Requirement for NBS1 in the S phase checkpoint response to DNA methylation combined with PARP inhibition

Treatment of PARP-1-expressing cells with the combination of a DNA methylating agent (MMS) and the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) leads to an ATR/Chk1-dependent S phase checkpoint and cell death by apoptosis. Activation of ATM/Chk2 is involved in sustaining the S phase checkpoint, and double strand break (DSB) accumulation was demonstrated. NBS1, part of the MRN complex that responds to DSBs, is known to modulate ATR- and ATM-dependent checkpoint responses to UV and IR, but a role in the response to PARP inhibition has not been addressed. Here we show that the S phase checkpoint observed 4-8h after MMS+4-AN treatment was absent in cells deficient in NBS1, but was present in NBS1-complemented (i.e., functionally wild-type) cells, indicating a critical role for NBS1 in this checkpoint response. NBS1 was phosphorylated in response to MMS+4-AN treatment, and this was partially ATR- and ATM-dependent, suggesting involvement of both upstream kinases. NBS1 expression had little effect on ATR-mediated phosphorylation of Chk1 and ATM-mediated phosphorylation of Chk2 in response to MMS+4-AN. Phosphorylation of SMC1 was also observed in response to MMS+4-AN treatment. In the absence of ATM and NBS1, phosphorylation of SMC1 was weak, especially at early times after MMS+4-AN treatment. In the absence of ATR activation, reduced SMC1 phosphorylation was seen over a 24h time course. These results suggested that both ATR and ATM phosphorylate SMC1 in response to MMS+4-AN and that this phosphorylation is enhanced by phospho-NBS1. The loss of the MMS+4-AN-induced S phase checkpoint in NBS1-deficient cells may be due to a reduced cellular level of the critical downstream effector, phospho-SMC1.     

Roles of the fusion and hemagglutinin-neuraminidase proteins in replication, tropism, and pathogenicity of avian paramyxoviruses

Virulent and moderately virulent strains of Newcastle disease virus (NDV), representing avian paramyxovirus serotype 1 (APMV-1), cause respiratory and neurological disease in chickens and other species of birds. In contrast, APMV-2 is avirulent in chickens. We investigated the role of the fusion (F) and hemagglutinin-neuraminidase (HN) envelope glycoproteins in these contrasting phenotypes by designing chimeric viruses in which the F and HN glycoproteins or their ectodomains were exchanged individually or together between the moderately virulent, neurotropic NDV strain Beaudette C (BC) and the avirulent APMV-2 strain Yucaipa. When we attempted to exchange the complete F and HN glycoproteins individually and together between the two viruses, the only construct that could be recovered was recombinant APMV-2 strain Yucaipa (rAPMV-2), containing the NDV F glycoprotein in place of its own. This substitution of NDV F into APMV-2 was sufficient to confer the neurotropic, neuroinvasive, and neurovirulent phenotypes, in spite of all being at reduced levels compared to what was seen for NDV-BC. When the ectodomains of F and HN were exchanged individually and together, two constructs could be recovered: NDV, containing both the F and HN ectodomains of APMV-2; and APMV-2, containing both ectodomains of NDV. This supported the idea that homologous cytoplasmic tails and matched F and HN ectodomains are important for virus replication. Analysis of these viruses for replication in vitro, syncytium formation, mean embryo death time, intracerebral pathogenicity index, and replication and tropism in 1-day-old chicks and 2-week-old chickens showed that the two contrasting phenotypes of NDV and APMV-2 could largely be transferred between the two backbones by transfer of homotypic F and HN ectodomains. Further analysis provided evidence that the homologous stalk domain of NDV HN is essential for virus replication, while the globular head domain of NDV HN could be replaced with that of APMV-2 with only a minimal attenuating effect. These results demonstrate that the F and HN ectodomains together determine the cell fusion, tropism, and virulence phenotypes of NDV and APMV-2 and that the regions of HN that are critical to replication and the species-specific phenotypes include the cytoplasmic tail and stalk domain but not the globular head domain.     

A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor

Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1–EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor’s amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer.     

A global review and meta-analysis of applications of the freshwater Fish Invasiveness Screening Kit

Reviews in Fish Biology and Fisheries - The freshwater Fish Invasiveness Screening Kit (FISK) has been applied in 35 risk assessment areas in 45 countries across the six inhabited continents (11...     

The effects of forest fragmentation on species richness on the Obudu Plateau,south‐eastern Nigeria

This study examines how fragmentation through changes in patch size and increasing distances between forest patches affects bird species richness. Numbers of bird species were higher in large forest patches and decrease with decreasing patch size. Bird species richness also decreased with increased isolation of forest patches. Suitable trees should be planted in the gaps between the forests that will create wildlife corridors to maintain connectivity.     

Validation of a novel index to assess insulin resistance of adipose tissue lipolytic activity in obese subjects

Insulin resistance in adipose tissue increases the release of free fatty acids into the circulation, which likely contributes to impaired insulin action in liver and skeletal muscle associated with obesity. However, reliable assessment of adipose tissue insulin resistance requires performing a hyperinsulinemic-euglycemic clamp procedure in conjunction with a fatty acid tracer infusion to determine insulin-mediated suppression of lipolytic rate. We developed a simpler method for evaluating adipose tissue insulin resistance in vivo, determined as the product of palmitate rate of appearance into the bloodstream and plasma insulin concentration during basal conditions. We validated our Adipose Tissue Insulin Resistance Index (ATIRI) by comparison with an assessment of adipose tissue insulin resistance determined by using the hyperinsulinemic-euglycemic clamp procedure in conjunction with a palmitate tracer infusion in 47 obese nondiabetic subjects (body mass index: 40.1 ± 9.3 kg/m(2)). We found the ATIRI correlated closely with adipose tissue insulin resistance assessed during the clamp procedure (r =-0.854, P < 0.001). These results demonstrate that the ATIRI provides a reliable index of adipose tissue insulin resistance in obese subjects.     

Archaeosortases and exosortases are widely distributed systems linking membrane transit with posttranslational modification

Multiple new prokaryotic C-terminal protein-sorting signals were found that reprise the tripartite architecture shared by LPXTG and PEP-CTERM: motif, TM helix, basic cluster. Defining hidden Markov models were constructed for all. PGF-CTERM occurs in 29 archaeal species, some of which have more than 50 proteins that share the domain. PGF-CTERM proteins include the major cell surface protein in Halobacterium, a glycoprotein with a partially characterized diphytanylglyceryl phosphate linkage near its C terminus. Comparative genomics identifies a distant exosortase homolog, designated archaeosortase A (ArtA), as the likely protein-processing enzyme for PGF-CTERM. Proteomics suggests that the PGF-CTERM region is removed. Additional systems include VPXXXP-CTERM/archeaosortase B in two of the same archaea and PEF-CTERM/archaeosortase C in four others. Bacterial exosortases often fall into subfamilies that partner with very different cohorts of extracellular polymeric substance biosynthesis proteins; several species have multiple systems. Variant systems include the VPDSG-CTERM/exosortase C system unique to certain members of the phylum Verrucomicrobia, VPLPA-CTERM/exosortase D in several alpha- and deltaproteobacterial species, and a dedicated (single-target) VPEID-CTERM/exosortase E system in alphaproteobacteria. Exosortase-related families XrtF in the class Flavobacteria and XrtG in Gram-positive bacteria mark distinctive conserved gene neighborhoods. A picture emerges of an ancient and now well-differentiated superfamily of deeply membrane-embedded protein-processing enzymes. Their target proteins are destined to transit cellular membranes during their biosynthesis, during which most undergo additional posttranslational modifications such as glycosylation.