We generated mice deficient in plakophilin-3 (PKP3), a member of the Armadillo-repeat family and a component of desmosomes and stress granules in epithelial cells. In these mice, several subsets of hair follicles (HFs) had morphological abnormalities, and the majority of awl and auchene hair shafts had fewer medullar air columns. Desmosomes were absent from the basal layer of the outer root sheath of HFs and from the matrix cells that are in contact with dermal papillae. In the basal layer of PKP3-null epidermis, densities of desmosomes and adherens junctions were remarkably altered. Compensatory changes in several junctional proteins were observed. PKP3-null mice housed in conventional facilities were prone to dermatitis. Our animal model provides in vivo evidence that PKP3 plays a critical role in morphogenesis of HFs and shafts and in limiting inflammatory responses in the skin.

We here describe the use of added reversed-phase chromatographic beads to concentrate peptides from highly diluted solutions. In the procedure developed, peptide-bead suspensions are dried under vacuum to complete dryness; peptides are subsequently eluted in a small volume of matrix-assisted laser desorption/ionization (MALDI)-matrix containing organic/aqueous solvent and transferred to a MALDI-target for mass analysis. We show that by using this bead-peptide concentration procedure, low femtomole amounts of peptides are efficiently concentrated, up to 1000 times, to volumes smaller than 0.7 microL. We have used this concentration procedure in combination with MALDI-post-source decay analysis to identify subpicomole amounts of proteins present in polyacrylamide gels. Furthermore, we show that the bead-peptide concentration method can be elegantly used to clean up samples contaminated with high concentrations of substances normally deleterious to MALDI-mass spectrometry (MS) experiments. We have found additionally that the bead-peptide concentration procedure can be successfully used to store low femtomole amounts of peptide for prolonged periods of time without severe losses of peptide material. This bead-peptide concentration procedure therefore seems to be a simple and convenient step in the MALDI-MS sample preparation process.

We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.

Plakophilin-3 (PKP3) is a member of the armadillo protein family, which is important inKeywords: Acute inflammation r Colitis r Dermatitis r Hematopoietic cell r Intestine r Plakophilin-3 r Sepsis r SkinAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe interaction of the organism with its environment requires the presence of a specialized "danger sensing" system. This protective system operates mostly at the surfaces between the outer and inner environments of the body, such as the skin, intestinal tract, and respiratory tract. From a classical point of view, the epithelial Correspondence: Mrs. Roosmarijn E. Vandenbroucke e-mail: Roosmarijn.Vandenbroucke@irc.VIB-UGent.be surfaces at these locations form a physical barrier that is impermeable to most infectious agents and thereby provide a first line of defense. More recently, subtle interactions between microbes and these epithelial surfaces have been shown to determine local immune reactions and disease. The barriers consists of three parts: an extensive epithelial cell surface exposed to the environment, a basement membrane that, together with various cell-junction structures, provides an extra level of separation, and a highly reactive lymphoid tissue underneath composed of antigen-presenting cells [1][2][3][4].C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 2898-2910 Innate immunity 2899Several inflammatory conditions, such as skin inflammation, inflammatory bowel disease (IBD) and the systemic inflammatory response syndrome (SIRS), are associated with disruption of the respective epithelial barriers. The skin is the largest organ of the body and is continuously exposed to microorganisms, ultraviolet radiation, toxic agents, and mechanical stress. Skin inflammation can be mimicked in the mouse by topical application of a single dose of 12-O-tetradecanoylphorbol-13-acetate, also known as phorbol-12-myristate-13-acetate (PMA). This induces an acute inflammatory reaction called irritant contact dermatitis (ICD) consisting of erythema, edema, and polymorphonuclear leukocyte (PMN) infiltration [5]. Skin inflammation is accompanied by epidermal barrier disruption because tight junctions between keratinocytes are disrupted and cytokines induce apoptosis or necrosis of keratinocytes [6]. Disruption of the epithelial barrier is also a key feature of IBD [7,8]. The intestinal barrier defects activate the underlying immune system, resulting in tissue damage. The dextran sulfate sodium (DSS) model of colitis correlates well with human IBD and is deemed suitable for investigating its pathogenesis [9]. SIRS is a lethal inflammatory disease caused by trauma, burns, infections, and other insults [10]. When SIRS is associated with hypotension, it is known as sepsis or septic shock. Several mouse models of sepsis are known [10]. The endotoxemia model involves the injection of bacterial lipopolysaccharides (LPS), which binds to the pathogen-recognition...