Selective 32P-labelling of individual species in a total tRNA population.

A simple procedure to label individual tRNA species in a total tRNA preparation has been developed. The principle of the method is as follows: total crude tRNA (from E. coli) is incubated in the presence of a crude aminoacyl-tRNA synthetase preparation, containing most aminoacyl-tRNA synthetases and only one specific amino acid corresponding to the tRNA species which is intended to be labelled. This achieves the purpose of charging the desired tRNA species thereby protecting its 3'OH-terminus; obviously all the other tRNA species will have a free 3'OH group. Periodate oxidation, followed by beta-elimination, destroys any free 3'OH. After deacylation of the specific aminoacylated tRNA at pH 8.8 the only free 3'OH group will be the one of the desired tRNA species. High specific activity (32P)-pCp is ligated to this 3'OH by means of T4-RNA ligase. Two-dimensional polyacrylamide gel electrophoresis (2D-PGE) and sequence analysis of the isolated tRNA show that the method is very specific. Individually labelled tRNA species can be used as probes for cloning tRNA genes.     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.     

Genetically augmenting tau levels does not modulate the onset or progression of Abeta pathology in transgenic mice

The two hallmark pathologies of Alzheimer's disease (AD) are amyloid plaques, composed of the small amyloid-beta (Abeta) peptide, and neurofibrillary tangles, comprised aggregates of the microtubule binding protein, tau. The molecular linkage between these two lesions, however, remains unknown. Based on human and mouse studies, it is clear that the development of Abeta pathology can trigger tau pathology, either directly or indirectly. However, it remains to be established if the interaction between Abeta and tau is bidirectional and whether the modulation of tau will influence Abeta pathology. To address this question, we used the 3xTg-AD mouse model, which is characterized by the age-dependent buildup of both plaques and tangles. Here we show that genetically augmenting tau levels and hyperphosphorylation in the 3xTg-AD mice has no effect on the onset and progression of Abeta pathology. These data suggest that the link between Abeta and tau is predominantly if not exclusively unidirectional, which is consistent with the Abeta cascade hypothesis and may explain why tauopathy-only disorders are devoid of any Abeta pathology.     

Probing the interaction between p53 and the bacterial protein azurin by single molecule force spectroscopy

p53 is a human tumour suppressor which regulates multiple cellular processes, including cell growth, genomic stability and cell death. Recent works have demonstrated the bacterial redox protein azurin to enter cancer cells and induce apoptosis through p53 stabilization, resulting in a tumour growth regression. Azurin has been shown to bind p53 although many details of the complex formed by these two proteins are still poorly characterized. Here, we get insight into the kinetics of this complex formation, by exploring the interaction between p53 and azurin in their environment by single molecule force spectroscopy. To this aim, azurin has been linked to the atomic force microscope tip, whereas p53 has been immobilized onto a gold substrate. Therefore, by performing force-distance cycles we have detected specific recognition events between p53 and azurin, displaying unbinding forces of around 70 pN for an applied loading rate of 3 nN s(-1). The specificity of these events has been assessed by the significant reduction of their frequency observed after blocking the p53 sample by an azurin solution. Moreover, by measuring the rupture force as a function of the loading rate we have determined the dissociation rate constant of this complex to be approximately 0.1 s(-1). Our findings are here discussed in connection with results obtained in bulk experiments, with the aim of clarifying some molecular details of the p53-azurin complex that may help designing new anticancer strategy.     

Alternative strategies of seed predator escape by early-germinating oaks in Asia and North America

Early germination of white oaks is widely viewed as an evolutionary strategy to escape rodent predation; yet, the mechanism by which this is accomplished is poorly understood. We report that chestnut oak Quercus montana (CO) and white oak Q. alba (WO) (from North America), and oriental cork oak Q. variabilis (OO) and Mongolian oak Q. mongolica (MO) (from Asia) can escape predation and successfully establish from only taproots. During germination in autumn, cotyledonary petioles of acorns of CO and WO elongate and push the plumule out of the cotyledons, whereas OO and MO extend only the hypocotyls and retain the plumule within the cotyledons. Experiments showed that the pruned taproots (>6 cm) of CO and WO acorns containing the plumule successfully germinated and survived, and the pruned taproots (≥12 cm) of OO and MO acorns without the plumule successfully regenerated along with the detached acorns, thus producing two seedlings. We argue that these two distinct regeneration morphologies reflect alternative strategies for escaping seed predation.     

Identification of trophic interactions between <Emphasis Type="Italic">Macrolophus caliginosus</Emphasis> (Heteroptera: Miridae) and <Emphasis Type="Italic">Myzus persicae</Emphasis> (Homoptera: Aphididae) using real time PCR

Understanding food web interactions in native or agricultural ecosystems is an important step towards establishing sustainable pest management strategies. While the role of generalist predators as biological control agents is increasingly appreciated, the study of trophic interactions between individual predator species and their prey provides practical difficulties. Recently, different approaches have been suggested to determine prey items from predator guts using molecular methods. Macrolophus caliginosus is a generalist predator active in herbaceous agro-ecosystems. We developed a system to identify the DNA of its prey after ingestion, using Myzus persicae as a model. Esterase (MpEST) and cytochrome oxidase I (MpCOI) genes were targeted in the aphid, while M. caliginosus COI gene was used as control for predator DNA. Real time PCR proved to be specific and sensitive enough to detect prey DNA upon ingestion after feeding experiments. The system provided a linear amplification response with only 10 fg of prey genomic DNA as template. The detection system of MpCOI gene was more sensitive than MpEST, while the detection period was similar for both genes. Possibilities for using the system in ecological and biosafety studies with regard to sustainable pest management are discussed.

Celiac anti-tissue transglutaminase antibodies interfere with the uptake of alpha gliadin peptide 31–43 but not of peptide 57–68 by epithelial cells

Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31–43 (but not peptide 57–68) uptake by cells, thereby impairing the ability of p31–43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31–43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.     

8,000 years of climate,vegetation, fire and land-use dynamics in the thermo-mediterranean vegetation belt of northern Sardinia (Italy)

Vegetation History and Archaeobotany - Knowledge about the vegetation history of Sardinia, the second largest island of the Mediterranean, is scanty. Here, we present a new sedimentary record...