Evaluation of concentration of heavy metals in animal rearing system

Animal manure is one of the diffusion routes of heavy metals and metalloids into the environment, where the soil can accumulate them. Heavy metals and metalloids can then be released into groundwater sources, be absorbed by crops, and enter the food chain with negative effects for human and animal health. The aim of this study was to evaluate the concentration of heavy metals and mineral nutrients from modern animal rearing systems in order to develop effective strategies to increase the sustainability. Samples of feed (n\ubc24: n\ubc16 from swine, n\ubc8 from cattle), faeces (n\ubc120: n\ubc80 from swine, n\ubc40 from cattle) and water (n\ubc8), were collected from eight typical intensive swine and cattle farms located in northern Italy. All samples were analysed for the humidity and the principal components. The samples were also dried, mineralised, and analysed by ICP-MS to detect the following elements: Na, Mg, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, and Pb. The swine diets represented the highest amounts of Zn and Cu, with an average concentration for the finishing and weaning phases of Zn: 1737.9 \ub1 301.3; 821.7 \ub1 301.3; Cu: 133.8 \ub1 11.6; 160.1 \ub1 11.6 mg/kg as fed, respectively. The faecal content reflected the heavy metal composition from feed. The average content of cattle diets of Zn and Cu did not result higher than the maximum permitted levels. We observed that the swine manure represented the sources of Zn and Cu output into the environment. The Zn and Cu content should be monitored strictly in line with agroecology principles

Microtubule depolymerization affects endocytosis and exocytosis in the tip and influences endosome movement in tobacco pollen tubes

Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments (AFs) and microtubules (MTs). AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane (PM). Secretory activity is balanced by PM retrieval that maintains cell membrane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exoendocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exoendocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since Nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. FRAP experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM

Bioaccumulation of heavy metals from wastewater through a Typha latifolia and Thelypteris palustris phytoremediation system

Animal production is a source of heavy metals in livestock wastewater and also a key link in the food chain, with negative impacts on human and animal health. In intensive animal production systems, the most critical elements are zinc and copper. In order to development of innovative non-invasive strategies to reduce the environmental impact of livestock, this study assessed the ability of two plants, Typha latifolia and Thelypteris palustris, to bioaccumulate the heavy metals used in animal nutrition, from wastewater. Four mesocosms (width 2.0\u202fm, length 2.0\u202fm, 695\u202fL of water, 210\u202fkg of soil) were assembled outdoors at the Botanical Garden. Two of them were planted with T. latifolia (TL treated, n\u202f=\u202f30; TL control, n\u202f=\u202f30) and two with T. palustris (TP treated, n\u202f=\u202f60; TP control, n\u202f=\u202f60). In T0 a solution of a mineral additive premix (Zn 44.02\u202fmg/L; Cu 8.63\u202fmg/L) was dissolved in the treated mesocosms. At T0, d 15 (T1) and d 45 (T2) samples of roots, leaves, stems, soil and water were collected, dried, mineralized and analyzed using ICP-MS in order to obtain HMs content. We found that T. latifolia and T. palustris accumulate and translocate Zn, Cu from contaminated wastewater into plant tissues in a way that is directly related to the exposure time (T2 for Zn: 271.64\u202f\ub1\u202f17.70, 409.26\u202f\ub1\u202f17.70 for Cu: 47.54\u202f\ub1\u202f3.56, 105.58\u202f\ub1\u202f3.56\u202fmg/kg of DM, respectively). No visual toxicity signs were observed during the experimental period. This phytoremediation approach could be used as an eco-sustainable approach to counteract the output of heavy metals

Morphological and proteomic responses of Eruca sativa exposed to silver nanoparticles or silver nitrate

Silver nanoparticles (AgNPs) are widely used in commercial products, and there are growing concerns about their impact on the environment. Information about the molecular interaction of AgNPs with plants is lacking. To increase our understanding of the mechanisms involved in plant responses to AgNPs and to differentiate between particle specific and ionic silver effects we determined the morphological and proteomic changes induced in Eruca sativa (commonly called rocket) in response to AgNPs or AgNO3. Seedlings were treated for 5 days with different concentrations of AgNPs or AgNO3. A similar increase in root elongation was observed when seedlings were exposed to 10 mg Ag L-1 of either PVP-AgNPs or AgNO3. At this concentration we performed electron microscopy investigations and 2-dimensional electrophoresis (2DE) proteomic profiling. The low level of overlap of differentially expressed proteins indicates that AgNPs and AgNO3 cause different plant responses. Both Ag treatments cause changes in proteins involved in the redox regulation and in the sulfur metabolism. These responses could play an important role to maintain cellular homeostasis. Only the AgNP exposure cause the alteration of some proteins related to the endoplasmic reticulum and vacuole indicating these two organelles as targets of the AgNPs action. These data add further evidences that the effects of AgNPs are not simply due to the release of Ag ions

Morphological evaluation of nicotiana tabacum plants transformed for the expression of verocytotoxic Escherichia coli antigens

Two transgenic Nicotiana tabacum plants carrying respectively the F18 and the B subunit of verocytotoxin genes from O138 verocytotoxic E.coli serotype (VTEC), were developed by agroinfection as model of edible vaccine. VTEC causes severe enterotoxaemia in weaned piglets and is responsible of important economic losses. For the construction of transgenic plants, the bacterial genes were placed under control of GLOB for the seed specific expression. Previous studies demonstrated that the Vt2e-B and FedA fimbrial genes were stably incorporated into tobacco plant genome by being transcribed through the nuclear apparatus of the plant for specific expression in the seeds, and that these genes are inherited by the next generation. The dietary administration of transgenic tobacco seeds promoted a significant increase in the number of intestinal mucosal IgA-producing cells in mice and showed a protective effect against VTEC strain in piglets. Agrobacterium tumefaciens binary vector system is an efficient tool to transform plant cells; however, the exogenous gene integrates at semi-random into the nuclear chromosomes. In other words, the insertion of a transgene into the plant genome inevitably disrupts the sequence of the endogenous plant DNA and may be accompanied by other mutations. For these reasons, the aim of this study is the morphological evaluation of Nicotiana tabacum plants transformed for the expression of F18 and Vt2e-B proteins with respect to Nicotiana tabacum wild type (WT). Three lines of tobacco seeds (F18, VT2e-B and WT) were seeded in homogeneous conditions and were harvested simultaneously. Tobacco plants were analysed by optical microscope in different phases of growth. Germination of transgenic seeds was delayed of three and five days compared to WT in two replicated experiments, suggesting that genetic manipulation influenced mechanisms leading to germination. The analysis of F18 and VT2e-B seed polypeptides, following two different methods of protein extraction showed differences in the electrophoretic profiles with respect to WT. Furthermore, morphological observations using optical microscope, showed no difference in the embryos of tree samples. On the contrary, a large amount of storage material (oleosomes or aleuron grains) are observed in the endosperm of F18 seeds, with respect to WT endosperm, in which storage proteins and lipids were already mobilized. This data could explain the delayed germination of transgenic lines

The imbalance between dynamic and stable microtubules underlies neurodegeneration induced by 2,5-hexanedione

Exposure to environmental toxins, including hydrocarbon solvents, increases the risk of developing Parkinson's disease. An emergent hypothesis considers microtubule dysfunction as one of the crucial events in triggering neuronal degeneration in Parkinson's disease. Here, we used 2,5-hexanedione (2,5-HD), the toxic metabolite of n-hexane, to analyse the early effects of toxin-induced neurodegeneration on the cytoskeleton in multiple model systems. In PC12 cells differentiated with nerve growth factor for 5 days, we found that 2,5-HD treatment affected all the cytoskeletal components. Moreover, we observed alterations in microtubule distribution and stability, in addition to the imbalance of post-translational modifications of \u3b1-tubulin. Similar defects were also found in vivo in 2,5-HD-intoxicated mice. Interestingly, we also found that 2,5-HD exposure induced significant changes in microtubule stability in human skin fibroblasts obtained from Parkinson's disease patients harbouring mutations in PRKN gene, whereas it was ineffective in healthy donor fibroblasts, suggesting that the genetic background may really make the difference in microtubule susceptibility to this environmental Parkinson's disease-related toxin. In conclusion, by showing the imbalance between dynamic and stable microtubules in hydrocarbon-induced parkinsonism, our data support the crucial role of microtubule defects in triggering neurodegeneration

Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold

In an attempt to dissect endocytosis in Nicotiana tabacum L. pollen tubes, two different probes \u2013 positively or negatively charged nanogold \u2013 were employed. The destiny of internalized plasma membrane domains, carrying negatively or positively charged residues, was followed at the ultrastructural level and revealed distinct endocytic pathways. Time-course experiments and electron microscopy showed internalization of subapical plasmamembrane domains that were mainly recycled to the secretory pathway through the Golgi apparatus and a second mainly degradative pathway involving plasma membrane retrieval at the tip. In vivo time-lapse experiments using FM4-64 combined with quantitative analysis confirmed the existence of distinct internalization regions. Ikarugamycin, an inhibitor of clathrin-dependent endocytosis, allowed us to further dissect the endocytic process: electron microscopy and time-lapse studies suggested that clathrin-dependent endocytosis occurs in the tip and subapical regions, because recycling of positively charged nanogold to the Golgi bodies and the consignment of negatively charged nanogold to vacuoles were affected. However, intact positively charged-nanogold transport to vacuoles supports the idea that an endocytic pathway that does not require clathrin is also present in pollen tubes

Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold

Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts

Papillary carcinoma of the thyroid: methylation is not involved in the regulation of MET expression

Hypomethylation has been reported to be responsible for the activation of several oncogenes. The possibility that hypomethylation is involved in the regulation of MET transcription was investigated through the analysis of the methylation status of one CpG island containing 43 CpGs in six cases of papillary carcinoma, in the corresponding normal thyroid tissue, and in two cases of hyperplastic goitre. Evidence of methylation was not found in any of the analysed CpG. © 2004 Cancer Research UK