transport them upward to their aerial parts. These functions are closely related to their anatomical structure; water and nutrients entering the root first move radially through several concentric layers of the epidermis, cortex, and endodermis before entering the central cylinder. The endodermis is the innermost cortical cell layer that features rings of hydrophobic cell wall material called the Casparian strips, which functionally resemble tight junctions in animal epithelia. Nutrient uptake from the soil can occur through three different routes that can be interconnected in various ways: the apoplastic route (through the cell wall), the symplastic route (through cellular connections), and a coupled trans-cellular route (involving polarized influx and efflux carriers). This Update presents recent advances in the radial transport of nutrients highlighting the coupled trans-cellular pathway and the roles played by the endodermis as a barrier.

Catheter entrapment in a pulmonary vein: a unique complication of pulmonary vein isolation.

Ablation strategies for the treatment of atrial fibrillation (AF) are associated with several potential complications. During electro-anatomic mapping of the left atrium (LA) before ablation, the ablation catheter was entrapped in the right inferior pulmonary vein (RIPV). After multiple unsuccessful gentle tractions, stronger maneuvers with rotation of the catheter slowly allowed its retrieval. Examination of the catheter showed a thin, translucent membrane covering its tip, suggesting complete stripping of a vein branch. Occlusion of the superior branch of the RIPV was confirmed by LA angiogram. During the following days, no pericardial effusion was noted, but the patient complained of light chest pain and mild hemoptysis, spontaneously resolving within 48 h. This case shows that catheter entrapment and mechanical disruption of a PV branch can be a rare potential complication of AF ablation. In this case, the outcome was spontaneously favorable and symptoms only included transient mild hemoptysis.

PCR-based isolation of multigene families: lessons from the avian MHC class IIB.

The amount of sequence data available today highly facilitates the access to genes from many gene families. Primers amplifying the desired genes over a range of species are readily obtained by aligning conserved gene regions, and laborious gene isolation procedures can often be replaced by quicker PCR-based approaches. However, in the case of multigene families, PCR-based approaches bear the often ignored risk of incomplete isolation of family members. This problem is most prominent in gene families with highly variable and thus unpredictable number of gene copies among species, such as in the major histocompatibility complex (MHC). In this study, we (i) report new primers for the isolation of the MHC class IIB (MHCIIB) gene family in birds and (ii) share our experience with isolating MHCIIB genes from an unprecedented number of avian species from all over the avian phylogeny. We report important and usually underappreciated problems encountered during PCR-based multigene family isolation and provide a collection of measures to help significantly improving the chance of successfully isolating complete multigene families using PCR-based approaches.

Natural genetic variation of root system architecture from Arabidopsis to Brachypodium: towards adaptive value.

Root system architecture is a trait that displays considerable plasticity because of its sensitivity to environmental stimuli. Nevertheless, to a significant degree it is genetically constrained as suggested by surveys of its natural genetic variation. A few regulators of root system architecture have been isolated as quantitative trait loci through the natural variation approach in the dicotyledon model, Arabidopsis. This provides proof of principle that allelic variation for root system architecture traits exists, is genetically tractable, and might be exploited for crop breeding. Beyond Arabidopsis, Brachypodium could serve as both a credible and experimentally accessible model for root system architecture variation in monocotyledons, as suggested by first glimpses of the different root morphologies of Brachypodium accessions. Whether a direct knowledge transfer gained from molecular model system studies will work in practice remains unclear however, because of a lack of comprehensive understanding of root system physiology in the native context. For instance, apart from a few notable exceptions, the adaptive value of genetic variation in root system modulators is unknown. Future studies should thus aim at comprehensive characterization of the role of genetic players in root system architecture variation by taking into account the native environmental conditions, in particular soil characteristics.

Alpha 3 domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells.

The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the alpha3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of alpha3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.