Characterization of immunostimulatory components of orf virus (parapoxvirus ovis).

Inactivated orf virus (ORFV, parapoxvirus ovis) induces antiviral activity in animal models of acute and chronic viral infections and exerts strong effects on human immune cells. ORFV activates antigen presenting cells (APC) via CD14 and, probably, Toll-like receptor signalling, and triggers the release of IFN-γ that has been identified as the key mediator of the antiviral activity. After delineating virus proteins as being the most likely active constituent, we aimed to characterize the ORFV proteins responsible for the therapeutic effect. By using a vaccinia virus/ORFV expression library we identified several multi-gene DNA fragments with strong immunomodulatory activity. Together these fragments contain 27 ORFs. The encoded proteins are related to virion structure and transcription but are otherwise unrelated. Two proteins were separately expressed and purified, and demonstrated immunostimulatory activity. Gene expression profiles induced by ORFV and the identified fragments were investigated by microarray analysis. Interestingly, all active fragments induced a similar gene-expression pattern, differing only in quantitative aspects. Obviously, several proteins of ORFV activate similar cellular pathways, modulating APC to generate a strong T-helper 1-dominated immune response. This was balanced by additional induction of immune dampening mechanisms, suggesting regulatory differences compared to single cytokine therapies. We conclude that ORFV may have the potential to enrich the armamentarium of antiviral therapies.

Gene expression analysis defines differences between region-specific GABAergic neurons.

Gamma-aminobutyric acid (GABA)ergic neurons are a diverse group of inhibitory neurons playing crucial roles in information processing. We analyzed the gene expression of regionally defined GABAergic neurons from the cortex, olfactory bulb, striatum, and cerebellum of glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mice. We introduce a generally applicable method for singularization of brain cells, flow cytometric enrichment, and global mRNA amplification for sensitive gene expression profiling. Systematic quantification elicited a high dynamic range of GABAergic cell numbers in different brain regions. Clustering of our gene expression results revealed major differences between hind and forebrain GABAergic neurons indicating that the development of GABAergic neurons depends on their regional location. While GABAergic neurons of the forebrain are characterized by three main groups of transcription factors of the Distal-less-family, the POU-family, and ETS/FOX family, specific members of the ZIC- and LHX-family of transcription factors appear to define hindbrain inhibitory neurons.

FGF signalling controls formation of the apical sensory organ in the cnidarian Nematostella vectensis.

Fibroblast growth factor (FGF) signalling regulates essential developmental processes in vertebrates and invertebrates, but its role during early metazoan evolution remains obscure. Here, we analyse the function of FGF signalling in a non-bilaterian animal, the sea anemone Nematostella vectensis. We identified the complete set of FGF ligands and FGF receptors, of which two paralogous FGFs (NvFGFa1 and NvFGFa2) and one FGF receptor (NvFGFRa) are specifically coexpressed in the developing apical organ, a sensory structure located at the aboral pole of ciliated larvae from various phyla. Morpholino-mediated knockdown experiments reveal that NvFGFa1 and NvFGFRa are required for the formation of the apical organ, whereas NvFGFa2 counteracts NvFGFRa signalling to prevent precocious and ectopic apical organ development. Marker gene expression analysis shows that FGF signalling regulates local patterning in the aboral region. Furthermore, NvFGFa1 activates its own expression and that of the antagonistic NvFGFa2, thereby establishing positive- and negative-feedback loops. Finally, we show that loss of the apical organ upon NvFGFa1 knockdown blocks metamorphosis into polyps. We propose that the control of the development of sensory structures at the apical pole of ciliated larvae is an ancestral function of FGF signalling.

The sulphur oxygenase reductase from Acidianus ambivalens is a multimeric protein containing a low-potential mononuclear non-haem iron centre.

The SOR (sulphur oxygenase reductase) is the initial enzyme in the sulphur-oxidation pathway of Acidianus ambivalens. Expression of the sor gene in Escherichia coli resulted in active, soluble SOR and in inclusion bodies from which active SOR could be refolded as long as ferric ions were present in the refolding solution. Wild-type, recombinant and refolded SOR possessed indistinguishable properties. Conformational stability studies showed that the apparent unfolding free energy in water is approx. 5 kcal x mol(-1) (1 kcal=4.184 kJ), at pH 7. The analysis of the quaternary structures showed a ball-shaped assembly with a central hollow core probably consisting of 24 subunits in a 432 symmetry. The subunits form homodimers as the building blocks of the holoenzyme. Iron was found in the wild-type enzyme at a stoichiometry of one iron atom/subunit. EPR spectroscopy of the colourless SOR resulted in a single isotropic signal at g=4.3, characteristic of high-spin ferric iron. The signal disappeared upon reduction with dithionite or incubation with sulphur at elevated temperature. Thus both EPR and chemical analysis indicate the presence of a mononuclear iron centre, which has a reduction potential of -268 mV at pH 6.5. Protein database inspection identified four SOR protein homologues, but no other significant similarities. The spectroscopic data and the sequence comparison led to the proposal that the Acidianus ambivalens SOR typifies a new type of non-haem iron enzyme containing a mononuclear iron centre co-ordinated by carboxylate and/or histidine ligands.

The ancestral role of Brachyury: expression of NemBra1 in the basal cnidarian Nematostella vectensis (Anthozoa).

The T-Box transcription factor Brachyury plays important roles in the development of all bilateral animals examined so far. In order to understand the ancestral function of Brachyury we cloned NemBra1, a Brachyury homolog from the anthozoan sea anemone Nematostella vectensis. Anthozoa are considered the basal group among the Cnidaria. First NemBra1 expression could be detected at the blastula/gastrula transition and gene activity persists until adulthood of the animals. In situ hybridization shows that NemBra1 expression in gastrulae and early planula larvae is restricted to a circle around the blastopore. When the larvae begin to metamorphose into primary polyps, the expression zone extends into the developing mesenteries. In adult polyps Brachyury expression persists in the mesenteries, but is excluded from the septal filament and the differentiated retractor muscles, which also develop from the mesenteries. We conclude that the ancestral function of Brachyury was in specifying the blastopore and its endodermal derivatives.

Origin and evolution of endoderm and mesoderm.

Germ layers are defined as cell layers that arise during early animal development, mostly during gastrulation, and that give rise to all tissues and organs in adults. The evolutionary origin of the inner germ layers, endoderm and mesoderm, and their relationship have been a matter of debate for decades. In this review we summarize the major modes of endoderm and mesoderm formation found in Metazoa and possible evolutionary scenarios to reconstruct the ancestral state. In the second part, we address the question whether endoderm as well as mesoderm are homologous among Bilateria. In this regard, we propose that the comparative analysis of some crucial transcription factors involved in the early specification and differentiation of these germ layers might provide cues for the level of homology. We focus on four classes of genes: the Zn-finger gene GATA 4-6, the bHLH gene twist, the Krüppel-like Zn-finger gene snail and the T-box gene brachyury. The role of each of these genes in mesendoderm formation is summarized and we propose that the specific function of each of these genes in endoderm and mesoderm formation evolved from the regulation of basic cellular features, such as cell adhesion, cell motility, cytoskeleton and cell cycle.