APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons

APOBEC3 proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and transposition of endogenous retroelements. One family member, APOBEC3A (hA3A), is an orphan, without any known antiviral activity. We show that hA3A is catalytically active and that it, but none of the other family members, potently inhibits replication of the parvovirus adeno-associated virus (AAV). hA3A was also a potent inhibitor of the endogenous LTR retroelements, MusD, IAP, and the non-LTR retroelement, LINE-1. Its function was dependent on the conserved amino acids of the hA3A active site, consistent with a role for cytidine deamination, although mutations in retroelement sequences were not found. These findings demonstrate the potent activity of hA3A, an APOBEC3 family member with no previously identified function. They also highlight the functional differences between APOBEC3 proteins. The APOBEC3 family members have distinct functions and may have evolved to resist various classes of genetic elements.     

A tale of two flatties: different responses of two terrestrial flatworms to past environmental climatic fluctuations at Tallaganda in montane southeastern Australia

Comparative phylogeographic studies of animals with low mobility and/or high habitat specificity remain rare, yet such organisms may hold fine-grained palaeoecological signal. Comparisons of multiple, codistributed species can elucidate major historical events. As part of a multitaxon programme, mitochondrial cytochrome oxidase I (COI) variation was analysed in two species of terrestrial flatworm, Artioposthia lucasi and Caenoplana coerulea. We applied coalescent demographic estimators and nested clade analysis to examine responses to past, landscape-scale, cooling-drying events in a model system of montane forest (Tallaganda). Correspondence of haplotype groups in both species to previously proposed microbiogeographic regions indicates at least four refuges from cool, dry conditions. The region predicted to hold the highest quality refuges (the Eastern Slopes Region), is indicated to have been a long-term refuge in both species, but so are several other regions. Coalescent analyses suggest that populations of A. lucasi are declining, while C. coerulea is expanding, although stronger population substructure in the former could yield similar patterns in the data. The differences in spatial and temporal genetic variation in the two species could be explained by differences in ecological attributes: A. lucasi is predicted to have lower dispersal ability but may be better able to withstand cold conditions. Thus, different contemporary population dynamics may reflect different responses to recent (Holocene) climate warming. The two species show highly congruent patterns of catchment-based local genetic endemism with one another and with previously studied slime-mould grazing Collembola.     

Biomimetic oxidative treatment of spruce wood studied by pyrolysis–molecular beam mass spectrometry coupled with multivariate analysis and 13C-labeled tetramethylammonium hydroxide thermochemolysis: implications for fungal degradation of wood

In this work, pyrolysis–molecular beam mass spectrometry analysis coupled with principal components analysis and ¹³C-labeled tetramethylammonium hydroxide thermochemolysis were used to study lignin oxidation, depolymerization, and demethylation of spruce wood treated by biomimetic oxidative systems. Neat Fenton and chelator-mediated Fenton reaction (CMFR) systems as well as cellulosic enzyme treatments were used to mimic the nonenzymatic process involved in wood brown-rot biodegradation. The results suggest that compared with enzymatic processes, Fenton-based treatment more readily opens the structure of the lignocellulosic matrix, freeing cellulose fibrils from the matrix. The results demonstrate that, under the current treatment conditions, Fenton and CMFR treatment cause limited demethoxylation of lignin in the insoluble wood residue. However, analysis of a water-extractable fraction revealed considerable soluble lignin residue structures that had undergone side chain oxidation as well as demethoxylation upon CMFR treatment. This research has implications for our understanding of nonenzymatic degradation of wood and the diffusion of CMFR agents in the wood cell wall during fungal degradation processes.     

Targeted mass spectrometric analysis of N-terminally truncated isoforms generated via alternative translation initiation

Alternative translation initiation is a mechanism whereby functionally altered proteins are produced from a single mRNA. Internal initiation of translation generates N-terminally truncated protein isoforms, but such isoforms observed in immunoblot analysis are often overlooked or dismissed as degradation products. We identified an N-terminally truncated isoform of human Dok-1 with N-terminal acetylation as seen in the wild-type. This Dok-1 isoform exhibited distinct perinuclear localization whereas the wild-type protein was distributed throughout the cytoplasm. Targeted analysis of blocked N-terminal peptides provides rapid identification of protein isoforms and could be widely applied for the general evaluation of perplexing immunoblot bands.     

Identification of Novel Pathways That Control Farnesoid X Receptor-mediated Hypocholesterolemia

Farnesoid X receptor (FXR) plays important regulatory roles in bile acid, lipoprotein, and glucose homeostasis. Here, we have utilized Fxr^−/− mice and mice deficient in scavenger receptor class B type I (SR-BI), together with an FXR-specific agonist and adenovirus expressing hepatocyte nuclear factor 4α or constitutively active FXR, to identify the mechanisms by which activation of FXR results in hypocholesterolemia. We identify a novel pathway linking FXR to changes in hepatic p-JNK, hepatocyte nuclear factor 4α, and finally SR-BI. Importantly, we demonstrate that the FXR-dependent increase in SR-BI results in both hypocholesterolemia and an increase in reverse cholesterol transport, a process involving the transport of cholesterol from peripheral macrophages to the liver for excretion into the feces. In addition, we demonstrate that FXR activation also induces an SR-BI-independent increase in reverse cholesterol transport and reduces intestinal cholesterol absorption. Together, these data indicate that FXR is a promising therapeutic target for treatment of hypercholesterolemia and coronary heart disease.     

Validation of an isocratic HPLC method to detect 2-fluoro-β-alanine for the analysis of dihydropyrimidine dehydrogenase activity

The efficacy of the chemotherapeutic drug 5′-fluorouracil is reduced by catabolism to 2′-fluoro-β-alanine (FBAL), a three-step reaction in which dihydropyrimidine dehydrogenase (DPD) catalyzes the rate-limiting step. To study in vitro DPD activity, we developed and validated an isocratic, reverse-phase HPLC method to detect and quantify FBAL without using multiple columns or radiolabeled substrates. Pre-column derivatization of FBAL was performed using o-phthalaldehyde in the presence of two sulfur donors, ethanthiol or β-mercaptoethanol, and the resulting products assayed. Calibration curves were linear over a range of 10–200 μg/ml and the method was successfully applied to the examination of DPD activity in cultured cells.     

Activin signaling and its role in regulation of cell proliferation, apoptosis, and carcinogenesis

Activins, cytokine members of the transforming growth factor-beta superfamily, have various effects on many physiological processes, including cell proliferation, cell death, metabolism, homeostasis, differentiation, immune responses endocrine function, etc. Activins interact with two structurally related serine/threonine kinase receptors, type I and type II, and initiate downstream signaling via Smads to regulate gene expression. Understanding how activin signaling is controlled extracellularly and intracellularly would not only lead to more complete understanding of cell growth and apoptosis, but would also provide the basis for therapeutic strategies to treat cancer and other related diseases. This review focuses on the recent progress on activin-receptor interactions, regulations of activin signaling by ligand-binding proteins, receptor-binding proteins, and nucleocytoplasmic shuttling of Smad proteins.     

Modelled and measured dynamics of viruses in Arctic winter sea-ice brines

We describe a model based on diffusion theory and the temperature-dependent mechanism of brine concentration in sea ice to argue that, if viruses partition with bacteria into sea-ice brine inclusions, contact rates between the two can be higher in winter sea ice than in seawater, increasing the probability of infection and possible virus production. To examine this hypothesis, we determined viral and bacterial concentrations in select winter sea-ice horizons using epifluorescence microscopy. Viral concentrations ranged from 1.6 to 82 x 10(6) ml(-1) of brine volume of the ice, with highest values in brines from coldest (-24 to -31 degrees C) ice horizons. Calculated virus-bacteria contact rates in underlying -1 degrees C seawater were similar to those in brines of -11 degrees C ice but up to 600 times lower than those in ice brines at or below -24 degrees C. We then incubated native bacterial and viral assemblages from winter sea ice for 8 days in brine at a temperature (-12 degrees C) and salinity ( approximately 160 psu) near expected in situ values, monitoring their concentrations microscopically. While different cores yielded different results, consistent with known spatial heterogeneity in sea ice, these experiments provided unambiguous evidence for viral persistence and production, as well as for bacterial growth, in -12 degrees C brine.     

Empirical array quality weights in the analysis of microarray data

Background  

Assessment of array quality is an essential step in the analysis of data from microarray experiments. Once detected, less reliable arrays are typically excluded or "filtered" from further analysis to avoid misleading results.