Arabidopsis GCP2 and GCP3 are part of a soluble gamma-tubulin complex and have nuclear envelope targeting domains

In higher plants, microtubules (MTs) are assembled in distinctive arrays in the absence of a defined organizing center. Three MT nucleation sites have been described: the nuclear surface, the cell cortex and cortical MT branch points. The Arabidopsis thaliana (At) genome contains putative orthologues encoding all the components of characterized mammalian nucleation complexes: gamma-tubulin and gamma-tubulin complex proteins GCP2 to GCP6. We have cloned the cDNA encoding AtGCP2, and show that gamma-tubulin, AtGCP2 and AtGCP3 are part of the same tandem affinity-purified complex and are present in a large membrane-associated complex. In addition, small soluble gamma-tubulin complexes of the size expected for a gamma-tubulin core complex are recruited to isolated nuclei. Using immunogold labelling, AtGCP3 is localized to both the nuclear envelope (NE) and the plasma membrane. To identify domains that could play a role in targeting complexes to these nucleation sites, truncated AtGCP2- and AtGCP3-green fluorescent protein fusion proteins were expressed in BY-2 cells. Several domains from AtGCP2 and AtGCP3 are capable of targeting fusions to the NE. We propose that regulated recruitment of soluble gamma-tubulin-containing complexes is responsible for nucleation at dispersed sites in plant cells and contributes to the formation and organization of the various MT arrays.     

Differential toxicities of air (mO-LDL) or copper-oxidized LDLs (Cu-LDL) toward endothelial cells.

In vivo low density protein (LDL) oxidation is a progressive phenomenon leading to the presence of minimally and highly oxidized LDLs in the subendothelial arterial space. Oxidized LDLs have been reported to be cytotoxic against endothelial cells. The goal of this study was to determine which of the minimally and highly oxidized LDLs were the most cytotoxic against bovine aortic endothelial cells (BAEC). Both the morphological aspect of the cells themselves, and LDH or MTT tests revealed that mO- or Cu-LDLs had similar cytotoxicity with up to 8 hours of oxidation, showing no relation with the level of LDL oxidation; for longer oxidation times, Cu-LDL cytotoxicity decreased. This phenomenon is linked to their different oxidation kinetics. Moreover, in the initial hours following BAEC incubation with mO- or Cu-LDLs, total cell glutathione dropped, whereas after 16 hours of incubation, highly oxidized Cu-LDL increased the glutathione level in the cell. The biphasic evolution of glutathione concentration corresponds to an autoprotective mechanism of cells against oxidized LDL cytotoxicity. This study suggests that the specific chemical characteristics of the different types of oxidized LDLs should always be precisely described in future assays devoted to studying the biological effects of what are known under the generic term as "oxidized LDLs". This precaution should prevent any confusion in interpreting different studies.     

Reduction of atherosclerosis by the peroxisome proliferator-activated receptor alpha agonist fenofibrate in mice

Several clinical and angiographic intervention trials have shown that fibrate treatment leads to a reduction of the coronary events associated to atherosclerosis. Fibrates are ligands for peroxisome proliferator-activated receptor alpha (PPARalpha) that modulate risk factors related to atherosclerosis by acting at both systemic and vascular levels. Here, we investigated the effect of treatment with the PPARalpha agonist fenofibrate (FF) on the development of atherosclerotic lesions in apolipoprotein (apo) E-deficient mice and human apoA-I transgenic apoE-deficient (hapoA-I Tg x apoE-deficient) mice fed a Western diet. In apoE-deficient mice, plasma lipid levels were increased by FF treatment with no alteration in the cholesterol distribution profile. FF treatment did not reduce atherosclerotic lesion surface area in the aortic sinus of 5-month-old apoE-deficient mice. By contrast, FF treatment decreased total cholesterol and esterified cholesterol contents in descending aortas of these mice, an effect that was more pronounced in older mice exhibiting more advanced lesions. Furthermore, FF treatment reduced MCP-1 mRNA levels in the descending aortas of apoE-deficient mice, whereas ABCA-1 expression levels were maintained despite a significant reduction of aortic cholesterol content. In apoE-deficient mice expressing a human apoA-I transgene, FF increased human apoA-I plasma and hepatic mRNA levels without affecting plasma lipid levels. This increase in human apoA-I expression was accompanied by a significant reduction in the lesion surface area in the aortic sinus. These data indicate that the PPARalpha agonist fenofibrate reduces atherosclerosis in these animal models of atherosclerosis.     

Combination of Insecticide Treated Nets and Indoor Residual Spraying in Northern Tanzania Provides Additional Reduction in Vector Population Density and Malaria Transmission Rates Compared to Insecticide Treated Nets Alone: A Randomised Control Trial

Indoor residual spraying (IRS) combined with insecticide treated nets (ITN) has been implemented together in several sub-Saharan countries with inconclusive evidence that the combined intervention provides added benefit. The impact on malaria transmission was evaluated in a cluster randomised trial comparing two rounds of IRS with bendiocarb plus universal coverage ITNs, with ITNs alone in northern Tanzania. From April 2011 to December 2012, eight houses in 20 clusters per study arm were sampled monthly for one night with CDC light trap collections. Anopheles gambiae s.l. were identified to species using real time PCR Taq Man and tested for the presence of Plasmodium falciparum circumsporozoite protein. ITN and IRS coverage was estimated from household surveys. IRS coverage was more than 85% in two rounds of spraying in January and April 2012. Household coverage with at least one ITN per house was 94.7% after the universal coverage net campaign in the baseline year and the proportion of household with all sleeping places covered by LLIN was 50.1% decreasing to 39.1% by the end of the intervention year. An.gambiae s.s. comprised 80% and An.arabiensis 18.3% of the anopheline collection in the baseline year. Mean An.gambiae s.l. density in the ITN+IRS arm was reduced by 84% (95%CI: 56%-94%, p = 0.001) relative to the ITN arm. In the stratum of clusters categorised as high anopheline density at baseline EIR was lower in the ITN+IRS arm compared to the ITN arm (0.5 versus 5.4 per house per month, Incidence Rate Ratio: 0.10, 95%CI: 0.01–0.66, p-value for interaction <0.001). This trial provides conclusive evidence that combining carbamate IRS and ITNs produces major reduction in Anopheles density and entomological inoculation rate compared to ITN alone in an area of moderate coverage of LLIN and high pyrethroid resistance in An.gambiae s.s.     

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.     

Nitrogen fluxes from treefrogs to tank epiphytic bromeliads: an isotopic and physiological approach

Diverse invertebrate and vertebrate species live in association with plants of the large Neotropical family Bromeliaceae. Although previous studies have assumed that debris of associated organisms improves plant nutrition, so far little evidence supports this assumption. In this study we used isotopic (¹⁵N) and physiological methods to investigate if the treefrog Scinax hayii, which uses the tank epiphytic bromeliad Vriesea bituminosa as a diurnal shelter, contributes to host plant nutrition. In the field, bromeliads with frogs had higher stable N isotopic composition (δ¹⁵N) values than those without frogs. Similar results were obtained from a controlled greenhouse experiment. Linear mixing models showed that frog feces and dead termites used to simulate insects that eventually fall inside the bromeliad tank contributed, respectively, 27.7% (±0.07 SE) and 49.6% (±0.50 SE) of the total N of V. bituminosa. Net photosynthetic rate was higher in plants that received feces and termites than in controls; however, this effect was only detected in the rainy, but not in the dry season. These results demonstrate for the first time that vertebrates contribute to bromeliad nutrition, and that this benefit is seasonally restricted. Since amphibian–bromeliad associations occur in diverse habitats in South and Central America, this mechanism for deriving nutrients may be important in bromeliad systems throughout the Neotropics.     

The distribution of coalescence times and distances between microsatellite alleles with changing effective population size

We investigate the effects of past changes of the effective population size on the present allelic diversity at a microsatellite marker locus. We first derive the analytical expression of the generating function of the joint probabilities of the time to the Most Recent Common Ancestor for a pair of alleles and of their distance (the difference in allele size). We give analytical solutions in the case of constant population size and the geometrical mutation model. Otherwise, numerical inversion allows the distributions to be calculated in general cases. The effects of population expansion or decrease and the possibility to detect an ancient bottleneck are discussed. The method is extended to samples of three and four alleles, which allows investigating the covariance structure of the frequencies f(k) of pairs of alleles with a size difference of k motifs, and suggesting some approaches to the estimation of past demography.