The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.     

Spatial patterns of acorn dispersal by rodents: do acorn crop size and ungulate presence matter?

Seed dispersal is qualitatively effective when it increases recruitment probability. A poorly studied factor likely affecting recruitment is the spatial distribution of dispersed seeds. Seed-caching animals are thought to disperse seeds in a way that reduces clumping and density to impede cache pilfering. Furthermore, dispersal might differ depending on whether the seed is immediately consumed versus cached for later consumption, and might differ depending on the ecological context. The main objectives of this study were to determine: 1) the spatial pattern of seed dispersal by rodents in a heterogeneous environment; 2) whether the patterns differ among years and among acorn competitor exclosure treatments, and 3) whether rodents create different spatial patterns of dispersal for acorns that are cached versus consumed immediately following dispersal. We studied the degree of spatial aggregation of acorn dispersal by rodents using two different estimators derived from the Ripley K and the Diggle G functions. We also analyzed various metrics of dispersal distances. For both analyses we used observed acorn dispersal patterns in two years differing in crop size and inside versus outside exclosures restricting access to acorn-consuming ungulates. During 2003, a year with a larger crop size, maximum seed dispersal distances were less, and the pattern of dispersed seeds was more clumped, than in 2004, a year with a smaller crop size. Median dispersal distances did not differ between years. In the presence of ungulates, seed dispersal was marginally sparser than in their absence. Cached acorns were dispersed more sparsely than acorns eaten immediately. These results have important implications for the quality of seed dispersal for oak recruitment that are likely relevant to other systems as well.     

BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions

Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER^-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER^-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER^-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.     

Identification and characterization of a novel O‐superfamily conotoxin from Conus litteratus

A novel conotoxin named lt6c, an O‐superfamily conotoxin, was identified from the cDNA library of venom duct of Conus litteratus. The full‐length cDNA contains an open reading frame encoding a predicted 22‐residue signal peptide, a 22‐residue proregion and a mature peptide of 28 amino acids. The signal peptide sequence of lt6c is highly conserved in O‐superfamily conotoxins and the mature peptide consists of six cysteines arranged in the pattern of C? C? CC? C? C that is defined the O‐superfamily of conotoxins. The mature peptide fused with thioredoxin, 6‐His tag, and a Factor Xa cleavage site was successfully expressed in Escherichia coli. About 12 mg lt6c was purified from 1L culture. Under whole‐cell patch‐clamp mode, lt6c inhibited sodium currents on adult rat dorsal root ganglion neurons. Therefore, lt6c is a novel O‐superfamily conotoxin that is able to block sodium channels. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Improving gene replacement by intracellular formation of linear homologous DNA

BACKGROUND: Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. METHODS: Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. RESULTS: Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. CONCLUSIONS: Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.     

Spatial variability of above‐ground net primary production in Uruguayan grasslands: a remote sensing approach

Question: How does above‐ground net primary production (ANPP) differ (estimated from remotely sensed data) among vegetation units in sub‐humid temperate grasslands? Location: Centre‐north Uruguay. Methods: A vegetation map of the study area was generated from LANDSAT imagery and the landscape configuration described. The functional heterogeneity of mapping units was analysed in terms of the fraction of photosynthetically active radiation absorbed by green vegetation (fPAR), calculated from the normalized difference vegetation index (NDVI) images provided by the moderate resolution imaging spectroradiometer (MODIS) sensor. Finally, the ANPP of each grassland class was estimated using NDVI and climatic data. Results: Supervised classification presented a good overall accuracy and moderate to good average accuracy for grassland classes. Meso‐xerophytic grasslands occupied 45% of the area, Meso‐hydrophytic grasslands 43% and Lithophytic steppes 6%. The landscape was shaped by a matrix of large, unfragmented patches of Meso‐xerophytic and Meso‐hydrophytic grasslands. The region presented the lowest anthropic fragmentation degree reported for the Rio de la Plata grasslands. All grassland units showed bimodal annual fPAR seasonality, with spring and autumn peaks. Meso‐hydrophytic grasslands showed a radiation interception 10% higher than the other units. On an annual basis, Meso‐hydrophytic grasslands produced 3800 kg dry matter (DM) ha^?1 yr^?1 and Meso‐xerophytic grasslands and Lithophytic steppes around 3400 kg·DM·ha^?1·yr^?1. Meso‐xerophytic grasslands had the largest spatial variation during most of the year. The ANPP temporal variation was higher than the fPAR variability. Conclusions: Our results provide valuable information for grazing management (identifying spatial and temporal variations of ANPP) and grassland conservation (identifying the spatial distribution of vegetation units).     

The Body Adiposity Index (Hip Circumference ÷ Height1.5) Is Not a More Accurate Measure of Adiposity Than Is BMI,Waist Circumference,or Hip Circumference

Based on cross‐sectional analyses, it was suggested that hip circumference divided by height^1.5 ?18 (the body adiposity index (BAI)), could directly estimate percent body fat without the need for further correction for sex or age. We compared the prediction of percent body fat, as assessed by dual‐energy X‐ray absorptiometry (PBF_DXA), by BAI, BMI, and circumference (waist and hip) measurements among 1,151 adults who had a total body scan by DXA and circumference measurements from 1993 through 2005. After accounting for sex, we found that PBF_DXA was related similarly to BAI, BMI, waist circumference, and hip circumference. In general, BAI underestimated PBF_DXA among men (2.5%) and overestimated PBF_DXA among women (4%), but the magnitudes of these biases varied with the level of body fatness. The addition of covariates and quadratic terms for the body size measures in regression models substantially improved the prediction of PBF_DXA, but none of the models based on BAI could more accurately predict PBF_DXA than could those based on BMI or circumferences. We conclude that the use of BAI as an indicator of adiposity is likely to produce biased estimates of percent body fat, with the errors varying by sex and level of body fatness. Although regression models that account for the nonlinear association, as well as the influence of sex, age, and race, can yield more accurate estimates of PBF_DXA, estimates based on BAI are not more accurate than those based on BMI, waist circumference, or hip circumference.     

Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control

Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning. Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 Mr polypeptides here designated 9 kd-1, 9 kd-2 and 9 kd-3. In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences. The E coding region specifies: an initiator of replication, which acts at the ori-2 site; a function that negatively regulates the expression of the E gene; and a function involved in mini-F copy number control. To assign one of these functions to one of the overlapping coding sequence, we have isolated, characterized and sequenced mutations mapping in the E coding region. In this paper, we analyse two mutations (cop5 and pla25) that abolish the repression of the E gene. As these mutations affect the primary structure of protein E itself but not the 9 kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis. In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function. The cop5 mutation causes an eightfold increase of the mini-F copy number. The pla25 mutation leads to the inability of the derived mini-F plasmid to give rise to plasmid-harbouring bacteria. The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence.     

G2 effects of DNA-repair inhibitors on chromatid-type aberrations in root-tip cells treated with maleic hydrazide and mitomycin C

In recent years the existence of a DNA-repair process in G2 has been proposed to explain the potentiating effects of DNA-repair inhibitors given in G2 on chromatid aberrations (CA) induced by S-dependent as well as S-independent DNA-damaging agents. In the present report, root-tip cells of Allium cepa were exposed to maleic hydrazide (MH) or mitomycin C (MMC) and post-treated in G2 with caffeine (Caff) and various inhibitors of DNA synthesis. No enhancement of chromosome damage was observed when Caff was present in G2, but hydroxyurea (HU) or 5-fluorodeoxyuridine (FdUrd) potentiated the frequencies of CA. A slight additional increase of CA frequencies was observed following treatment with Ara C and excess thymidine in G2. When MH-damaged cells were pulse-treated with Caff earlier during recovery, the yield of CA was enhanced. The earlier Caff was present following MH treatment, the stronger was the potentiation.