The pro-oxidant role of protein SH groups of hemoglobin in rat erythrocytes exposed to menadione.

Menadione is selectively toxic to erythrocytes. Although GSH is considered a primary target of menadione, intraerythrocyte thiolic alterations consequent to menadione exposure are only partially known. In this study alterations of GSH and protein thiols (PSH) and their relationship with methemoglobin formation were investigated in human and rat red blood cells (RBC) exposed to menadione. In both erythrocyte types, menadione caused a marked increase in methemoglobin associated with GSH depletion and increased oxygen consumption. However, in human RBC, GSH formed a conjugate with menadione, whereas, in rat RBC it was converted to GSSG, concomitantly with a loss of protein thiols (corresponding to menadione arylation), and an increase in glutathione-protein mixed disulfides (GS-SP). Such differences were related to the presence of highly reactive cysteines, which characterize rat hemoglobin (cys beta125). In spite of the greater thiol oxidation in rat than in human RBC, methemoglobin formation and the rate of oxygen consumption elicited by menadione in both species were rather similar. Moreover, in repeated experiments under N2 or CO-blocked heme, it was found that menadione conjugation (arylation) in both species was not dependent on the presence of oxygen or the status of heme. Therefore, we assumed that GSH (human RBC) and protein (rat RBC) arylation was equally responsible for increased oxygen consumption and Hb oxidation. Moreover, thiol oxidation of rat RBC was strictly related to methemoglobin formation.     

Expression of rolB in apomictic Hieracium piloselloides Vill. causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency

The effects of the Agrobacterium rhizogenes rolB oncogene on apomixis were examined in the facultative apomictic plant Hieracium piloselloides because the oncogene has been shown to alter plant growth, morphogenesis and cellular sensitivity to auxin. Introduction of rolB under the control of either its own promoter or the CaMV35S promoter induced ectopic meristem formation from the inflorescence, confirming in planta a meristem-inducing role for this oncogene previously observed only in tissue culture. These ectopic meristems formed vegetative rosettes and floral plant organs. Upon immersion in water these meristems generated roots, suggesting that meristem commitment towards the generation of a specific organ type is a separate and later event that is dependent upon the developmental context. Ovule identity and form was altered in ectopically induced florets in plants expressing the CaMV35S::rolB construct. In contrast to the ovules of untransformed apomictic plants, the sexual process ceased earlier, prior to meiosis, yet surprisingly, apomixis initiated from a greater number of cells, and embryos and endosperm continued to develop in the structurally altered ovules. The alternative possibilities that the effects on reproduction might result from rolB influencing cellular response to auxin, or from alterations in cell signaling caused by changes in ovule morphology that are induced because of the expression of the oncogene are discussed.     

Accumulation of copper and zinc from liquid manure in agricultural soils and crop plants

In Northern Italy it is a common practice to utilise slurries and other manure from intensive animal farming to fertilise agricultural soils. Due to the relatively high copper and zinc contents of these materials, this practice could lead to contamination of soils and the crops grown on them. In this study we have evaluated the extent of copper and zinc contamination of soils subjected to different levels of fertilisation with liquid manure and the copper and zinc concentrations in the edible tissues of three crops (maize, sugar beet and lucerne) grown on the same soils. There was a direct correlation between soil copper and zinc contents and the levels of application of animal wastes and copper concentrations were high enough to represent a risk, according to current European legislation. The metal contents in the edible tissues of the three crops were relatively low and variable, but showed no clear correlations with the intensity of liquid manure applications. Overall, there was no risk of contamination of the food chain, as all concentrations were well below levels considered to be toxic to animals.     

The Interrelationships of the Gastrotricha Using Nuclear Small rRNA Subunit Sequence Data, with an Interpretation Based on Morphology

Gastrotrichs are meiobenthic invertebrates of obscure origin and unclear phylogenetic alliances. Uncertainties also plague the intra-group relationship with major contrasts between the evolutionary scenarios inferred from morphology or molecules. In this study we analysed partial sequences of the 18S rDNA gene of 18 taxa (14 new and 4 published) to test morphological estimates of gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data are due to poor sampling. Data were analysed using both maximum parsimony and maximum likelihood. MP topology was then forced to reflect published morphological estimates and the most parsimonious solutions from each constraint analysis was statistically compared against the unconstrained solution. MP analysis yielded a single tree with few nodes well supported by bootstrap resampling. These included the monophyly of the Chaetonotidae and the internal relationships of the members of this family, with Aspidiophorus appearing as the most basal member. The monophyly of the Turbanellidae was also well supported with some suggestion that its sister group might be Mesodasys. Lepidodasyidae was found to be an unnatural taxon with Lepidodasys forming a separated clade but unrelated also to the Thaumastodermatidae. With the exception of genera Lepidodasys and Neodasys, the Macrodasyida appeared to be resolved separately from the Chaetonotida, and Dactylopodola was resolved as the most basal macrodasyid. ML analysis yielded a tree not too dissimilar from MP, although Dactylopodola and Xenodasys were resolved as a clade. Statistics indicate that the output from our MP analysis is compatible with the classical view placing representatives of the two orders within two distinct evolutionary lines. Most of the constrained solutions, except the shortest, corroborate the monophyly of the two orders, whereas all five constrained solutions support also the notion that sees Neodasys as an early divergent clade along the Chaetonotida branch. Thus, results are generally compatible with the hypothesised evolutionary scenario based on morphological data, but are in contrast with previous findings from molecules. Future research should consider using the complete SSU rDNA gene sequence in their analysis and additional genes for deeper resolution.     

Degradation and Transformability of DNA from Transgenic Leaves

The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.     

AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death.

In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (Gln-Val-Val-Ala-Gly). Recombinant A. thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified. AtCYS1 inhibits the catalytic activity of papain (Kd = 4.0 x 10-2 micro m, at pH 7.0 and 25 degrees C), generally taken as a molecular model of cysteine proteases. The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex. AtCYS1 is constitutively expressed in roots and in developing siliques of A. thaliana. In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A. thaliana suspension cultured cells and in transgenic tobacco plants. The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.     

Home range dynamics of male roe deerCapreolus capreolus in a mountainous habitat

Male spacing behaviour of roe deer Capreolus capreolus (Linnaeus, 1758) was studied in a wooded mountainous habitat in the Casentinesi Forest National Park, Italy. Data were collected using radio-tracking techniques from March 1997 to February 1998. Annual, seasonal, and bimonthly home ranges were analysed. Different factors may influence male spacing behaviour throughout the year. Winter home range sizes may be dependent on environmental conditions, while social factors could determine a high level of individual variability during the territorial and reproductive period. Prime age males showed great spatial stability, in contrast to the ranging movements of yearlings.