The solution structure and activation of visual arrestin studied by small-angle X-ray scattering.

Visual arrestin is converted from a 'basal' state to an 'activated' state by interaction with the phosphorylated C-terminus of photoactivated rhodopsin (R*), but the conformational changes in arrestin that lead to activation are unknown. Small-angle X-ray scattering (SAXS) was used to investigate the solution structure of arrestin and characterize changes attendant upon activation. Wild-type arrestin forms dimers with a dissociation constant of 60 micro m. Small conformational changes, consistent with local movements of loops or the mobile N- or C-termini of arrestin, were observed in the presence of a phosphopeptide corresponding to the C-terminus of rhodopsin, and with an R175Q mutant. Because both the phosphopeptide and the R175Q mutation promote binding to unphosphorylated R*, we conclude that arrestin is activated by subtle conformational changes. Most of the arrestin will be in a dimeric state in vivo. Using the arrestin structure as a guide [Hirsch, J.A., Schubert, C., Gurevich, V.V. & Sigler, P.B. (1999) Cell 97, 257-269], we have identified a model for the arrestin dimer that is consistent with our SAXS data. In this model, dimerization is mediated by the C-terminal domain of arrestin, leaving the N-terminal domains free for interaction with phosphorylated R*.     

Acid phosphatase activity in Golgi apparatus and related structures in pars recta of rat kidney studied by thin and semi-thin section cytochemistry

Summary Using 0.5 thick (i.e., semi-thin) and conventional thin sections, observations have been made on the localization of acid phosphatase in the Golgi apparatus and related structures in the pars recta of rat kidney. In thin sections one or two Golgi cisternae located at the concave (basilar) aspect of the stack had enzymic activity. The periphery of these cisternae may be fenestrated. Coated vesicles were seen apparently free in the cytoplasm and in continuity with both reactive Golgi cisternae and smooth tubular elements. Smooth vesicular profiles with electron-lucent matrices and low enzymic activity were seen, apparently free, in the central Golgi zone. Semi-thin sections demonstrated more fully the extent of the reactive Golgi elements, their architecture and their relationships with other organelles. Within a stack the reactive Golgi cisternae were continuous with one another. A network of anastomosing tubular elements formed the periphery of the cisternae and linked some adjacent cisternae. Tubules extended considerable distances from this network in apical and lateral directions. Vesicular protuberances formed ends to the tubular extensions but free vesicles were not obvious. Apparent continuity was seen between reactive tubules and dense bodies (secondary lysosomes). Vesicular profiles with electron-lucent matrices andlow enzymic activity appeared to be continuous with the periphery of reactive Golgi cisternae and may represent the formation of primary lysosomes. This study demonstrates that semi-thin sections could be used to great advantage in the study of organelle interactions in both normal and pathological states.     

Rotational echo double resonance detection of cross-links formed in mussel byssus under high-flow stress.

13C2H rotational echo double resonance NMR has been used to provide the first evidence for the formation of quinone-derived cross-links in mussel byssal plaques. Labeling of byssus was achieved by allowing mussels to filter feed from seawater containing L-[phenol-4-13C]tyrosine and L-[ring-d4]tyrosine for 2 days. Plaques and threads were harvested from two groups of mussels over a period of 28 days. One group was maintained in stationary water while the other was exposed to turbulent flow at 20 cm/s. The flow-stressed byssal plaques exhibited significantly enhanced levels of 5, 5'-di-dihydroxyphenylalanine cross-links. The average concentration of di-dihydroxyphenylalanine cross-links in byssal plaques is 1 per 1800 total protein amino acid residues.     

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

Discovery of a potent and selective aurora kinase inhibitor

This communication describes the discovery of a novel series of Aurora kinase inhibitors. Key SAR and critical binding elements are discussed. Some of the more advanced analogues potently inhibit cellular proliferation and induce phenotypes consistent with Aurora kinase inhibition. In particular, compound 21 (SNS-314) is a potent and selective Aurora kinase inhibitor that exhibits significant activity in pre-clinical in vivo tumor models.     

High affinity glycosaminoglycan and autoantigen interaction explains joint specificity in a mouse model of rheumatoid arthritis

In the K/BxN mouse model of rheumatoid arthritis, autoantibodies specific for glucose-6-phosphate isomerase (GPI) can transfer joint-specific inflammation to most strains of normal mice. Binding of GPI and autoantibody to the joint surface is a prerequisite for joint-specific inflammation. However, how GPI localizes to the joint remains unclear. We show that glycosaminoglycans (GAGs) are the high affinity (83 nm) joint receptors for GPI. The binding affinity and structural differences between mouse paw/ankle GAGs and elbows/knee GAGs correlated with the distal to proximal disease severity in these joints. We found that cartilage surface GPI binding was greatly reduced by either chondroitinase ABC or beta-glucuronidase treatment. We also identified several inhibitors that inhibit both GPI/GAG interaction and GPI enzymatic activities, which suggests that the GPI GAG-binding domain overlaps with the active site of GPI enzyme. Our studies raise the possibility that GAGs are the receptors for other autoantigens involved in joint-specific inflammatory responses.     

Changes in Escherichia coli O157:H7 numbers during holding on excised lean, fascia and fat beef surfaces at different temperatures

Aim:  To investigate changes in Escherichia coli O157:H7 numbers on excised beef carcass surfaces over 72 h at different temperatures.
Methods and Results:  Excised lean meat, fascia and fat were inoculated with E. coli O157:H7 and held in an environmental chamber for 72 h, at air speed 0·5 m s⁻¹, relative humidity (RH) 90%, and temperatures 4, 8 and 12°C. On lean, pathogen counts increased significantly at 12°C. On fascia, significant reductions in counts occurred at 4 and 8°C. Pathogen numbers were significantly reduced on fat at 4, 8 and 12°C (64 h). Counts on fat were significantly less at all temperatures, compared to lean or fascia and surface water activity, a_w, decreased significantly over time on fat at 4°C. Significant decreases in surface pH values were recorded on all meat substrates.
Conclusions:  The survival of E. coli O157:H7 varied in relation to the meat substrate and the holding temperature. Reductions in counts on fat surfaces appeared to be related to low surface a_w values. No relationship between pathogen survival and surface pH was established.
Significance and Impact of the Study:  The use of excised meat pieces in an environmental cabinet offers a more flexible approach to determining the use of different chilling regimes in the production of safe meat.     

Biological Nitrogen Fixation in Two Tropical Forests: Ecosystem-Level Patterns and Effects of Nitrogen Fertilization

Humid tropical forests are often characterized by large nitrogen (N) pools, and are known to have large potential N losses. Although rarely measured, tropical forests likely maintain considerable biological N fixation (BNF) to balance N losses. We estimated inputs of N via BNF by free-living microbes for two tropical forests in Puerto Rico, and assessed the response to increased N availability using an on-going N fertilization experiment. Nitrogenase activity was measured across forest strata, including the soil, forest floor, mosses, canopy epiphylls, and lichens using acetylene (C₂H₂) reduction assays. BNF varied significantly among ecosystem compartments in both forests. Mosses had the highest rates of nitrogenase activity per gram of sample, with 11 ± 6 nmol C₂H₂ reduced/g dry weight/h (mean ± SE) in a lower elevation forest, and 6 ± 1 nmol C₂H₂/g/h in an upper elevation forest. We calculated potential N fluxes via BNF to each forest compartment using surveys of standing stocks. Soils and mosses provided the largest potential inputs of N via BNF to these ecosystems. Summing all components, total background BNF inputs were 120 ± 29 μg N/m²/h in the lower elevation forest, and 95 ± 15 μg N/m²/h in the upper elevation forest, with added N significantly suppressing BNF in soils and forest floor. Moisture content was significantly positively correlated with BNF rates for soils and the forest floor. We conclude that BNF is an active biological process across forest strata for these tropical forests, and is likely to be sensitive to increases in N deposition in tropical regions.