In Arabidopsis, the EMBYRONIC FLOWER2 (EMF2), VERNALISATION2 (VRN2) and FERTILISATION INDEPENDENT ENDOSPERM2 (FIS2) genes encode related Polycomb-group (Pc-G)proteins. Their homologues in animals act together with other Pc-G proteins as part of a multimeric complex, Polycomb Repressive Complex 2 (PRC2), which functions as a histone methyltransferase. Despite similarities between the fis2 mutant phenotype and those of some other plant Pc-G members, it has remained unclear how the FIS2/EMF2/VRN2 class Pc-G genes interact with the others. We have identified a weak emf2 allele that reveals a novel phenotype with striking similarity to that of severe mutations in another Pc-G gene, CURLY LEAF (CLF), suggesting that the two genes may act in a common pathway. Consistent with this, we demonstrate that EMF2 and CLF interact genetically and that this reflects interaction of their protein products through two conserved motifs, the VEFS domain and the C5 domain. We show that the full function of CLF is masked by partial redundancy with a closely related gene, SWINGER(SWN), so that null clf mutants have a much less severe phenotype than emf2 mutants. Analysis in yeast further indicates a potential for the CLF and SWN proteins to interact with the other VEFS domain proteins VRN2 and FIS2. The functions of individual Pc-G members may therefore be broader than single mutant phenotypes reveal. We suggest that plants have Pc-G protein complexes similar to the Polycomb Repressive Complex2 (PRC2) of animals, but the duplication and subsequent diversification of components has given rise to different complexes with partially discrete functions.

Genes of the FERTILISATION INDEPENDENT SEED (FIS) class regulate cell proliferation during reproductive development in Arabidopsis [1-5]. The FIS genes FERTILISATION INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA) encode homologs of animal Polycomb group (Pc-G) proteins, transcriptional regulators that modify chromatin structure and are thought to form multimeric complexes [3-11]. To test whether similarities in fis mutant phenotypes reflect interactions between their protein products, we characterised FIE RNA and protein localisation in vivo, and FIE protein interactions in yeast and in vitro. Expression of FIE mRNA overlaps with that of MEA during embryo sac and seed development and is unaffected in mea mutants. Results from the yeast two-hybrid system and an in vitro pull-down assay indicate that MEA and FIE proteins interact. The relevance of this interaction in vivo is supported by the finding that FIE and MEA co-localise in the nucleus in transfected plant cells. Interaction of MEA and FIE is mediated by the amino-terminal region of MEA. Despite sequence divergence in this domain, MEA can interact with its corresponding animal partner Extrasexcombs (ESC) in the yeast two-hybrid system. We conclude that FIE and MEA act together as part of a multimeric complex and that this accounts for the similarities in mutant phenotypes. We propose that an ancient mechanism for chromatin modification has been independently recruited to different developmental processes in the two kingdoms.

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Despite recent developments, there is a high medical need for new treatment options for chronic inflammatory dermatoses like allergic contact dermatitis (ACD) and psoriasis. Particularly, more predictive skin inflammation models are required to facilitate the process of drug discovery. Murine contact hypersensitivity (CHS) models adequately reflect ACD and are also used to characterize therapeutic approaches for psoriasis. Using the hapten 2,4-dinitrofluorobenzene (DNFB), we established new subacute and subchronic DNFB-induced CHS models in C57BL/6 mice, which more closely reflect the characteristics of chronic T-cell-dependent inflammatory dermatoses as pronounced keratinocyte proliferation, strong hypervascularization, immune cell infiltration and overexpression of T cell and inflammatory cytokines. For the subacute DNFB model, we demonstrated anti-inflammatory activity of the glucocorticoid, prednisolone, as well as of neutralization of TNFα, IL-12/IL-23 or IL-18. In the subchronic DNFB-induced CHS model, deficiency for MyD88 and IL-12/IL-35 p35 chain but not IL-12/IL-23 p40 chain led to decreased skin inflammation. Furthermore, as exemplified by the dose-dependently effective therapeutic prednisolone treatment, the subchronic model allows the continuous therapy of a pre-established stable contact dermatitis. Altogether, prolonged DNFB-induced mouse CHS models closely reflect ACD sensitive to glucocorticoids as standard therapy, reveal a more chronic skin inflammation and are responsive to cytokine antagonization.

T-cell-mediated processes play an essential role in the pathogenesis of several inflammatory skin diseases such as atopic dermatitis, allergic contact dermatitis and psoriasis. The aim of this study was to investigate the role of the IL-2-inducible tyrosine kinase (Itk), an enzyme acting downstream of the T-cell receptor (TCR), in T-cell-dependent skin inflammation using three approaches. Itk knockout mice display significantly reduced inflammatory symptoms in mouse models of acute and subacute contact hypersensitivity (CHS) reactions. Systemic administration of a novel small molecule Itk inhibitor, Compound 44, created by chemical optimization of an initial high-throughput screening hit, inhibited Itk's activity with an IC50 in the nanomolar range. Compound 44 substantially reduced proinflammatory immune responses in vitro and in vivo after systemic administration in two acute CHS models. In addition, our data reveal that human Itk, comparable to its murine homologue, is expressed mainly in T cells and is increased in lesional skin from patients with atopic dermatitis and allergic contact dermatitis. Finally, silencing of Itk by RNA interference in primary human T cells efficiently blocks TCR-induced lymphokine secretion. In conclusion, Itk represents an interesting new target for the therapy of T-cell-mediated inflammatory skin diseases.

Mn:PIN-PMN-PT piezocrystals are under consideration for potential use in miniaturised ultrasonic scalpels for robotic minimally-invasive surgery where small size and light weight may be advantageous. Electromechanical coupling coefficient k > 0.9 was calculated for both for both [001] and [011] poled Mn:PIN-PMN-PT was calculated, confirming the well-recognized higher efficiency of this material when compared to standard piezoceramics. Novel transducer design strategies have been explored, and outcomes are discussed. The introduction of components with additional compliance in a standard d31 mode transducer has been shown to drop the resonant frequency of the first longitudinal mode by more than 17%, with more than 75% improvement in tip/blade displacement. Results suggest that the combination of high performance piezocrystals with highly compliant components may be a useful route to follow to achieve our miniaturisation target.

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