Regulation of Escherichia coli polynucleotide phosphorylase by ATP

Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.     

Influence of the anionic ligands on the anticancer activity of Ru(II)-dmso complexes: Kinetics of aquation and in vitro cytotoxicity of new dicarboxylate compounds in comparison with their chloride precursors

We performed extensive studies on the kinetics of hydrolysis of a series of Ru(II)-dmso complexes containing dicarboxylate ligands, such as oxalate, malonate, succinate and 1,1-cyclobutane dicarboxylate (cbdc), derived from anticancer-active Ru(II)-dmso-Cl precursors. The in vitro antitumor activity of those compounds in comparison with their chloride precursors was evaluated against two tumor cell lines, the human KB oral carcinoma and the murine B16-F10 melanoma. The aim of this study was to assess how the nature of the anionic ligands (i.e. dicarboxylates vs. chlorides) affects the chemical behavior and the in vitro antitumor activity of Ru(II)-dmso complexes. Among the tested compounds only one complex, the dimer [fac-Ru(dmso-S)(3)(H(2)O)(mu-cbdc)](2) (5), exhibited moderate activity against both cell lines. Interestingly, this compound is the most kinetically stable in aqueous solution among those investigated. Despite the moderate in vitro activity, in an in vivo test, complex 5 exhibited no activity against both the primary tumor growth and the formation of spontaneous metastases on the MCa mammary carcinoma model.     

Fasciola hepatica: Surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78

The tegumental alterations in adult Fasciola hepatica induced by the experimental fasciolide OZ78 were investigated utilizing scanning electron microscopy (SEM). Twelve weeks post-infection with F. hepatica, rats were treated with a single 100mg/kg oral dose of OZ78 and flukes were recovered from the bile ducts after 24-72 h. In vitro F. hepatica were incubated with OZ78 for 48 h at a concentration of 10 microg/ml in the absence or presence of haemin. Twenty-four and 48 h post-treatment of rats disruption of the tegument of F. hepatica as blebbing, swelling and furrowing was evident. The recovery of flukes 72 h post-treatment was low. Flukes examined at this time point showed an increasing severity of tegumental damage as sloughing and absence of spines, in particular in the tail region. SEM analysis of F. hepatica incubated in the presence of OZ78 without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing or blebbing, in particular in the anterior part, was observed. In conclusion, our experiments confirm the interesting fasciocidal properties of OZ78. The tegument of adult F. hepatica might play a role in the action of this drug.     

In vitro and in vivo studies of F0F1ATP synthase regulation by inhibitor protein IF1 in goat heart

A method has been developed to allow the level of F₀F₁ATP synthase capacity and the quantity of IF₁ bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF₁ content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF₁ antibodies.Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF₁ content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF₁. In addition, both in vivo and in vitro, 1.3-1.4 mol of IF₁ was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF₁ in the F₀ sector.     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Induced Effects of Sodium Ions on Dopaminergic G-Protein Coupled Receptors

G-protein coupled receptors, the largest family of proteins in the human genome, are involved in many complex signal transduction pathways, typically activated by orthosteric ligand binding and subject to allosteric modulation. Dopaminergic receptors, belonging to the class A family of G-protein coupled receptors, are known to be modulated by sodium ions from an allosteric binding site, although the details of sodium effects on the receptor have not yet been described. In an effort to understand these effects, we performed microsecond scale all-atom molecular dynamics simulations on the dopaminergic D₂ receptor, finding that sodium ions enter the receptor from the extracellular side and bind at a deep allosteric site (Asp2.50). Remarkably, the presence of a sodium ion at this allosteric site induces a conformational change of the rotamer toggle switch Trp6.48 which locks in a conformation identical to the one found in the partially inactive state of the crystallized human β₂ adrenergic receptor. This study provides detailed quantitative information about binding of sodium ions in the D₂ receptor and reports a possibly important sodium-induced conformational change for modulation of D₂ receptor function.     

Weather-Related Flood and Landslide Damage: A Risk Index for Italian Regions

The frequency of natural hazards has been increasing in the last decades in Europe and specifically in Mediterranean regions due to climate change. For example heavy precipitation events can lead to disasters through the interaction with exposed and vulnerable people and natural systems. It is therefore necessary a prevention planning to preserve human health and to reduce economic losses. Prevention should mainly be carried out with more adequate land management, also supported by the development of an appropriate risk prediction tool based on weather forecasts. The main aim of this study is to investigate the relationship between weather types (WTs) and the frequency of floods and landslides that have caused damage to properties, personal injuries, or deaths in the Italian regions over recent decades. In particular, a specific risk index (WT-FLARI) for each WT was developed at national and regional scale. This study has identified a specific risk index associated with each weather type, calibrated for each Italian region and applicable to both annual and seasonal levels. The risk index represents the seasonal and annual vulnerability of each Italian region and indicates that additional preventive actions are necessary for some regions. The results of this study represent a good starting point towards the development of a tool to support policy-makers, local authorities and health agencies in planning actions, mainly in the medium to long term, aimed at the weather damage reduction that represents an important issue of the World Meteorological Organization mission.