High-resolution melting analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine

The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D. pinea in Austrian pine (Pinus nigra). HRMA exploits differences in the melting behaviour of PCR products to rapidly identify DNA sequence variants without the need for cumbersome post-PCR methods. We developed a HRMA method to detect specific fungal sequences in the mitochondrial small subunit ribosome gene (mt SSU rDNA). The reliability of this technique was firstly assessed on DNA extracted from pure cultures of D. pinea and closely related species. Amplicon differences were screened by HRMA and the results confirmed by direct DNA sequencing. Subsequently, HRMA was tested on DNA from symptomatic and symptomless pine shoots, and the presence of the fungus was also confirmed by both conventional and molecular quantitative approaches. The HRMA allowed the distinction of D.?pinea from closely related species, showing specific melting profiles for the each pathogen. This new molecular technique, here tested in a plant-fungus pathosystem for the first time, was very reliable in both symptomatic and symptomless shoots. HRMA is therefore a highly effective and accurate technique that permits the rapid screening of pathogens in the host.     

D-Aspartic acid and L-amino acids in the neural system of the amphioxus <Emphasis Type="Italic">Branchiostoma lanceolatum</Emphasis>

Summary. The lancelet (amphioxus), a cephalochordate, is the closest invertebrate relative to vertebrates, with a simple vertebrate-like body plan and a prototypical genome. We have determined D-aspartic acid (D-Asp) and major free L-amino acids (L-AAs) content in the nervous system (neural tube) of the European amphioxus Branchiostoma lanceolatum, and have compared these values with those of molluscs and human brain. The B. lanceolatum neural tube contains relatively high amounts of L-Glu, L-Asp, L-Ala and L-Gly. Thus, the amphioxus neural tube has in common with the molluscan and human nervous systems the presence of appreciable amounts of L-Glu and L-Asp, which suggests that they are the most common neurotransmitters among these phylogenetically distant animal groups. The relatively high concentration of L-Ala in amphioxus is consistent with that found in molluscs and the low concentration of taurine is consistent with that described in the human brain. The D-Asp concentration, very high in the molluscan nervous system, was rather low in amphioxus, although a little higher than the extremely low amounts observed in the human brain. Our data on free amino acids composition is in agreement with the intermediate phylogenetic position of cephalochordates, in terms of the evolutionary transition from simple to complex neural systems.     

Modulation of an AMPA-like glutamate receptor (SqGluR) gating by L- and D-aspartic acids

Summary. L- and D-aspartic acids (L-Asp and D-Asp) are present in the majority of nervous systems. In phylogeny, significant levels have been reported in mollusc brains, particularly cephalopods. To examine the role of L- and D-Asp on a cephalopod receptor, we studied ligand gating of a squid glutamate receptor (SqGluR) expressed in HEK 239 (human embryonic kidney) cells. Under voltage clamp, application of L-glutamate (L-Glu; 1–30 mM), but not D-glutamate (D-Glu), or L- or D-Asp, evoked an inward current of 0.1 nA. L- or D-Asp (200 μM) applied with 20 mM L-Glu, slowed the time course of activation and inactivation of the L-Glu gated current (time constant increased from 1 s (L-Glu alone) to 3 s (D-Asp and L-Glu) and to 19 s (L-Asp and L-Glu)). Our results suggest that in molluscan systems, aspartic acid could act as a neuromodulator during glutamatergic transmission and could significantly alter synaptic integration by slowing glutamate receptor gating.     

Effect of low doses of red wine and pure resveratrol on circulating endothelial progenitor cells

Circulating endothelial progenitor cells (EPCs) play a significant role in neovascularization of ischaemic tissues and in re-endothelization of injured blood vessels. Identification of compounds able to enhance EPC levels and improve their functional activity, noticeably compromised by risk factors for coronary heart disease, is of clinical interest. This study evaluates the effects of red wine on EPCs. After being isolated from total peripheral blood mononuclear cells, EPC phenotype was confirmed by the presence of double positive cells for DiLDL uptake and lectin binding and by expression of CD34, CD133 and VE-cadherin cell surface markers. Long-term culture in the presence of red wine (1 microl/ml), containing resveratrol (Resv) at physiological concentration (nM), determined a time-dependent amelioration of cell number (P < 0.05). The presence of red wine prevented the TNF-alpha-induced reduction of EPC number (P < 0.05) and this effect was accompanied by reduced p38-phosphorylation expression levels (P < 0.05) and increased NOx levels (P < 0.05) Indeed, pure Resv alone significantly improved the TNF-alpha reduced EPC number (P < 0.05). This evidence indicates novel beneficial effects of red wine and Resv in the positive modulation of EPCs levels.