Skin mucus proteins of lumpsucker (Cyclopterus lumpus)

Fish skin mucus serves as a first line of defense against pathogens and external stressors. In this study the proteomic profile of lumpsucker skin mucus was characterized using 2D gels coupled with tandem mass spectrometry. Mucosal proteins were identified by homology searches across the databases SwissProt, NCBInr and vertebrate EST. The identified proteins were clustered into ten groups based on their gene ontology biological process in PANTHER (www.patherdb.org). Calmodulin, cystatin-B, histone H2B, peroxiredoxin1, apolipoprotein A1, natterin-2, 14-3-3 protein, alfa enolase, pentraxin, warm temperature acclimation 65 kDa (WAP65kDa) and heat shock proteins were identified. Several of the proteins are known to be involved in immune and/or stress responses. Proteomic profile established in this study could be a benchmark for differential proteomics studies.     

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments

Humanized Fab′ fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab′ yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab′ expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab′, DsbC had the most significant impact, increasing humanized Fab′ production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored “wild type” cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD₆₀₀ 105), 40 h post Fab′ induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab′ fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212–220, 2017     

Synthesis and spectroscopic studies of antimony pentachloride complexes with neutral oxygen, sulfur and selenium ligands

The compounds [SbCl₅(R₃EY)] (R = Me or Ph; E = P or As; Y = O or S) have been prepared from SbCl₅ and the appropriate ligand in CH₂Cl₂ or CCl₄ solutions, and characterised by analysis, IR, ¹H, ³¹P{¹H}, ¹²¹Sb NMR spectroscopy and conductance measurements. The [SbCl₅(μ-L-L)SbCl₅] L-L = Ph₂P(O)CH₂P(O)Ph₂, Ph₂P(O)(CH₂)₂P(O)Ph₂, Ph₂P(S)CH₂P(S)Ph₂, Ph₂As(O)CH₂As(O)Ph₂, and o-C₆H₄(P(O)Ph₂)₂ have been synthesised and similarly characterised. The unstable [SbCl₅(R₃PSe)] have been prepared at low temperatures and characterised by IR spectroscopy. In solution in chlorocarbons they decompose rapidly to Se and R₃PCl₂. The reactions of R₃SbS with SbCl₅ produced R₃SbCl₂.     

A shorter peptide model from staphylococcal nuclease for the folding-unfolding equilibrium of a beta-hairpin shows that unfolded state has significant contribution from compact conformational states

It is important to understand the conformational features of the unfolded state in equilibrium with folded state under physiological conditions. In this paper, we consider a short peptide model LMYKGQPM from staphylococcal nuclease to model the conformational equilibrium between a hairpin conformation and its unfolded state using molecular dynamics simulation under NVT conditions at 300K using GROMOS96 force field. The free energy landscape has overall funnel-like shape with hairpin conformations sampling the minima. The "unfolded" state has a higher free energy of approximately 12kJ/mol with respect to native hairpin minimum and occupies a plateau region. We find that the unfolded state has significant contributions from compact conformations. Many of these conformations have hairpin-like topology. Further, these compact conformational forms are stabilized by hydrophobic interactions. Conversion between native and non-native hairpins occurs via unfolded states. Frequent conversions between folded and unfolded hairpins are observed with single exponential kinetics. We compare our results with the emerging picture of unfolded state from both experimental and theoretical studies.     

CD8alpha and CD8beta in Atlantic halibut, Hippoglossus hippoglossus: cloning, characterization and gene expression during viral and bacterial infection

CD8 is expressed on cytotoxic T-cells where it functions as a co-receptor for the TCR by binding to MHC class I proteins that present peptides on the cell surface. In this study we describe the cloning and sequencing of full length cDNAs encoding CD8alpha and CD8beta from Atlantic halibut (Hippoglossus hippoglossus L.) and subsequent isolation and characterization of the CD8alpha and CD8beta genes. The predicted halibut CD8alpha and CD8beta proteins are similar to those of mammals and other fish. Real time RT-PCR revealed that the highest levels of CD8 mRNA were found in the thymus, while some expression was also seen in the spleen, the gills, and the anterior and posterior kidney. In situ hybridization confirmed that the halibut thymus contained numerous CD8alpha and CD8beta expressing cells, while the anterior kidney had no CD8alpha positive cells but only a few CD8beta expressing cells. Only moderate changes in CD8 mRNA expression in other organs during either nodavirus or Vibrio anguillarum infection were observed. Both CD8alpha and CD8beta were significantly (P<0.05) down-regulated in spleen at 48h compared to their levels at 12h post-infection with nodavirus and V. anguillarum.     

Role of Epac1, an exchange factor for Rap GTPases, in endothelial microtubule dynamics and barrier function

Rap1 GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to colocalize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8-pCPT-2'O-Me-cAMP resulted in a net increase in the length of microtubules. This did not require cell-cell interactions or Rap GTPase activation, and it was attributed to microtubule growth as assessed by time-lapse microscopy of human umbilical vein endothelial cell expressing fluorophore-linked microtubule plus-end marker end-binding protein 3. An intact microtubule network was required for Epac-mediated changes in cortical actin and barrier enhancement, but it was not required for Rap activation. Finally, Epac activation reversed microtubule-dependent increases in vascular permeability induced by tumor necrosis factor-alpha and transforming growth factor-beta. Thus, Epac can directly promote microtubule growth in endothelial cells. This, together with Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability.     

Modulation of TNF release by choline requires alpha7 subunit nicotinic acetylcholine receptor-mediated signaling

The alpha7 subunit-containing nicotinic acetylcholine receptor (alpha7nAChR) is an essential component in the vagus nerve-based cholinergic anti-inflammatory pathway that regulates the levels of TNF, high mobility group box 1 (HMGB1), and other cytokines during inflammation. Choline is an essential nutrient, a cell membrane constituent, a precursor in the biosynthesis of acetylcholine, and a selective natural alpha7nAChR agonist. Here, we studied the anti-inflammatory potential of choline in murine endotoxemia and sepsis, and the role of the alpha7nAChR in mediating the suppressive effect of choline on TNF release. Choline (0.1-50 mM) dose-dependently suppressed TNF release from endotoxin-activated RAW macrophage-like cells, and this effect was associated with significant inhibition of NF-kappaB activation. Choline (50 mg/kg, intraperitoneally [i.p.]) treatment prior to endotoxin administration in mice significantly reduced systemic TNF levels. In contrast to its TNF suppressive effect in wild type mice, choline (50 mg/kg, i.p.) failed to inhibit systemic TNF levels in alpha7nAChR knockout mice during endotoxemia. Choline also failed to suppress TNF release from endotoxin-activated peritoneal macrophages isolated from alpha7nAChR knockout mice. Choline treatment prior to endotoxin resulted in a significantly improved survival rate as compared with saline-treated endotoxemic controls. Choline also suppressed HMGB1 release in vitro and in vivo, and choline treatment initiated 24 h after cecal ligation and puncture (CLP)-induced polymicrobial sepsis significantly improved survival in mice. In addition, choline suppressed TNF release from endotoxin-activated human whole blood and macrophages. Collectively, these data characterize the anti-inflammatory efficacy of choline and demonstrate that the modulation of TNF release by choline requires alpha7nAChR-mediated signaling.     

A quantitative analysis of kinase inhibitor selectivity

Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.