N‐nitrosomelatonin enhances photic synchronization of mammalian circadian rhythms

Most physiological processes in mammals are synchronized to the daily light:dark cycle by a circadian clock located in the hypothalamic suprachiasmatic nucleus. Signal transduction of light‐induced phase advances of the clock is mediated through a neuronal nitric oxide synthase‐guanilyl cyclase pathway. We have employed a novel nitric oxide‐donor, N‐nitrosomelatonin, to enhance the photic synchronization of circadian rhythms in hamsters. The intraperitoneal administration of this drug before a sub‐saturating light pulse at circadian time 18 generated a twofold increase of locomotor rhythm phase‐advances, having no effect over saturating light pulses. This potentiation was also obtained even when inhibiting suprachiasmatic nitric oxide synthase activity. However, N‐nitrosomelatonin had no effect on light‐induced phase delays at circadian time 14. The photic‐enhancing effects were correlated with an increased suprachiasmatic immunoreactivity of FBJ murine osteosarcoma viral oncogene and period1. Moreover, in vivo nitric oxide release by N‐nitrosomelatonin was verified by measuring nitrate and nitrite levels in suprachiasmatic nuclei homogenates. The compound also accelerated resynchronization to an abrupt 6‐h advance in the light:dark cycle (but not resynchronization to a 6‐h delay). Here, we demonstrate the chronobiotic properties of N‐nitrosomelatonin, emphasizing the importance of nitric oxide‐mediated transduction for circadian phase advances.

     

The productive traits,fatty acid profile and nutritional indices of three lupin (Lupinus spp.) species cultivated in a Mediterranean environment for the livestock

The aim of this study was to investigate the productive traits and fatty acid profiles of eight sweet varieties of Lupinus albus, Lupinus angustifolius and Lupinus luteus cultivated in a Mediterranean environment (Sicily, southern Italy). This is being done to identify the most suitable species, from an agronomical and nutritional point of view, for supporting animal husbandry in this area. Seed yield, oil quantity and oil quality were considerably different depending on the genotype. L. albus showed the highest yield (2.0–2.4 t/ha), 100 seed weight (27.0 g, on average), oil content (70.4–75.5 g/kg), and n?3/n?6 polyunsaturated fatty acid ratio (0.39–0.51), L. angustifolius showed the highest content of saturated fatty acids (21.03–26.66 g/100 g) and L. luteus showed the highest levels of n?6 polyunsaturated fatty acids (47.56–50.85 g/100 g), unsaturated/saturated (4.93–5.62) and polyunsaturated/saturated (3.33–3.99) fatty acid ratios and the best atherogenic (0.06) and thrombogenic (0.09–0.11) indices. The data obtained suggests L. albus as the most interesting lupin species for aiding the crop-livestock food chain and L. luteus as a promising crop due to its high nutritive traits for the Mediterranean environment.     

Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice

Prion diseases are caused by a conformational modification of the cellular prion protein (PrP^C) into disease-specific forms, termed PrP^Sc, that have the ability to interact with PrP^C promoting its conversion to PrP^Sc. In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP^C region involved in the interaction with PrP^Sc thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP^Sc in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.     

In vitro physical mutagenesis of giant reed (Arundo donax L.)

Giant reed (Arundo donax L.) is a C₃ perennial, warm‐season, rhizomatous grass of emerging interest for bioenergy and biomass derivatives production, and for phytoremediation. It only propagates vegetatively and very little genetic variation is found among ecotypes, basically precluding breeding efforts. With the objective to increase the genetic variation in this species, we developed and applied a mutagenesis protocol based on γ‐irradiation of in vitro cell cultures from which regenerants were obtained. Based on a radiosensitivity test, the irradiation dose reducing to 50% the number of regenerants per callus (RD₅₀) was estimated at 35 Gy. A large mutagenic experiment was carried out by irradiating a total of 3120 calli with approx. 1×, 1.5× and 2× RD₅₀. A total of 1004 regenerants from irradiated calli were hardened in pots and transplanted to the field. Initial phenotypic characterization of the collection showed correlated responses of biomass‐related quantitative traits to irradiation doses. Approx. 10% of field‐grown clones showed remarkable morphological aberrations including dwarfism, altered tillering, abnormal inflorescence, leaf variegation and others, which were tested for stability over generations. Clone lethality reached 0.4%. Our results show for the first time that physical mutagenesis can efficiently induce new genetic and phenotypic variation of agronomic and prospective industrial value in giant reed. The methodology and the plant materials described here may contribute to the domestication and the genetic improvement of this important biomass species.     

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.     

On the use of 2,1,3-benzothiadiazole derivatives as selective live cell fluorescence imaging probes

Newly designed 2,1,3-benzothiadiazole-containing fluorescent probes with four excited state intramolecular proton transfer (ESIPT) sites were successfully tested in live cell-imaging assays using a confluent monolayer of human stem-cells (tissue). All tested dyes were compared with the commercially available DAPI and gave far better results.     

New Insights in Trichoderma harzianum Antagonism of Fungal Plant Pathogens by Secreted Protein Analysis

Trichoderma harzianum ALL42 were capable of overgrowing and degrading Rhizoctonia solani and Macrophomina phaseolina mycelia, coiling around the hyphae with formation of apressoria and hook-like structures. Hyphae of T. harzianum ALL42 did not show any coiling around Fusarium sp. hyphae suggesting that mycoparasitism may be different among the plant pathogens. In this study, a secretome analysis was used to identify some extracellular proteins secreted by T. harzianum ALL42 after growth on cell wall of M. phaseolina, Fusarium sp., and R. solani. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. A total of 60 T. harzianum ALL42 secreted proteins excised from the gel were analyzed from the three growth conditions. While seven cell wall-induced proteins were identified, more than 53 proteins spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced. Endochitinase, β-glucosidase, α-mannosidase, acid phosphatase, α-1,3-glucanase, and proteases were identified in the gel and also detected in the supernatant of culture.