An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5′ end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.     

Expression,purification, phosphorylation and characterization of recombinant human statherin

This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.     

Phosphorylation pattern of the NDUFS4 subunit of complex I of the mammalian respiratory chain

The NDUFS4 subunit of complex I of the mammalian respiratory chain has a fully conserved carboxy-terminus with a canonical RVSTK phosphorylation site. Immunochemical analysis with specific antibodies shows that the serine in this site of the protein is natively present in complex I in both the phosphorylated and non-phosphorylated state. Two-dimensional IEF/SDS–PAGE electrophoresis, ³²P labelling and immunodetection show that “in vitro” PKA phosphorylates the serine in the C-terminus of the NDUFS4 subunit in isolated bovine complex I. ³²P labelling and TLC phosphoaminoacid mapping show that PKA phosphorylates serine and threonine residues in the purified heterologous human NDUFS4 protein.     

Elevation-induced variations of pollen assemblages in the North-western Alps: An analysis of their value as temperature indicators

Seventy-seven modern pollen samples from various elevations (350-2680 m a.s.l.) in two different areas of the north-western Alps (the Aosta Valley, Italy and the Taillefer Massif, France) were statistically analyzed to derive correlations between pollen assemblages, elevation and temperature at the sampled points. Numerical classifications were performed on pollen data to judge similarities between the two areas. The results show that a strong relationship exists between altitude and variations in pollen taxa percentages despite some floristic differences between the two areas. As a test, transfer functions from pollen percentages to elevation and temperature were calculated from pollen data. The reconstruction appears to be reliable, with a higher reliability at sites located over 1000m. This analysis aims to serve as a basis for further quantitative reconstruction of temperature changes during the Holocene based on fossil pollen data from sensitive regions that encompass a significant altitudinal gradient.     

Response of Tumor Spheroids to Radiation: Modeling and Parameter Estimation

We propose a spatially distributed continuous model for the spheroid response to radiation, in which the oxygen distribution is represented by means of a diffusion-consumption equation and the radiosensitivity parameters depend on the oxygen concentration. The induction of lethally damaged cells by a pulse of radiation, their death, and the degradation of dead cells are included. The compartments of lethally damaged cells and of dead cells are subdivided into different subcompartments to simulate the delays that occur in cell death and cell degradation, with a gain in model flexibility. It is shown that, for a single irradiation and under the hypothesis of a sufficiently small spheroid radius, the model can be reformulated as a linear stationary ordinary differential equation system. For this system, the parameter identifiability has been investigated, showing that the set of unknown parameters can be univocally identified by exploiting the response of the model to at least two different radiation doses. Experimental data from spheroids originated from different cell lines are used to identify the unknown parameters and to test the predictive capability of the model with satisfactory results.     

Beans in Europe: origin and structure of the European landraces of Phaseolus vulgaris L.

This study focuses on the expansion of Phaseolus vulgaris in Europe. The pathways of distribution of beans into and across Europe were very complex, with several introductions from the New World that were combined with direct exchanges between European and other Mediterranean countries. We have analyzed here six chloroplast microsatellite (cpSSR) loci and two unlinked nuclear loci (for phaseolin types and Pv-shatterproof1). We have assessed the genetic structure and level of diversity of a large collection of European landraces of P. vulgaris (307) in comparison to 94 genotypes from the Americas that are representative of the Andean and Mesoamerican gene pools. First, we show that most of the European common bean landraces (67%) are of Andean origin, and that there are no strong differences across European regions for the proportions of the Andean and Mesoamerican gene pools. Moreover, cytoplasmic diversity is evenly distributed across European regions. Secondly, the cytoplasmic bottleneck that was due to the introduction of P. vulgaris into the Old World was very weak or nearly absent. This is in contrast to evidence from nuclear analyses that have suggested a bottleneck of greater intensity. Finally, we estimate that a relatively high proportion of the European bean germplasm (about 44%) was derived from hybridization between the Andean and Mesoamerican gene pools. Moreover, although hybrids are present everywhere in Europe, they show an uneven distribution, with high frequencies in central Europe, and low frequencies in Spain and Italy. On the basis of these data, we suggest that the entire European continent and not only some of the countries therein can be regarded as a secondary diversification center for P. vulgaris. Finally, we outline the relevance of these inter-gene pool hybrids for plant breeding.     

Interaction between double helix DNA fragments and a new topopyrone acting as human topoisomerase I poison

A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 °C. Structural models of the complex with d(CGTACG)₂ were developed on the basis of DOSY, 2D NOESY and ³¹P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.     

Asymptotic behaviour of solutions to abstract logistic equations

We analyze the asymptotic behaviour of solutions of the abstract differential equation u'(t)=Au(t)-F(u(t))u(t)+f. Our results are applicable to models of structured population dynamics in which the state space consists of population densities with respect to the structure variables. In the equation the linear term A corresponds to internal processes independent of crowding, the nonlinear logistic term F corresponds to the influence of crowding, and the source term f corresponds to external effects. We analyze three separate cases and show that for each case the solutions stabilize in a way governed by the linear term. We illustrate the results with examples of models of structured population dynamics -- a model for the proliferation of cell lines with telomere shortening, a model of proliferating and quiescent cell populations, and a model for the growth of tumour cord cell populations.     

Photosynthesis responses to ozone in young trees of three species with different sensitivities, in a 2-year open-top chamber experiment (Curno, Italy)

This paper reports the findings of an open-top chamber experiment carried out in northern Italy (Forest nursery at Curno), during the 2004 and 2005 growth seasons, on Fagus sylvatica and Quercus robur seedlings and on Populus nigra cuttings, in order to test their photosynthesis response to ambient ozone. The experimental protocols were non-filtered air (NF), charcoal-filtered air (CF) and open air (OA). Tests performed included morphological features of leaves; development of foliar symptoms; chlorophyll content, determined by non-destructive means; chlorophyll fluorescence (direct fluorescence and JIP test) and gas exchanges and net photosynthesis (P_N). Main findings were as follows: (1) symptoms occurred early and were extensive in P. nigra , and they occurred later in F. sylvatica , whereas early degeneration of chlorophyll occurred in late summer in Q. robur ; (2) in conditions of ozone exposure, the three species all presented a decline in photosynthesis efficiency and a decrease in P_N, regardless of the symptomatology they displayed; (3) leaf traits are predictors of species-specific sensitivity to ozone—the high density of Q. robur foliar tissues prevents this species from developing visible symptoms and reduces the extent of physiological responses and (4) physiological responses varied from year to year in the same species—responses were lower in the second year of the experiment, when plants had become better acclimatized to plot conditions.