Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants

The term 'RNA silencing' describes a process that results in the specific degradation of an RNA target. In plants, silenced tissues can initiate the spreading of the process into non-silenced regions by a mobile signal that can be transmitted over long distances. In the present work, we made use of a modified grafting approach to elucidate the driving force behind long-distance transport of the silencing signal. We made reciprocal grafts of two GFP-transgenic Nicotiana benthamiana lines, the non-silenced line 16c (sensor) and the silenced line 6.4 (inducer). We show that the direction of systemic spread of silencing from inducer to sensor can be manipulated by altering sink/source relations in the plant. Using radioactive phosphate as a phloem tracer, we demonstrated that plants that transmitted silencing from silenced scion to non-silenced rootstock had developed a persisting phloem flow from scion to rootstock. These data provide experimental proof of what has been hypothesized so far, that the silencing signal travels via phloem from source to sink. We present here evidence that the appearance of systemic silencing is not an accidental stochastic process, but can be predicted on the basis of the direction of phloem flow.     

Identification of genes differentially expressed in dorsal and ventral chick midbrain during early Development

Background  

During the development of the central nervous system (CNS), patterning processes along the dorsoventral (DV) axis of the neural tube generate different neuronal subtypes. As development progresses these neurons are arranged into functional units with varying cytoarchitecture, such as laminae or nuclei for efficient relaying of information. Early in development ventral and dorsal regions are similar in size and structure. Different proliferation rates and cell migration patterns are likely to result in the formation of laminae or nuclei, eventually. However, the underlying molecular mechanisms that establish these different structural arrangements are not well understood.     

INCITE: A randomised trial comparing constraint induced movement therapy and bimanual training in children with congenital hemiplegia

Background  

Congenital hemiplegia is the most common form of cerebral palsy (CP) accounting for 1 in 1300 live births. These children have limitations in capacity to use the impaired upper limb and bimanual coordination deficits which impact on daily activities and participation in home, school and community life. There are currently two diverse intensive therapy approaches. Traditional therapy has adopted a bimanual approach (BIM training) and recently, constraint induced movement therapy (CIMT) has emerged as a promising unimanual approach. Uncertainty remains about the efficacy of these interventions and characteristics of best responders. This study aims to compare the efficacy of CIMT to BIM training to improve outcomes across the ICF for school children with congenital hemiplegia.     

Hierarchical analysis of piecewise affine models of gene regulatory networks

A key point in the analysis of dynamical models of biological systems is to handle systems of relatively high dimensions. In the present paper we propose a method to hierarchically organize a certain type of piecewise affine (PWA) differential systems. This specific class of systems has been extensively studied for the past few years, as it provides a good framework to model gene regulatory networks. The method, shown on several examples, allows a qualitative analysis of the asymptotic behavior of a PWA system, decomposing it into several smaller subsystems. This technique, based on the well-known strongly connected components decomposition, is not new. However, its adaptation to the non-smooth PWA differential equations turns out to be quite relevant because of the strong discrete structure underlying these equations. Its biological relevance is shown on a 7-dimensional PWA system modeling the gene network responsible for the carbon starvation response in Escherichia coli.

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Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

Background  

The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene.     

Assembly of turnip yellow mosaic virus replication complexes: interaction between the proteinase and polymerase domains of the replication proteins

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like supergroup, encodes two nonstructural replication proteins (140K and 66K), both of which are required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase activities, while the 66K protein encompasses the RNA-dependent RNA polymerase domain. Recruitment of the 66K protein to the sites of viral replication, located at the periphery of chloroplasts, is dependent upon the expression of the 140K protein. Using antibodies raised against the 140K and 66K proteins and confocal microscopy, we report the colocalization of the TYMV replication proteins at the periphery of chloroplasts in transfected or infected cells. The replication proteins cofractionated in functional replication complexes or with purified chloroplast envelope membranes prepared from infected plants. Using a two-hybrid system and coimmunoprecipitation experiments, we also provide evidence for a physical interaction of the TYMV replication proteins. In contrast to what has been found for other members of the alphavirus-like supergroup, the interaction domains were mapped to the proteinase domain of the 140K protein and to a large region encompassing the core polymerase domain within the 66K protein. Coexpression and colocalization experiments confirmed that the helicase domain of the 140K protein is unnecessary for the proper recruitment of the 66K protein to the chloroplast envelope, while the proteinase domain appears to be essential for that process. These results support a novel model for the interaction of TYMV replication proteins and suggest that viruses in the alphavirus-like supergroup may have selected different pathways to assemble their replication complexes.     

Anthrax edema toxin cooperates with lethal toxin to impair cytokine secretion during infection of dendritic cells

Bacillus anthracis secretes two critical virulence factors, lethal toxin (LT) and edema toxin (ET). In this study, we show that murine bone marrow-derived dendritic cells (DC) infected with B. anthracis strains secreting ET exhibit a very different cytokine secretion pattern than DC infected with B. anthracis strains secreting LT, both toxins, or a nontoxinogenic strain. ET produced during infection selectively inhibits the production of IL-12p70 and TNF-alpha, whereas LT targets IL-10 and TNF-alpha production. To confirm the direct role of the toxins, we show that purified ET and LT similarly disrupt cytokine secretion by DC infected with a nontoxinogenic strain. These effects can be reversed by specific inhibitors of each toxin. Furthermore, ET inhibits in vivo IL-12p70 and IFN-gamma secretion induced by LPS. These results suggest that ET produced during infection impairs DC functions and cooperates with LT to suppress the innate immune response. This may represent a new strategy developed by B. anthracis to escape the host immune response.     

The intercentriolar linkage is critical for the ability of heterologous centrosomes to induce parthenogenesis in Xenopus

Centrosomes isolated from various sources, including human cells, have the capacity to induce parthenogenetic development when injected into unfertilized amphibian eggs. We recently isolated calf thymus centrosomes and showed that they differ structurally and functionally from previously isolated centrosomes of KE37 cells, in that the two centrioles in calf thymocytes are linearly associated by their proximal ends through a mass of electron dense material and nucleate few microtubules from their distal ends (Komesli, S., F. Tournier, M. Paintrand, R. Margolis, D. Job, and M. Bornens. 1989. J. Cell Biol. 109:2869-2878). We report here that these centrosomes are also unable to induce egg cleavage and examine the various possibilities which could account for this lack of competence. The results show that: (a) the kinetics of microtubule assembly on calf thymus centrosomes in Xenopus extracts are comparable to those of KE37 centrosomes; (b) centrosomes isolated from thymus of calves raised under controlled conditions (without anabolic agents) also lack competence; (c) centrosomes isolated from bovine cells of other tissues are competent; (d) centrosomes isolated from thymus of three other species (rat, mouse, and human) are competent. Since the lack of activity of calf thymus centrosomes apparently was not linked to species or tissue differences, we compared the ultrastructure of the centrosomes in the various centrosome preparations. The results show a strict correlation between the linear arrangement of centrioles and the lack of activity of the centrosomes. They suggest that the centrosome cycle can be blocked when the centrioles are prevented from separating into a nonlinear configuration, a step which might be critical for the initiation of procentriole budding. They also indicate that the centrosome may be involved in the G0-G1 transition.