Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing

RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.     

Identification of a series of 1,3,4-oxadiazol-2-amines as potent alpha-7 agonists with efficacy in the novel object recognition model of cognition

A series of α7 nicotinic acetylcholine receptor full-agonists with a 1,3,4-oxadiazol-2-amine core has been discovered. Systematic exploration of the structure-activity relationships for both α7 potency and selectivity with respect to interaction with the hERG channel are described. Further profiling led to the identification of compound 22, a potent full agonist showing efficacy in the novel object recognition model of cognition enhancement.     

Estimating seed dispersal distance: A comparison of methods using animal movement and plant genetic data on two primate‐dispersed Neotropical plant species

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Expression of cancer related BRCA1 missense variants decreases MMS-induced recombination in Saccharomyces cerevisiae without altering its nuclear localization

BRCA1 tumor suppressor gene is found mutated in familial breast and ovarian cancer. Most cancer related mutations were found located at the RING (Really Interesting New Gene) and at the BRCT (BRca1 C-Terminal) domain. However, 20 y after its identification, the biological role of BRCA1 and which domains are more relevant for tumor suppression are still being elucidated. We previously reported that expression of BRCA1 cancer related variants in the RING and BRCT domain increases spontaneous homologous recombination in yeast indicating that BRCA1 may interact with yeast DNA repair/recombination. To finally demonstrate whether BRCA1 interacts with yeast DNA repair, we exposed yeast cells expressing BRCA1wt, the cancer-related variants C-61G and M1775R to different doses of the alkylating agent methyl methane-sulfonate (MMS) and then evaluated the effect on survival and homologous recombination. Cells expressing BRCA1 cancer variants were more sensitive to MMS and less inducible to recombination as compared to cell expressing BRCA1wt. Moreover, BRCA1-C61G and -M1775R did not change their nuclear localization form as compared to the BRCA1wt or the neutral variant R1751Q indicating a difference in the DNA damage processing. We propose a model where BRCA1 cancer variants interact with the DNA double strand break repair pathways producing DNA recombination intermediates, that maybe less repairable and decrease MMS-induced recombination and survival. Again, this study strengthens the use of yeast as model system to characterize the mechanisms leading to cancer in humans carrying the BRCA1 missense variant.     

Production of recombinant proteins and metabolites in yeasts

Recombinant DNA (rDNA) technologies allow the production of a wide range of peptides, proteins and metabolites from naturally non-producing cells. Since human insulin was the first heterologous compound produced in a laboratory in 1977, rDNA technology has become one of the most important technologies developed in the 20th century. Recombinant protein and metabolites production is a multi-billion dollar market. The development of a new product begins with the choice of the cell factory. The final application of the compound dictates the main criteria that should be taken into consideration: (1) quality, (2) quantity, (3) yield and (4) space time yield of the desired product. Quantity and quality are the most predominant requirements that must be considered for the commercial production of a protein. Quantity and yield are the requirements for the production of a metabolite. Finally, space time yield is crucial for any production process. It therefore becomes clear why the perfect host does not exist yet, and why—despite important advances in rDNA applications in higher eukaryotic cells—microbial biodiversity continues to represent a potential source of attractive cell factories. In this review, we compare the advantages and limitations of the principal yeast and bacterial workhorse systems.     

Pyrrolizidine alkaloids from Anchusa strigosa and their antifeedant activity

The pyrrolizidine alkaloid (PA) content of flowers, leaves, and roots of Anchusa strigosa (Boraginaceae) was analysed by ESI-LC-MS. Six PAs, including two new natural compounds, were detected, characterized by NMR spectroscopy, and quantified in each plant organ. The results indicated that the highest total concentration of PAs was in the leaves (23.63 mg/g of dried part), followed by the flowers (19.77 mg/g), and finally by the roots (1.80 mg/g). All PAs isolated were subjected to Spodoptera exigua and Pieris brassicae larvae. Feeding activity by both herbivore species using a bioassay was inhibited up to circa 75% depending on PA and applied concentration.     

Selective gamma-hydroxybutyric acid receptor ligands increase extracellular glutamate in the hippocampus, but fail to activate G protein and to produce the sedative/hypnotic effect of gamma-hydroxybutyric acid

Two gamma-hydroxybutyric acid (GHB) analogues, trans-gamma-hydroxycrotonic acid (t-HCA) and gamma-(p-methoxybenzyl)-gamma-hydroxybutyric acid (NCS-435) displaced [3H]GHB from GHB receptors with the same affinity as GHB but, unlike GHB, failed to displace [3H]baclofen from GABAB receptors. The effect of the GHB analogues, GHB and baclofen, on G protein activity and hippocampal extracellular glutamate levels was compared. While GHB and baclofen stimulated 5'-O-(3-[35S]thiotriphospate) [35S]GTPgammaS binding both in cortex homogenate and cortical slices, t-HCA and NCS-435 were ineffective up to 1 mm concentration. GHB and baclofen effect was suppressed by the GABAB antagonist CGP 35348 but not by the GHB receptor antagonist NCS-382. Perfused into rat hippocampus, 500 nm and 1 mm GHB increased and decreased extracellular glutamate levels, respectively. GHB stimulation was suppressed by NCS-382, while GHB inhibition by CGP 35348. t-HCA and NCS-435 (0.1-1000 microm) locally perfused into hippocampus increased extracellular glutamate; this effect was inhibited by NCS-382 (10 microm) but not by CGP 35348 (500 microm). The results indicate that GHB-induced G protein activation and reduction of glutamate levels are GABAB-mediated effects, while the increase of glutamate levels is a GHB-mediated effect. Neither t-HCA nor NCS-435 reproduced GHB sedative/hypnotic effect in mice, confirming that this effect is GABAB-mediated. The GHB analogues constitute important tools for understanding the physiological role of endogenous GHB and its receptor.     

Multiplex primer-extension assay for identification of Yersinia species

A multiplex primer-extension reaction (PER) assay, was specifically designed for the identification of ten Yersinia species. The assay, directed towards the tufA (elongation factor Tu) gene, was tested on a total of 42 samples representing Yersinia species and non-Yersinia species. The primers used in the preliminary PCR, designed in highly conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 587 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of Yersinia spp. in food samples and to evaluate the potential hazard for consumers.     

Long-term release of clodronate from biodegradable microspheres

This paper describes the formulation of a biodegradable microparticulate drug delivery system containing clodronate, a bisphosphonate intended for the treatment of bone diseases. Microspheres were prepared with several poly(D,L-lactide-co-glycolide) (PLGA) copolymers of various molecular weights and molar compositions and 1 poly(D,L-lactide) (PDLLA) homopolymer by a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation procedure. Critical process parameters and formulation variables (ie, addition of stabilizing agents) were evaluated for their effect on drug encapsulation efficiency and clodronate release rate from microparticles Well-formed clodronate-loaded microspheres were obtained for all polymers by selecting suitable process parameters (inner water/oil volume ratio 1∶16, temperature-raising rate in the solvent evaporation step 1°C/min, 2% wt/vol NaCl in the external aqueous phase). Good yields were obtained in all batches of clodronate microspheres (above 60%); drug encapsulation efficiencies ranged between 49% and 75% depending on the polymer used. Clodronate release from all copolymer microspheres was completed in about 48 hours, while those from PDLLA microspheres required about 20 days. The change of microsphere composition by adding a surfactant such as Span 20 or a viscosing agent such as carboxymethylcellulose extended the long-term release up to 3 months. Clodronate was successfully entrapped in PLGA and PDLLA microspheres, and drug release could be modulated from 48 hours up to 3 months by suitable selection of polymer, composition, additives, and manufacturing conditions. Published: July 11, 2001.