Terephthalamide-containing ligands: fast removal of iron from transferrin

The mechanism and effectiveness of iron removal from transferrin by three series of new potential therapeutic iron sequestering agents have been analyzed with regard to the structures of the chelators. All compounds are hexadentate ligands composed of a systematically varied combination of methyl-3,2-hydroxypyridinone (Me-3,2-HOPO) and 2,3-dihydroxyterephthalamide (TAM) binding units linked to a polyamine scaffold through amide linkers; each series is based on a specific backbone: tris(2-aminoethyl)amine, spermidine, or 5-LIO(TAM), where 5-LIO is 2-(2-aminoethoxy)ethylamine. Rates of iron removal from transferrin were determined spectrophotometrically for the ten ligands, which all efficiently acquire ferric ion from diferric transferrin with a hyperbolic dependence on ligand concentration (saturation kinetics). The effect of the two iron-binding subunits Me-3,2-HOPO and TAM and of the scaffold structures on iron removal ability is discussed. At the low concentrations corresponding to therapeutic dose, TAM-containing ligands exhibit the fastest rates of iron removal, which correlates with their high affinity for ferric ion and suggests the insertion of such binding units into future therapeutic chelating agents. In addition, urea polyacrylamide gel electrophoresis was used to measure the individual microscopic rates of iron removal from the three iron-bound transferrin species (diferric transferrin, N-terminal monoferric transferrin, C-terminal monoferric transferrin) by the representative chelators 5-LIO(Me-3,2-HOPO)(2)(TAM) and 5-LIO(TAMmeg)(2)(TAM), where TAMmeg is 2,3-dihydroxy-1-(methoxyethylcarbamoyl)terephthalamide. Both ligands show preferential removal from the C-terminal site of the iron-binding protein. However, cooperative effects between the two binding sites differ with the chelator. Replacement of hydroxypyridinone moieties by terephthalamide groups renders the N-terminal site more accessible to the ligand and may represent an advantage for iron chelation therapy.     

Sisyphus, the Drosophila myosin XV homolog, traffics within filopodia transporting key sensory and adhesion cargos

Unconventional myosin proteins of the MyTH-FERM superclass are involved in intrafilopodial trafficking, are thought to be mediators of membrane-cytoskeleton interactions, and are linked to several forms of deafness in mammals. Here we show that the Drosophila myosin XV homolog, Sisyphus, is expressed at high levels in leading edge cells and their cellular protrusions during the morphogenetic process of dorsal closure. Sisyphus is required for the correct alignment of cells on opposing sides of the fusing epithelial sheets, as well as for adhesion of the cells during the final zippering/fusion phase. We have identified several putative Sisyphus cargos, including DE-cadherin (also known as Shotgun) and the microtubule-linked proteins Katanin-60, EB1, Milton and aPKC. These cargos bind to the Sisyphus FERM domain, and their binding is in some cases mutually exclusive. Our data suggest a mechanism for Sisyphus in which it maintains a balance between actin and microtubule cytoskeleton components, thereby contributing to cytoskeletal cross-talk necessary for regulating filopodial dynamics during dorsal closure.     

Nonparametric variance estimation in the analysis of microarray data: a measurement error approach

We investigate the effects of measurement error on the estimationof nonparametric variance functions. We show that either ignoringmeasurement error or direct application of the simulation extrapolation,SIMEX, method leads to inconsistent estimators. Nevertheless,the direct SIMEX method can reduce bias relative to a naiveestimator. We further propose a permutation SIMEX method thatleads to consistent estimators in theory. The performance ofboth the SIMEX methods depends on approximations to the exactextrapolants. Simulations show that both the SIMEX methods performbetter than ignoring measurement error. The methodology is illustratedusing microarray data from colon cancer patients.     

Joint modelling of paired sparse functional data using principal components

We propose a modelling framework to study the relationship betweentwo paired longitudinally observed variables. The data for eachvariable are viewed as smooth curves measured at discrete time-pointsplus random errors. While the curves for each variable are summarizedusing a few important principal components, the associationof the two longitudinal variables is modelled through the associationof the principal component scores. We use penalized splinesto model the mean curves and the principal component curves,and cast the proposed model into a mixed-effects model frameworkfor model fitting, prediction and inference. The proposed methodcan be applied in the difficult case in which the measurementtimes are irregular and sparse and may differ widely acrossindividuals. Use of functional principal components enhancesmodel interpretation and improves statistical and numericalstability of the parameter estimates.     

Copper-induced structural changes in the ovine prion protein are influenced by a polymorphism at codon 112

Prion diseases are associated with conformational change in the copper-binding protein PrP. The copper-binding sites in PrP are located in the N-terminal region of the molecule and comprise a series of tandem repeats of the sequence PHGGGWGQ together with two histidines at residues 96 and 111 (human PrP numbering). The co-ordination of copper ions within the non-octapeptide repeat metal ion-binding site involves Met109 (human numbering, which corresponds with Met112 in ovine PrP) and the binding of copper to this site leads to an increase in beta-sheet formation in PrP. Here we have investigated the influence of the M112T polymorphism on copper-induced structural changes in ovine recombinant PrP. M112ARQ and T112ARQ ovine PrP show similar secondary structure although M112ARQ appears more thermostable than T112ARQ. Following treatment with copper, M112ARQ showed a greater increase in beta-sheet content than did T112ARQ when measured by CD spectroscopy and by ELISA using anti-PrP monoclonal antibodies. These biochemical and biophysical differences between M112ARQ and T112ARQ correlate with similar differences seen between allelic variants of ovine PrP associated with susceptibility and resistance to classical scrapie. These observations suggest that T112ARQ may provide a measure of resistance to classical scrapie pathogenesis compared to M112ARQ.     

The denitrification capability of cluster 1 Defluviicoccus vanus-related glycogen-accumulating organisms

The denitrification capability of Cluster 1 Defluviicoccus vanus-related glycogen-accumulating organisms (DvGAOs) is investigated. A sequencing batch reactor (SBR) fed with acetate as the sole carbon source was operated under alternating anaerobic-aerobic conditions to enrich Cluster 1 DvGAOs. Fluorescence in situ hybridization (FISH) showed that more than 85% of the bacterial population present in the reactor bound to the probes previously designed for Cluster 1 DvGAOs. A series of batch tests were performed to evaluate the capability of the community to reduce nitrate and nitrite. The tests were carried out both before and after the adaptation of the culture to anoxic conditions, and with both the intracellularly stored carbon and acetate as the electron donors. It was found that Cluster 1 DvGAOs were able to reduce nitrate but most likely unable to reduce nitrite. When un-adapted Cluster 1 DvGAOs were exposed to nitrate for the first time, a lag phase of approximately 4 h occurred, which was likely required for the synthesis of the necessary enzymes.     

Monitoring of native chemical ligation on solid substrate by surface plasmon resonance

During the last years native chemical ligation (NCL) gained in popularity as a method allowing the chemical synthesis of large peptides and entire proteins. NCL is particularly well-suited for chemoselective and nondenaturing attachment of biomolecules on solid substrates. In the present work, we show the feasibility of monitoring of peptide synthesis, NCL and its catalysis on silicon oxide modified gold surfaces by surface plasmon resonance (SPR). NCL of a model peptide-bradykinin thioester-was carried out and monitored with a custom-built SPR apparatus. Solid-phase produced bradykinin thioester was ligated to the surface in the presence of variable concentrations of 4-mercaptophenylacetic acid as transthioesterification catalyst. At catalyst concentration of 48 mM and above, the NCL reaction was maximal and identical to the reaction of the purified peptide-mercaptophenylacetic acid thioester. SPR curves indicate typical first-order kinetics with t(1/2) of 81 s for this aryl thioester, but of 104 min for the primary alkyl thioester.     

Chemical ablation of the Purkinje system causes early termination and activation rate slowing of long-duration ventricular fibrillation in dogs

Endocardial mapping has suggested that Purkinje fibers may play a role in the maintenance of long-duration ventricular fibrillation (LDVF). To determine the influence of Purkinje fibers on LDVF, we chemically ablated the Purkinje system with Lugol solution and recorded endocardial and transmural activation during LDVF. Dog hearts were isolated and perfused, and the ventricular endocardium was exposed and treated with Lugol solution (n = 6) or normal Tyrode solution as a control (n = 6). The left anterior papillary muscle endocardium was mapped with a 504-electrode (21 x 24) plaque with electrodes spaced 1 mm apart. Transmural activation was recorded with a six-electrode plunge needle on each side of the plaque. Ventricular fibrillation (VF) was induced, and perfusion was halted. LDVF spontaneously terminated sooner in Lugol-ablated hearts than in control hearts (4.9 +/- 1.5 vs. 9.2 +/- 3.2 min, P = 0.01). After termination of VF, both the control and Lugol hearts were typically excitable, but only short episodes of VF could be reinduced. Endocardial activation rates were similar during the first 2 min of LDVF for Lugol-ablated and control hearts but were significantly slower in Lugol hearts by 3 min. In control hearts, the endocardium activated more rapidly than the epicardium after 4 min of LDVF with wave fronts propagating most often from the endocardium to epicardium. No difference in transmural activation rate or wave front direction was observed in Lugol hearts. Ablation of the subendocardium hastens VF spontaneous termination and alters VF activation sequences, suggesting that Purkinje fibers are important in the maintenance of LDVF.     

Role of heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetylcholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2-/-), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 microM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2-/- mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2-/- mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2-/- mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2-/- mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation.     

Liposomal glucocorticoids as tumor-targeted anti-angiogenic nanomedicine in B16 melanoma-bearing mice

This study evaluates whether the inhibitory effects of prednisolone phosphate (PLP) encapsulated in long-circulating liposomes (LCL-PLP) on tumor growth and tumor angiogenesis described previously can be generalized to other types of glucocorticoids (GC) encapsulated in LCL (LCL-GC). Four types of synthetic GC, i.e. budesonide disodium phosphate (BUP), dexamethasone disodium phosphate (DXP), methylprednisolone disodium phosphate (MPLP), and PLP, were selected based on the difference in their potency to activate the human glucocorticoid receptor. The effects of all LCL-GC on the production of angiogenic/inflammatory factors in vivo in the B16.F10 murine melanoma model as well as on the viability and proliferation of tumor cells and endothelial cells in vitro were investigated. Our results show that all four selected LCL-GC formulations inhibit tumor growth, albeit to different degrees. The differences in antitumor activity of LCL-GC correlate with their efficacy to suppress tumor angiogenesis and inflammation. The strongest antitumor effect is achieved by LCL-encapsulated BUP (LCL-BUP), due to the highest potency of BUP versus the other three GC types. The in vitro results presented herein suggest that LCL-BUP has strong cytotoxic effects on B16.F10 melanoma cells and the anti-proliferative effects of all LCL-GC towards angiogenic endothelial cells play a role in their antitumor activity.