CCL1-CCR8 interactions: an axis mediating the recruitment of T cells and Langerhans-type dendritic cells to sites of atopic skin inflammation

Atopic dermatitis represents a chronically relapsing skin disease with a steadily increasing prevalence of 10-20% in children. Skin-infiltrating T cells, dendritic cells (DC), and mast cells are thought to play a crucial role in its pathogenesis. We report that the expression of the CC chemokine CCL1 (I-309) is significantly and selectively up-regulated in atopic dermatitis in comparison to psoriasis, cutaneous lupus erythematosus, or normal skin. CCL1 serum levels of atopic dermatitis patients are significantly higher than levels in healthy individuals. DC, mast cells, and dermal endothelial cells are abundant sources of CCL1 during atopic skin inflammation and allergen challenge, and Staphylococcus aureus-derived products induce its production. In vitro, binding and cross-linking of IgE on mast cells resulted in a significant up-regulation of this inflammatory chemokine. Its specific receptor, CCR8, is expressed on a small subset of circulating T cells and is abundantly expressed on interstitial DC, Langerhans cells generated in vitro, and their monocytic precursors. Although DC maintain their CCR8+ status during maturation, brief activation of circulating T cells recruits CCR8 from intracytoplamic stores to the cell surface. Moreover, the inflammatory and atopy-associated chemokine CCL1 synergizes with the homeostatic chemokine CXCL12 (SDF-1alpha) resulting in the recruitment of T cell and Langerhans cell-like DC. Taken together, these findings suggest that the axis CCL1-CCR8 links adaptive and innate immune functions that play a role in the initiation and amplification of atopic skin inflammation.     

Novel Humanized and Highly Efficient Bispecific Antibodies Mediate Killing of Prostate Stem Cell Antigen-Expressing Tumor Cells by CD8+ and CD4+ T Cells

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3-anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8(+) and CD4(+) T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4(+) T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.     

Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif

Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function at the cleavage site of the signal peptides (ASAFAP), which is particularly important for successful plastid targeting. Screening genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan, tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and putative receptor proteins involved in targeting, we constructed presequence:GFP fusion proteins with or without modifications of the “ASAFAP”-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import, as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus of the transit peptide together with a cleavable signal peptide is crucial for plastid import. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. Gruber and S. Vugrinec contributed equally to this work.     

Effect of water content on the structural reorganization and elastic properties of biopolymer films: a comparative study

In this work, the effect of water uptake on the structural reorganization and elastic properties of three types of biopolymer films was studied. The water-biopolymer interaction for hydroxypropyl cellulose (HPC), gelatin, and cassava starch films prepared from aqueous solutions was studied and compared using Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction, dynamic vapor sorption (DVS), and dynamic mechanical thermal analysis with humidity generator and controller (DMTA) techniques. The FTIR spectral variations due to the water sorption were generalized into two-dimensional (2D) correlation graphs for each biopolymer, and the effect of water on the molecular conformation was compared. The water sorption isotherms were fitted with Guggenheim-Anderson-De Boer (GAB) and D'Arcy and Watt models. The water content in the mono- and multilayers predicted by both models for each biopolymer was discussed and compared. The correlation of the fitted data obtained from the sorption isotherms to the DMTA data allowed us to conclude that the elastic properties of the HPC films depended on the total water content in contrast to the elastic properties of the gelatin and cassava starch films, which decrease only with the appearance of multilayer water.     

Translocation of a phycoerythrin alpha subunit across five biological membranes

Cryptophytes, unicellular algae, evolved by secondary endosymbiosis and contain plastids surrounded by four membranes. In contrast to cyanobacteria and red algae, their phycobiliproteins do not assemble into phycobilisomes and are located within the thylakoid lumen instead of the stroma. We identified two gene families encoding phycoerythrin alpha and light-harvesting complex proteins from an expressed sequence tag library of the cryptophyte Guillardia theta. The proteins bear a bipartite topogenic signal responsible for the transport of nuclear encoded proteins via the ER into the plastid. Analysis of the phycoerythrin alpha sequences revealed that more than half of them carry an additional, third topogenic signal comprising a twin arginine motif, which is indicative of Tat (twin arginine transport)-specific targeting signals. We performed import studies with several derivatives of one member using a diatom transformation system, as well as intact chloroplasts and thylakoid vesicles isolated from pea. We demonstrated the different targeting properties of each individual part of the tripartite leader and show that phycoerythrin alpha is transported across the thylakoid membrane into the thylakoid lumen and protease-protected. Furthermore, we showed that thylakoid transport of phycoerythrin alpha takes place by the Tat pathway even if the 36 amino acid long bipartite topogenic signal precedes the actual twin arginine signal. This is the first experimental evidence of a protein being targeted across five biological membranes.     

Protein-poor diet reduces host-specific immune gene expression in Bombus terrestris

Parasites infect hosts non-randomly as genotypes of hosts vary in susceptibility to the same genotypes of parasites, but this specificity may be modulated by environmental factors such as nutrition. Nutrition plays an important role for any physiological investment. As immune responses are costly, resource limitation should negatively affect immunity through trade-offs with other physiological requirements. Consequently, nutritional limitation should diminish immune capacity in general, but does it also dampen differences among hosts? We investigated the effect of short-term pollen deprivation on the immune responses of our model host Bombus terrestris when infected with the highly prevalent natural parasite Crithidia bombi. Bumblebees deprived of pollen, their protein source, show reduced immune responses to infection. They failed to upregulate a number of genes, including antimicrobial peptides, in response to infection. In particular, they also showed less specific immune expression patterns across individuals and colonies. These findings provide evidence for how immune responses on the individual-level vary with important elements of the environment and illustrate how nutrition can functionally alter not only general resistance, but also alter the pattern of specific host–parasite interactions.     

NMR for direct determination of Km and Vmax of enzyme reactions based on the Lambert W function-analysis of progress curves

¹H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K_m and V_max, were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8 mM. Using the Lambert W function the parameters K_m and V_max were fitted to obtain the experimental progress curve and resulted in K_m = 28 mM and V_max = 13 μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K_m = 379 μM and k_cat = 0.04 s^− 1. The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.     

N-terminal amino acid sequences of the hemopexins from chicken, rat and rabbit

The N-terminal amino acid sequences of the hemopexins purified from the plasma of rat, rabbit and chicken were compared with each other and with that of human hemopexin. Although the N-terminal sequences differ among these species, residues 2, 3 and 14 are identical in all four hemopexins. Ten of the first 28 residues are identical in all but the chicken protein. When introducing gaps into the sequence, a much greater homology is observed between the human and rat or rabbit hemopexins (60%) than when the sequences were compared directly (40%).     

Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development

An increased rate of lipid synthesis in cancerous tissues has long been recognised as an important aspect of the rewired metabolism of transformed cells. However, the contribution of lipids to cellular transformation, tumour development and tumour progression, as well as their potential role in facilitating the spread of cancerous cells to secondary sites, are not yet fully understood. In this article, we review the recent findings that support the importance of lipid synthesis and metabolism in tumorigenesis. Specifically, we explore the role of aberrant lipid biosynthesis in cancer cell migration and invasion, and in the induction of tumour angiogenesis. These processes are crucial for the dissemination of tumour cells and formation of metastases, which constitute the main cause of cancer mortality.     

The Deubiquitinating Enzyme AMSH1 and the ESCRT-III Subunit VPS2.1 Are Required for Autophagic Degradation in Arabidopsis

In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit VACUOLAR PROTEIN SORTING2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.