Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar ‘Villafranca’) cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml^?1, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml^?1 and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin‐induced NO production was sensitive to sodium azide and N^G‐monomethyl‐l ‐arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.     

Partial migration in roe deer: migratory and resident tactics are end points of a behavioural gradient determined by ecological factors

Ungulate populations exhibiting partial migration present a unique opportunity to explore the causes of the general phenomenon of migration. The European roe deer Capreolus capreolus is particularly suited for such studies due to a wide distribution range and a high level of ecological plasticity. In this study we undertook a comparative analysis of roe deer GPS location data from a representative set of European ecosystems available within the EURODEER collaborative project. We aimed at evaluating the ecological factors affecting migration tactic (i.e. occurrence) and pattern (i.e. timing, residence time, number of migratory trips). Migration occurrence varied between and within populations and depended on winter severity and topographic variability. Spring migrations were highly synchronous, while the timing of autumn migrations varied widely between regions, individuals and sexes. Overall, roe deer were faithful to their summer ranges, especially males. In the absence of extreme and predictable winter conditions, roe deer seemed to migrate opportunistically, in response to a tradeoff between the costs of residence in spatially separated ranges and the costs of migratory movements. Animals performed numerous trips between winter and summer ranges which depended on factors influencing the costs of movement such as between‐range distance, slope and habitat openness. Our results support the idea that migration encompasses a behavioural continuum, with one‐trip migration and residence as its end points, while commuting and multi‐trip migration with short residence times in seasonal ranges are intermediate tactics. We believe that a full understanding of the variation in tactics of temporal separation in habitat use will provide important insights on migration and the factors that influence its prevalence.     

Phytotoxicity, not nitrogen immobilization, explains plant litter inhibitory effects: evidence from solid-state 13C NMR spectroscopy

Litter decomposition provides nutrients that sustain ecosystem productivity, but litter may also hamper root proliferation. The objectives of this work were to assess the inhibitory effect of litter decomposition on seedling growth and root proliferation; to study the role of nutrient immobilization and phytotoxicity; and to characterize decomposing litter by (13)C NMR spectroscopy. A litter-bag experiment was carried out for 180 d with 16 litter types. Litter inhibitory effects were assessed by two bioassays: seed germination and root proliferation bioassays. Activated carbon (C) and nutrient solutions were used to evaluate the effects of phytotoxic factors and nutrient immobilization. An inhibitory effect was found for all species in the early phase of decomposition, followed by a decrease over time. The addition of activated C to litter removed this inhibition. No evidence of nutrient immobilization was found in the analysis of nitrogen dynamics. NMR revealed consistent chemical changes during decomposition, with a decrease in O-alkyl and an increase in alkyl and methoxyl C. Significant correlations were found among inhibitory effects, the litter decay rate and indices derived from NMR. The results show that it is possible to predict litter inhibitory effects across a range of litter types on the basis of their chemical composition.     

Sequential depolarization of root cortical and stelar cells induced by an acute salt shock - implications for Na(+) and K(+) transport into xylem vessels

Early events in NaCl-induced root ion and water transport were investigated in maize (Zea mays L) roots using a range of microelectrode and imaging techniques. Addition of 100 mm NaCl to the bath resulted in an exponential drop in root xylem pressure, rapid depolarization of trans-root potential and a transient drop in xylem K(+) activity (A(K+) ) within ～1 min after stress onset. At this time, no detectable amounts of Na(+) were released into the xylem vessels. The observed drop in A(K+) was unexpected, given the fact that application of the physiologically relevant concentrations of Na(+) to isolated stele has caused rapid plasma membrane depolarization and a subsequent K(+) efflux from the stelar tissues. This controversy was explained by the difference in kinetics of NaCl-induced depolarization between cortical and stelar cells. As root cortical cells are first to be depolarized and lose K(+) to the environment, this is associated with some K(+) shift from the stelar symplast to the cortex, resulting in K(+) being transiently removed from the xylem. Once Na(+) is loaded into the xylem (between 1 and 5 min of root exposure to NaCl), stelar cells become more depolarized, and a gradual recovery in A(K+) occurs.     

Muscular laminopathies: role of prelamin A in early steps of muscle differentiation

Lamin A is a nuclear envelope constituent involved in a group of human disorders, collectively referred to as laminopathies, which include Emery-Dreifuss muscular dystrophy. Because increasing evidence suggests a role of lamin A precursor in nuclear functions, we investigated the processing of prelamin A along muscle differentiation. Both protein levels and cellular localization of prelamin A appears to be modulated during C2C12 mouse myoblasts activation. Similar changes also occur in the expression of two lamin A-binding proteins: emerin and LAP2α. Furthermore prelamin A forms a complex with LAP2α in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affects LAP2α and PCNA amount and increases caveolin 3 mRNA and protein levels, whilst accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor inhibits caveolin 3 expression. These data provide evidence for a critical role of lamin A precursor in the early steps of muscle cell differentiation. In fact the post-translational processing of prelamin A affects caveolin 3 expression and influences the myoblast differentiation process. Thus, altered lamin A processing could affect myoblast differentiation and/or muscle regeneration and might contribute to the myopathic phenotype.     

Effect of the point mutation H148G on GFPmut2 unfolding kinetics by fluorescence spectroscopy

We have used fluorescence spectroscopy techniques such as fluorescence correlation spectroscopy and fluorescence anisotropy decay on a wide time range, from nanoseconds to seconds, to investigate the unfolding kinetics induced by guanidinium chloride of GFPMut2 and its point mutation H148G, which has proved to be relevant for GFP photochemistry and photophysics. The mutation affects the unfolding kinetics of GFP leading to a much faster process at alkaline pH values, where protonation dynamics is negligible, that can be ascribed to a twofold role of His148, either as a proton shutter towards the chromophore and as a conformation stabiliser. For both mutants a soft region located near beta-strand 3 is found that starts to gain flexibility in the ns range at denaturant concentrations far lower than those required to turn off the chromophore fluorescence, as derived from the anisotropy decay of an extrinsic probe covalently bound to the proteins.     

Cytochrome P-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3A-null (Cyp3a(-/-)) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a(-/-) DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a(-/-) DA. However, responses of RA-treated WT and Cyp3a(-/-) mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a(-/-) newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally.