Latent transforming growth factor beta-binding proteins-2 and -3 inhibit the proprotein convertase 5/6A

The basic amino acid-specific proprotein convertase 5/6 (PC5/6) is an essential secretory protease, as knock-out mice die at birth and exhibit multiple homeotic transformation defects, including impaired bone morphogenesis and lung structure. Some of the observed defects were attributed to impaired processing of the TGFβ-like growth differentiating factor 11 precursor (proGdf11). In this work we present evidence that the latent TGFβ-binding proteins 2 and 3 (LTBP-2 and -3) inhibit the extracellular processing of proGdf11 by PC5/6A. This is partly due to the binding of LTBPs in the endoplasmic reticulum to the zymogen proPC5/6A, thus allowing the complex to exit the endoplasmic reticulum and be sequestered as an inactive zymogen in the extracellular matrix but not at the cell surface. This results in lower levels of PC5/6A in the media, without affecting those of PACE4, Furin, or a soluble form of PC7. The secreted soluble protease-specific activity of PC5/6A or a variant lacking the C-terminal Cys-rich domain (PC5/6-ΔCRD) is significantly decreased when co-expressed with LTBPs in cells. A similar enzymatic inhibition seems to apply to PACE4 and Furin. In situ hybridization analyses revealed extensive co-localization of PC5/6 and LTBP-3 mRNAs in mice at embryonic day 15.5 and post partum day 1. In conclusion, this is the first time that a zymogen of the proprotein convertases was shown to exit the endoplasmic reticulum in the presence of LTBPs, representing a potential novel mechanism for the regulation of PC5/6A activity, e.g. in tissues such as bone and lung where LTBP-3 and PC5/6 co-localize.     

Single Cell Wall Nonlinear Mechanics Revealed by a Multiscale Analysis of AFM Force-Indentation Curves

Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions.     

Wild European Apple (Malus sylvestris (L.) Mill.) Population Dynamics: Insight from Genetics and Ecology in the Rhine Valley. Priorities for a Future Conservation Programme

The increasing fragmentation of forest habitats and the omnipresence of cultivars potentially threaten the genetic integrity of the European wild apple (Malus sylvestris (L.) Mill). However, the conservation status of this species remains unclear in Europe, other than in Belgium and the Czech Republic, where it has been declared an endangered species. The population density of M. sylvestris is higher in the forests of the upper Rhine Valley (France) than in most European forests, with an unbalanced age-structure, an overrepresentation of adults and a tendency to clump. We characterize here the ecology, age-structure and genetic diversity of wild apple populations in the Rhine Valley. We use these data to highlight links to the history of this species and to propose guidelines for future conservation strategies. In total, 255 individual wild apple trees from six forest stands (five floodplain forests and one forest growing in drier conditions) were analysed in the field, collected and genotyped on the basis of data for 15 microsatellite markers. Genetic analyses showed no escaped cultivars and few hybrids with the cultivated apple. Excluding the hybrids, the genetically “pure” populations displayed high levels of genetic diversity and a weak population structure. Age-structure and ecology studies of wild apple populations identified four categories that were not randomly distributed across the forests, reflecting the history of the Rhine forest over the last century. The Rhine wild apple populations, with their ecological strategies, high genetic diversity, and weak traces of crop-to-wild gene flow associated with the history of these floodplain forests, constitute candidate populations for inclusion in future conservation programmes for European wild apple.     

Patterns of climber distribution in the temperate forests of the Americas

Aims and Methods We propose a standard protocol at the landscape to continental scale for examining to what extent the range of ecological conditions found in temperate latitudes explains the variations in climber species richness and traits. The protocol was tested in forests of the two Americas. The data set included 151 climber species. We selected four categorical traits and grouped these species into six clusters with regard to these traits. Floristic records of American forests were first gathered into alliances, second combined with bioclimatic indices (rainfall, temperature, continentality). We obtained a total of 59 vegetational units in which we calculated values of climber species richness and proportion of clusters. Vegetational units were ultimately gathered into five forest formations (characterized by leaf longevity). Wetlands and uplands were considered separately.Important findings Our results emphasize clear trends in large-scale patterns of climber distribution, independently of taxonomy. Climber species richness (in particular woody climbers) peaks in moist and warm upland forests with oceanic climates, and where conifers are rare. In flooded areas, climber richness is also very high and peaks in seasonally flooded large floodplains. In ecological conditions of frost, dryness or lack of nutrients, climber species richness, abundance and trait diversity decline, resulting in the dominance of small, twining and deciduous life traits.     

Insights into the Vitis complex in the Danube floodplain (Austria)

European grapevine populations quickly disappeared from most of their range, massively killed by the spread of North American grapevine pests and diseases. Nowadays taxonomic pollution represents a new threat. A large Vitis complex involves escaped cultivars, rootstocks, and wild grapevines. The study aimed to provide insight into the Vitis complex in the Danube region through field and genetic analyses. Among the five other major rivers in Europe which still host wild grapevine populations, the Danube floodplain is the only one benefiting from an extensive protected forest area (93 km²) and an relatively active dynamic flood pulse. The Donau‐Auen National Park also regroups the largest wild grapevine population in Europe. Ninety‐two percent of the individuals collected in the park were true wild grapevines, and 8% were hybrids and introgressed individuals of rootstocks, wild grapevines, and cultivars. These three groups are interfertile acting either as pollen donor or receiver. Hybrids were established within and outside the dykes, mostly in anthropized forest edges. The best‐developed individuals imply rootstock genes. They establish in the most erosive parts of the floodplain. 42% of the true wild grapevines lived at the edges of forest/meadow, 33.3% at the edges forest/channels, and 23.9% in forest gaps. DBH (Diameter Breast Height) varied significantly with the occurrence of flooding. Clones were found in both true wild and hybrids/introgressed grapevines. The process of cloning seemed to be prevented in places where flooding dynamics is reduced. The current global distribution of true wild grapevines shows a strong tendency toward clustering, in sites where forestry practices were the most extensive. However, the reduced flooding activity is a danger for long‐term sustainability of the natural wild grapevine population.     

<Emphasis Type="Italic">Pcsk5</Emphasis> is required in the early cranio-cardiac mesoderm for heart development

Background

Loss of proprotein convertase subtilisin/kexin type 5 (Pcsk5) results in multiple developmental anomalies including cardiac malformations, caudal regression, pre-sacral mass, renal agenesis, anteroposterior patterning defects, and tracheo-oesophageal and anorectal malformations, and is a model for VACTERL/caudal regression/Currarino syndromes (VACTERL association - Vertebral anomalies, Anal atresia, Cardiac defects, Tracheoesophageal fistula and/or Esophageal atresia, Renal & Radial anomalies and Limb defects).

Results

Using magnetic resonance imaging (MRI), we examined heart development in mouse embryos with zygotic and cardiac specific deletion of Pcsk5. We show that conditional deletion of Pcsk5 in all epiblastic lineages recapitulates all developmental malformations except for tracheo-esophageal malformations. Using a conditional deletion strategy, we find that there is an essential and specific requirement for Pcsk5 in the cranio-cardiac mesoderm for cardiogenesis, but not for conotruncal septation or any other aspect of embryonic development. Surprisingly, deletion of Pcsk5 in cardiogenic or pharyngeal mesodermal progenitors that form later from the cranio-cardiac mesoderm does not affect heart development. Neither is Pcsk5 essential in the neural crest, which drives conotruncal septation.

Conclusions

Our results suggest that Pcsk5 may have an essential and early role in the cranio-cardiac mesoderm for heart development. Alternatively, it is possible that Pcsk5 may still play a critical role in Nkx2.5-expressing cardiac progenitors, with persistence of mRNA or protein accounting for the lack of effect of deletion on heart development.

     

Isolation of Agrobacterium Ti-plasmid regulatory mutants

Summary Crown gall tumors incited by Agrobacterium tumefaciens synthesize basic amino acid derivatives called opines. Opine production in tumours and opine catabolism by A. tumefaciens are coded by Ti-plasmids which confer oncogenicity on this bacterium. Catabolism of opines is inducible, and a method for isolation of regulatory mutants is described. From octopine-type bacteria, by plating on non-inducing substrates (noroctopine, noroctopine acid, D-histopine) we have isolated regulatory mutants of three types: constitutive, partially constitutive, and fully inducible by the analogue. From nopaline-type bacteria, by plating on octopine (a non inducing substrate) we have isolated analogous regulatory mutants.Synthetic opines, in which the amino acid moiety has been replaced by toxic arginine analogues, are toxic for these regulatory mutants. We isolated mutants resistant to such synthetic opines, and found that some had lost the capacity to utilize octopine. A survey of a large number of such mutants revealed that all of them still incited octopine synthetizing tumors.Mutants constitutive for octopine catabolism are in some instances also constitutive for Ti-plasmid transfer. A simple method for screening regulatory mutants for constitutive Ti-plasmid transfer is described.This work has been supported in part by grants from the Centre National de la Recherche Scientifique (contrats ATP 2814 and 3363).     

Correlative Association between Resident Plasmids and the Host Chromosome in a Diverse Agrobacterium Soil Population

Soil samples collected from a fallow field which had not been cultivated for 5 years harbored a population of Agrobacterium spp. estimated at 3 × 10⁷ CFU/g. Characterization of 72 strains selected from four different isolation media showed the presence of biovar 1 (56%) and bv. 2 (44%) strains. Pathogenicity assays on five different test plants revealed a high proportion (33%) of tumorigenic strains in the resident population. All tumorigenic strains belonged to bv. 1. Differentiation of the strains by restriction fragment length polymorphism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cellular proteins, and utilization patterns of 95 carbon substrates (Biolog GN microplate) revealed a diversified bv. 1 population, composed of five distinct chromosomal backgrounds (chr A, C, D, E, and F), and a homogeneous bv. 2 population (chr B). chr A, B, C, and D were detected at similar levels throughout the study site. According to opine metabolism, pathogenicity, and agrocin sensitivity, chr A strains carried a nopaline Ti plasmid (pTi), whereas chr C strains had an octopine pTi. In addition, four of six nontumorigenic bv. 1 strains (two chr D, one chr E, and one chr F) had distinct and unusual opine catabolism patterns. chr B (bv. 2) strains were nonpathogenic and catabolized nopaline. Although agrocin sensitivity is a pTi-borne trait, 14 chr B strains were sensitive to agrocin 84, apparently harboring a defective nopaline pTi similar to pAtK84b. The other two chr B strains were agrocin resistant. The present analysis of chromosomal and plasmid phenotypes suggests that in this Agrobacterium soil population, there is a preferential association between the resident plasmids and their bacterial host.