NAADP+ is an agonist of the human P2Y11 purinergic receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is an intracellular second messenger releasing Ca2+ from intracellular stores in different cell types. In addition, it is also active in triggering [Ca2+](i) increase when applied extracellularly and various underlying mechanisms have been proposed. Here, we used hP2Y(11)-transfected 1321N1 astrocytoma cells to unequivocally establish whether extracellular NAADP+ is an agonist of the P2Y(11) receptor, as previously reported for beta-NAD+ [I. Moreschi, S. Bruzzone, R.A. Nicholas, et al., Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes, J. Biol. Chem. 281 (2006) 31419-31429]. Extracellular NAADP+ triggered a concentration-dependent two-step elevation of [Ca2+](i) in 1321N1-hP2Y(11) cells, but not in wild-type 1321N1 cells, secondary to the intracellular production of IP(3), cAMP and cyclic ADP-ribose (cADPR). Specifically, the transient [Ca2+](i) rise proved to be related to IP(3) overproduction and to consequent Ca2+ mobilization, while the sustained [Ca2+](i) elevation was caused by the cAMP/ADP-ribosyl cyclase (ADPRC)/cADPR signalling cascade and by influx of extracellular Ca2+. In human granulocytes, endogenous P2Y(11) proved to be responsible for the NAADP+-induced cell activation (as demonstrated by the use of NF157, a selective and potent inhibitor of P2Y(11)), unveiling a role of NAADP+ as a pro-inflammatory cytokine. In conclusion, we provide unequivocal evidence for the activation of a member of the P2Y receptor subfamily by NAADP+.     

Soluble M1 protein of Streptococcus pyogenes triggers potent T cell activation

Streptococcus pyogenes of the M1 serotype is commonly associated with large outbreaks of invasive streptococcal infections and development of streptococcal toxic shock syndrome (STSS). The pathogenesis behind these infections is believed to involve bacterial superantigens that induce potent inflammatory responses, but the reason why strains of the M1 serotype are over-represented in STSS is still not understood. In the present investigation, we show that a highly purified soluble form of the M1 protein from S. pyogenes , which lacks the membrane-spanning region, is a potent inducer of T cell proliferation and release of Th1 type cytokines. M1 protein-evoked T cell proliferation was HLA class II-dependent but not MHC-restricted, did not require intracellular processing and was Vβ-restricted. Extensive mass spectrometry studies indicated that there were no other detectable proteins in the preparation. Taken together, our data demonstrate that soluble M1 protein is a novel streptococcal superantigen, which likely contributes to the excessive T cell activation and hyperinflammatory response seen in severe invasive streptococcal infections.     

Weak functional coupling of the melanocortin-1 receptor expressed in human adipocytes

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40-49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-alpha), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-alpha upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R-effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.     

A novel betaproteobacterial agent of gill epitheliocystis in seawater farmed Atlantic salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although 'Candidatus Piscichlamydia salmonis', previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as 'Candidatus Branchiomonas cysticola', was the cyst-forming agent in these samples. While histology and ultrastructure of 'Ca. B. cysticola' cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae.     

Silintaphin-1--interaction with silicatein during structure-guiding bio-silica formation

Silicateins are unique enzymes of sponges (phylum Porifera) that template and catalyze the polymerization of nanoscale silicate to siliceous skeletal elements. These multifunctional spicules are often elaborately shaped, with complex symmetries. They carry an axial proteinaceous filament, consisting of silicatein and the scaffold protein silintaphin-1, which guides silica deposition and subsequent spicular morphogenesis. In vivo, the synthesis of the axial filament very likely proceeds in three steps: (a) assembly of silicatein monomers to form one pentamer; (b) assembly of pentamers to form fractal-like structures; and finally (c) assembly of fractal-like structures to form filaments. The present study was aimed at exploring the effect of self-assembled complexes of silicatein and silintaphin-1 on biosilica synthesis in vitro. Hence, in a comparative approach, recombinant silicatein and recombinant silintaphin-1 were used at different stoichiometric ratios to form axial filaments and to synthesize biosilica. Whereas recombinant silicatein-α reaggregates to randomly organized structures, coincubation of silicatein-α and silintaphin-1 (molecular ratio 4 : 1) resulted in synthetic filaments via fractal-like patterned self-assemblies, as observed by electron microscopy. Concurrently, owing to the concerted action of both proteins, the enzymatic activity of silicatein-α strongly increased by 5.3-fold (with the substrate tetraethyl orthosilicate), leading to significantly enhanced synthesis of biosilica. These results indicate that silicatein-α-mediated biosilicification depends on the concomitant presence of silicatein-α and silintaphin-1. Accordingly, silintaphin-1 might not only enhance the enzymatic activity of silicatein-α, but also accelerate the nonenzymatic polycondensation of the silica product before releasing the fully synthesized biosiliceous polymer.     

Chlamydia pneumoniae infection acts as an endothelial stressor with the potential to initiate the earliest heat shock protein 60-dependent inflammatory stage of atherosclerosis

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.     

Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8(+) T cells sensitizing them to apoptotic cell death

The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23～24～27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.     

The Ovicidal,Larvacidal and Adulticidal Properties of 5,5′-Dimethyl-2,2′-Bipyridyl against Drosophila melanogaster

Insecticide resistance has limited the number of available chemical options for insect pest control. Hence there is a need for new chemistries with novel modes of action. Here we investigate the mode of action for an insecticide that has not yet been released for commercial use. The ovicidal, larvacidal and adulticidal effects of 5,5′-dimethyl -2, 2′-dipyridyl (termed Ha44), which is being developed as a treatment for head lice, were evaluated in the Drosophila melanogaster model system. Ha44 demonstrated significant activity against embryos and was capable of arresting development at a number of stages of embryogenesis. The effects of Ha44 on embryos was shown to be reversible following the addition of the metal ions Fe(II) and Fe(III), Cu and Zn. When larvae were exposed to Ha44, lethality was recorded at similar concentrations to those observed for embryos. Using an eYFP reporter system it was shown that Ha44 was able to reduce the levels of both copper and zinc in the digestive tract, confirming the binding of Ha44 to these metals in vivo. Ha44 has further been shown to inhibit a zinc containing metalloproteinase in vitro. Exposure of adult flies to Ha44 resulted in lethality, but at higher concentrations than those observed for embryos and larvae. The median lethal dose in adult flies was shown to be associated with the type of exposure, with an LD-50 of 1.57 mM being recorded following the direct contact of flies with Ha44, while an LD-50 of 12.29 mM was recorded following the ingestion of the compound. The capacity of Ha44 to act on all stages of the life-cycle and potentially via a range of targets suggests that target site resistance is unlikely to evolve.