Further characterization and mode of action of dactylomelin-P, an antibacterial protein isolated from the ink of the sea hare Aplysia dactylomela (Rang, 1828)

Sea hares are well known, nearly shell-less, marine opisthobranchs that use a complex repertoire of chemicals for defense and communication instead of a conventional gastropod shell. The most conspicuous characteristic of these invertebrates is the secretion of ink, which is rich in bioactive proteins. Many of these proteins belong to a family of L-amino acid oxidases (L-AAOs). In the current study, we aimed to determine whether dactylomelin-P, an antibacterial protein isolated from the ink of Aplysia dactylomela, could act as an L-AAO. We also investigated its biochemical properties and antibacterial mechanism of action. We found that dactylomelin-P is an acidic protein (pI = 5.0), rich in glutamic acid/glutamine, aspartic acid/asparagine, tyrosine, serine, and proline. It was stable under a broad pH range (3.0-12.0), after heating to 55 °C for 30 min, and after treating with protease. Its N-terminal amino acid sequence was DGVCSNRRQCNKEVCGSSYDVAIVGA and showed high similarity to other sea hare proteins previously identified as L-AAOs. The L-AAO activity was confirmed in an enzymatic assay, which showed that dactylomelin-P could oxidize L-lysine and L-arginine. We also demonstrated that the bacteriostatic activity of dactylomelin-P was mediated by hydrogen peroxide generated in the enzymatic reaction, but it acted as a bactericide in the presence of L-lysine and L-arginine. Transmission electron microscopy analyses showed that dactylomelin-P bound to growth-phase bacteria without causing morphological alterations to the cells. The bactericidal effect seems to involve H₂O₂ and other reactive components since it was not counteracted by H₂O₂ scavengers. Our findings showed biochemical, functional, and phylogenetic similarities among L-AAOs isolated from sea hares; this offers new insight into the evolution of these proteins and their roles in chemical defense.     

The 1,25D3-MARRS protein: contribution to steroid stimulated calcium uptake in chicks and rats

There are currently two main candidates for the membrane receptor for 1,25(OH)2D3: the 1,25D3-MARRS protein/ERp57; and the classical VDR. The 1,25D3-MARRS protein is essential for hormone-stimulated phosphate and calcium uptake in chick intestinal cells, whereas the VDR is not. The 1,25D3-MARRS protein also shows a high degree of correlation with growth periods in which bone is rapidly formed, whereas the VDR does not. However, in rat enterocytes, both the 1,25D3-MARRS protein and the VDR play a role in the rapid, steroid-mediated uptake of phosphate or calcium. Therefore, the theory that alternate binding sites on the VDR for various analogs account for all membrane-initiated phenomena, is incorrect.     

Antiviral and regulatory T cell immunity in a patient with stromal interaction molecule 1 deficiency

Stromal interaction molecule 1 (STIM1) deficiency is a rare genetic disorder of store-operated calcium entry, associated with a complex syndrome including immunodeficiency and immune dysregulation. The link from the molecular defect to these clinical manifestations is incompletely understood. We report two patients with a homozygous R429C point mutation in STIM1 completely abolishing store-operated calcium entry in T cells. Immunological analysis of one patient revealed that despite the expected defect of T cell proliferation and cytokine production in vitro, significant antiviral T cell populations were generated in vivo. These T cells proliferated in response to viral Ags and showed normal antiviral cytotoxicity. However, antiviral immunity was insufficient to prevent chronic CMV and EBV infections with a possible contribution of impaired NK cell function and a lack of NKT cells. Furthermore, autoimmune cytopenia, eczema, and intermittent diarrhea suggested impaired immune regulation. FOXP3-positive regulatory T (Treg) cells were present but showed an abnormal phenotype. The suppressive function of STIM1-deficient Treg cells in vitro, however, was normal. Given these partial defects in cytotoxic and Treg cell function, impairment of other immune cell populations probably contributes more to the pathogenesis of immunodeficiency and autoimmunity in STIM1 deficiency than previously appreciated.     

MHC motif viewer

In vertebrates, the major histocompatibility complex (MHC) presents peptides to the immune system. In humans, MHCs are called human leukocyte antigens (HLAs), and some of the loci encoding them are the most polymorphic in the human genome. Different MHC molecules present different subsets of peptides, and knowledge of their binding specificities is important for understanding the differences in the immune response between individuals. Knowledge of motifs may be used to identify epitopes, to understand the MHC restriction of epitopes, and to compare the specificities of different MHC molecules. Algorithms that predict which peptides MHC molecules bind have recently been developed and cover many different alleles, but the utility of these algorithms is hampered by the lack of tools for browsing and comparing the specificity of these molecules. We have, therefore, developed a web server, MHC motif viewer, that allows the display of the likely binding motif for all human class I proteins of the loci HLA A, B, C, and E and for MHC class I molecules from chimpanzee (Pan troglodytes), rhesus monkey (Macaca mulatta), and mouse (Mus musculus). Furthermore, it covers all HLA-DR protein sequences. A special viewing feature, MHC fight, allows for display of the specificity of two different MHC molecules side by side. We show how the web server can be used to discover and display surprising similarities as well as differences between MHC molecules within and between different species. The MHC motif viewer is available at .     

Filamentous network mechanics and active contractility determine cell and tissue shape

For both cells and tissues, shape is closely correlated with function presumably via geometry-dependent distribution of tension. In this study, we identify common shape determinants spanning cell and tissue scales. For cells whose sites of adhesion are restricted to small adhesive islands on a micropatterned substrate, shape resembles a sequence of inward-curved circular arcs. The same shape is observed for fibroblast-populated collagen gels that are pinned to a flat substrate. Quantitative image analysis reveals that, in both cases, arc radii increase with the spanning distance between the pinning points. Although the Laplace law for interfaces under tension predicts circular arcs, it cannot explain the observed dependence on the spanning distance. Computer simulations and theoretical modeling demonstrate that filamentous network mechanics and contractility give rise to a modified Laplace law that quantitatively explains our experimental findings on both cell and tissue scales. Our model in conjunction with actomyosin inhibition experiments further suggests that cell shape is regulated by two different control modes related to motor contractility and structural changes in the actin cytoskeleton.     

Calcineurin-induced energy wasting in a transgenic mouse model of heart failure

Overexpression of calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P-NMR spectroscopy as ATP and phosphocreatine concentrations ([ATP] and [PCr]) and free energy of ATP hydrolysis (|Delta G( approximately ATP)|). Mitochondrial and glycolytic enzyme activities, myocardial O2 consumption, and myocyte ultrastructure were determined. In transgenic (TG) hearts at all levels of work, indexes of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower than in non-TG hearts. This is the first report showing that myocardial [ATP] is lower in a TG mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because Pi concentration remained low, despite lower [ATP] and [PCr], |Delta G( approximately ATP)| was normal; however, chemical energy did not translate to systolic performance. This was most apparent with beta-adrenergic stimulation of TG hearts, during which, for similar changes in |Delta G( approximately ATP)|, systolic pressure decreased, rather than increased. Structural abnormalities observed for sarcomeres and mitochondria likely contribute to decreased contractile performance. On the basis of the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor cyclosporin A, we also conclude that CLN directed inhibition of ATP-producing pathways in non-TG and TG hearts.     

Seasonal and spatial variation of carbohydrates in mistletoes (Viscum album) and the xylem sap of its hosts (Populus x euamericana and Abies alba)

In the present field study we analysed the seasonal pattern of carbohydrate composition and contents in the xylem sap of Viscum album and the xylem sap of a deciduous ( Populus × euramericana ) and a coniferous ( Abies alba ) host tree species. The results were compared with the soluble carbohydrate composition and contents of mistletoe tissues. On both hosts significant amounts of glucose, fructose, and sucrose were found in the xylem sap of Viscum throughout the seasons. The general seasonal pattern of sugar contents, i.e. high concentrations in spring and lower concentrations in other seasons on Populus , and intermediate concentrations throughout the year on Abies , largely reflected the xylem sap carbohydrate composition and contents of the respective host. These observations provide indirect evidence for carbohydrate flux from the xylem sap of the host into the mistletoe. However, in both hosts xylem sap seems to be deviated into the mistletoe without specific control of carbohydrate flux. Differences observed between the seasonal pattern of xylem sap carbohydrate concentrations in Viscum on Populus and Abies may originate from the different time of leaf development of these species. A clear-cut seasonal pattern of soluble carbohydrates was not observed in the leaves of Viscum on both hosts. Still soluble carbohydrates seem to be reallocated from the senescing to the newly developed leaves of Viscum indicating that the seasonal requirement of carbohydrate for growth and development can only completely be met by carbohydrate acquisition from the host and their own photosynthesis.