ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

Replication of the ~30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

Domains in three-dimensional ferroelectric nanostructures

We report the experimental measurement of domains in single- crystal nanocolumns of ferroelectric BaTiO3, together with a theory of domain size scaling in three- dimensional structures which explains the observations.

Wall thickness dependence of the scaling law for ferroic stripe domains

The periodicity of 180 degrees. stripe domains as a function of crystal thickness scales with the width of the domain walls, both for ferroelectric and for ferromagnetic materials. Here we derive an analytical expression for the generalized ferroic scaling factor and use this to calculate the domain wall thickness and gradient coefficients ( exchange constants) in some ferroelectric and ferromagnetic materials. We then use these to discuss some of the wider implications for the physics of ferroelectric nanodevices and periodically poled photonic crystals.

BACE1 Polymorphisms Do Not Influence Platelet Membrane β-secretase Activity or Genetic Susceptibility for Alzheimer’s Disease in the Northern Irish Population

ß-Site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a biological and positional candidate gene for Alzheimer’s disease (AD). BACE1 is a protease that catalyses APP cleavage at the ß-secretase site. We evaluated all common and putatively functional polymorphisms in the genomic region encompassing BACE1 for an association with AD, and for functional effects on platelet ß-secretase activity. Tag SNPs (n = 10) derived from phase II of the International HapMap Project, and a nonsynonymous variant, were successfully genotyped in 901 Caucasian individuals from Northern Ireland using Sequenom iPLEX and TaqMan technologies. APOE genotyping was performed by PCR-RFLP. Platelet membrane ß-secretase activity was assayed in a subset of individuals (n = 311). Hardy–Weinberg equilibrium was observed for all variants. Evidence for an association with AD was observed with multi-marker haplotype analyses (P = 0.01), and with rs676134 when stratified for APOE genotype (P = 0.02), however adjusting for multiple testing negated the evidence for association of this variant with AD. ?2 analysis of genotype and allele frequencies in cases versus controls for individual SNPs revealed no evidence for association (5% level). No genetic factors were observed that significantly influenced platelet membrane ß-secretase activity. We have selected an appropriate subset of variants suitable for comprehensive genetic investigation of the BACE1 gene. Our results suggest that common BACE1 polymorphisms and putatively functional variants have no significant influence on genetic susceptibility to AD, or platelet ß-secretase activity, in this Caucasian Northern Irish population.

AINT/ERIC/TACC: an expanding family of proteins with C-terminal coiled coil domains:an expanding family of proteins with C-terminal coiled coil domains

The AINT/ERIC/TACC genes encode novel proteins with a coiled coil domain at their C-terminus. The founding member of this expanding family of genes, transforming acidic coiled coil 1 (TACC1), was isolated from a BAC contig spanning the breast cancer amplicon-1 on 8p11. Transfection of cells in vitro with TACC1 resulted in anchorage-independent growth consistent with a more "neoplastic" phenotype. Database searches employing the human TACC1 sequence revealed other novel genes, TACC2 and TACC3, with substantial sequence homology particularly in the C-terminal regions encoding the coiled coil domains. TACC2, located at 10q26, is similar to anti-zuai-1 (AZU-1), a candidate breast tumour suppressor gene, and ECTACC, an endothelial cell TACC which is upregulated by erythropoietin (Epo). The murine homologue of TACC3, murine erythropoietin-induced cDNA (mERIC-1) was also found to be upregulated by Epo in the Friend virus anaemia (FVA) model by differential display-PCR. Human ERIC-1, located at 4p16.3, has been cloned and encodes an 838-amino acid protein whose N- and C-terminal regions are highly homologous to the shorter 558-amino acid murine protein, mERIC-1. In contrast, the central portions of these proteins differ markedly. The murine protein contains four 24 amino acid imperfect repeats. ARNT interacting protein (AINT), a protein expressed during embryonic development in the mouse, binds through its coiled coil region to the aryl hydrocarbon nuclear translocator protein (ARNT) and has a central portion that contains seven of the 24 amino acid repeats found in mERIC-1. Thus mERIC-1 and AINT appear to be developmentally regulated alternative transcripts of the gene. Most members of the TACC family discovered so far contain a novel nine amino acid putative phosphorylation site with the pattern [R/K]-X(3)-[E]-X(3)-Y. Genes with sequence homology to the AINT/ERIC/TACC family in other species include maskin in Xenopus, D-TACC in Drosophila and TACC4 in the rabbit. Maskin contains a peptide sequence conserved among eIF-4E binding proteins that is involved in oocyte development. D-TACC cooperates with another conserved microtubule-associated protein Msps to stabilise spindle poles during cell division. The diversity of function already attributed to this protein family, including both transforming and tumour suppressor properties, should ensure that a new and interesting narrative is about to unfold.

Origin of Ferroelastic domains in Free-Standing Single Crystal Ferroelectric Films

The origin of the unusual 90 degrees ferroelectric/ferroelastic domains, consistently observed in recent studies on mesoscale and nanoscale free-standing single crystals of BaTiO3 [Schilling , Phys. Rev. B 74, 024115 (2006); Schilling , Nano Lett. 7, 3787 (2007)], has been considered. A model has been developed which postulates that the domains form as a response to elastic stress induced by a surface layer which does not undergo the paraelectric-ferroelectric cubic-tetragonal phase transition. This model was found to accurately account for the changes in domain periodicity as a function of size that had been observed experimentally. The physical origin of the surface layer might readily be associated with patterning damage, seen in experiment; however, when all evidence of physical damage is removed from the BaTiO3 surfaces by thermal annealing, the domain configuration remains practically unchanged. This suggests a more intrinsic origin, such as the increased importance of surface tension at small dimensions. The effect of surface tension is also shown to be proportional to the difference in hardness between the surface and the interior of the ferroelectric. The present model for surface-tension induced twinning should also be relevant for finely grained or core-shell structured ceramics.

Advanced glycation end product (AGE) accumulation on Bruch's membrane: links to age-related RPE dysfunction.

PURPOSE: Advanced glycation end products (AGEs) accumulate during aging and have been observed in postmortem eyes within the retinal pigment epithelium (RPE), Bruch's membrane, and subcellular deposits (drusen). AGEs have been associated with age-related dysfunction of the RPE-in particular with development and progression to age-related macular degeneration (AMD). In the present study the impact of AGEs at the RPE-Bruch's membrane interface was evaluated, to establish how these modifications may contribute to age-related disease. METHODS: AGEs on Bruch's membrane were evaluated using immunohistochemistry. A clinically relevant in vitro model of substrate AGE accumulation was established to mimic Bruch's membrane ageing. Responses of ARPE-19 growing on AGE-modified basement membrane (AGE-BM) for 1 month were investigated by using a microarray approach and validated by quantitative (q)RT-PCR. In addition to identified AGE-related mRNA alterations, lysosomal enzyme activity and lipofuscin accumulation were also studied in ARPE-19 grown on AGE-BM. RESULTS: Autofluorescent and glycolaldehyde-derived AGEs were observed in clinical specimens on Bruch's membrane and choroidal extracellular matrix. In vitro analysis identified a range of dysregulated mRNAs in ARPE-19 exposed to AGE-BM. Altered ARPE-19 degradative enzyme mRNA expression was observed on exposure to AGE-BM. AGE-BM caused a significant reduction in cathepsin-D activity in ARPE-19 (P

Imaging domains in BaTiO3 single crystal nanostructures: comparing information from transmission electron microscopy and piezo-force microscopy

This article compares and contrasts information
obtained, using transmission electron microscopy (TEM)
and piezo-force microscopy (PFM), on domain configurations
adopted in single crystal lamellae of BaTiO3, that had
been cut directly from bulk using a focused ion beam
microscope with top and bottom surfaces parallel to
{100}pseudocubic. Both forms of imaging reveal domain
walls parallel to {110}pseudocubic, consistent with sets of 90
domains with dipoles oriented parallel to the two
\001[pseudocubic directions in the plane of the lamellae.
However, the domain width was observed to be dramatically
larger using PFM than it was using TEM. This suggests
significant differences in the surface energy densities
that drive the domain formation in the first place, that could
relate to differences in the boundary conditions in the two
modes of imaging (TEM samples are imaged under high
vacuum, whereas PFM imaging was performed in air).
Attempts were made to map local dipole orientations
directly, using a form of ‘vector’ PFM. However, information
inferred was largely inconsistent with the known
crystallography of the samples, raising concern about the
levels of care needed for accurate interpretation of PFM
images.

Domains in Ferroelectric Nanodots

Almost free-standing single crystal mesoscale and nanoscale dots of ferroelectric BaTiO3 have been made by direct focused ion beam patterning of bulk single crystal material. The domain structures which appear in these single crystal dots, after cooling through the Curie temperature, were observed to form into quadrants, with each quadrant consisting of fine 90° stripe domains. The reason that these rather complex domain configurations form is uncertain, but we consider and discuss three possibilities for their genesis: first, that the quadrant features initially form to facilitate field-closure, but then develop 90° shape compensating stripe domains in order to accommodate disclination stresses; second, that they are the result of the impingement of domain packets which nucleate at the sidewalls of the dots forming “Forsbergh” patterns (essentially the result of phase transition kinetics); and third, that 90° domains form to conserve the shape of the nanodot as it is cooled through the Curie temperature but arrange into quadrant packets in order to minimize the energy associated with uncompensated surface charges (thus representing an equilibrium state). While the third model is the preferred one, we note that the second and third models are not mutually exclusive.