RNA as a new target for toxic and protective agents

In contrast to damage of genomic DNA and despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome, such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human chronic diseases. We investigated whether RNA damage could be related to the exposure of particular xenobiotics by testing the RNA-damaging activity of a series of chemicals with different mechanisms of action. Cultured human T-lymphoblastoid cells were treated with ethyl methanesulfonate (EMS), H(2)O(2), doxorubicin, spermine, or S-nitroso-N-acetylpenicillamine (SNAP). Furthermore, we studied the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate can be mediated by alterations of the rates of toxic agent absorption and uptake, by trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA. We used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of pomegranate. We demonstrated that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H(2)O(2), spermine and SNAP. However, EMS, a well-known DNA-damaging agent, was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties, were able to damage RNA. Pomegranate reduced the RNA-damaging effect of doxorubicin, H(2)O(2), and spermine. Its inhibitory activity could be related with its ability to forms complexes with doxorubicin and H(2)O(2), or interacts with the intracellular formation of reactive species mediating their toxicity. For spermine, an alteration of the rates of spermine absorption and uptake can also be involved.     

Distribution and evolution of circular miniproteins in flowering plants

Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids ( approximately 125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.     

Mitigation measures for pandemic influenza in Italy: an individual based model considering different scenarios

Background

Individual-based models can provide the most reliable estimates of the spread of infectious diseases. In the present study, we evaluated the diffusion of pandemic influenza in Italy and the impact of various control measures, coupling a global SEIR model for importation of cases with an individual based model (IBM) describing the Italian epidemic.

Methodology/Principal Findings

We co-located the Italian population (57 million inhabitants) to households, schools and workplaces and we assigned travel destinations to match the 2001 census data. We considered different R₀ values (1.4; 1.7; 2), evaluating the impact of control measures (vaccination, antiviral prophylaxis -AVP-, international air travel restrictions and increased social distancing). The administration of two vaccine doses was considered, assuming that first dose would be administered 1-6 months after the first world case, and different values for vaccine effectiveness (VE). With no interventions, importation would occur 37–77 days after the first world case. Air travel restrictions would delay the importation of the pandemic by 7–37 days. With an R₀ of 1.4 or 1.7, the use of combined measures would reduce clinical attack rates (AR) from 21–31% to 0.3–4%. Assuming an R₀ of 2, the AR would decrease from 38% to 8%, yet only if vaccination were started within 2 months of the first world case, in combination with a 90% reduction in international air traffic, closure of schools/workplaces for 4 weeks and AVP of household and school/work close contacts of clinical cases. Varying VE would not substantially affect the results.

Conclusions

This IBM, which is based on country-specific demographic data, could be suitable for the real-time evaluation of measures to be undertaken in the event of the emergence of a new pandemic influenza virus. All preventive measures considered should be implemented to mitigate the pandemic.     

Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease

In prion disease, the abnormal conformer of the cellular prion protein, PrP(Sc), deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP(Sc) to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrP(Sc) C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrP(Sc) or PrP27-30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrP(Sc) in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrP(Sc) and indicate that the characterization of truncated PrP(Sc) forms may further improve molecular typing in CJD.     

Schistosoma mansoni: lack of correlation between praziquantel-induced intra-worm calcium influx and parasite death

The schistosomicidal activity of praziquantel (PZQ) is accompanied by a large influx of calcium into the worms, suggesting that this phenomenon could be the source of the observed muscular contraction, surface disruption and eventual death of the parasite. We have incubated live adult schistosomes in a medium containing radioactive calcium and we were able to confirm that PZQ does indeed stimulate calcium entry into the parasite. An even higher calcium uptake, however, occurred in schistosomes exposed to PZQ after pre-incubation with cytochalasin D, a condition that suppresses PZQ schistosomicidal effects and allows the complete survival of the parasites. The calcium blockers nicardipine and nifedipine also failed to prevent the calcium influx induced by PZQ. Similarly, a large calcium influx occurred in 28-day-old worms exposed to PZQ, in spite of the fact that these immature worms are largely insensitive to the schistosomicidal effects of the drug. Schistosomes incubated overnight with radioactive calcium and PZQ and then returned to normal medium, retained a calcium content higher than worms pre-incubated with cytochalasin D, but the difference could be a consequence—rather than a cause—of schistosomicidal effects. These results suggest that calcium accumulation by itself, at least as measured in whole parasites maintained in vitro, may not represent an exhaustive explanation for the schistosomicidal effects of PZQ.     

Oxidative stress-induced risk factors associated with the metabolic syndrome: a unifying hypothesis

Although the biochemical steps linking insulin resistance with the metabolic syndrome have not been completely clarified, mounting experimental and clinical evidence indicate oxidative stress as an attractive candidate for a central pathogenic role since it potentially explains the appearance of all risk factors and supports the clinical manifestations. In fact, metabolic syndrome patients exhibit activation of biochemical pathways leading to increased delivery of reactive oxygen species, decreased antioxidant protection and increased lipid peroxidation. The described associations between increased abdominal fat storage, liver steatosis and systemic oxidative stress, the diminished concentration of nitric oxide derivatives and antioxidant vitamins and the endothelial oxidative damages observed in subjects with the metabolic syndrome definitively support oxidative stress as the common second-level event in a unifying pathogenic view. Moreover, it has been observed that oxidative stress regulates the expression of genes governing lipid and glucose metabolism through activation or inhibition of intracellular sensors. Diet constituents can modulate redox reactions and the oxidative stress extent, thus also acting on nuclear gene expression. As a consequence of the food-gene interaction, metabolic syndrome patients may express different disease features and extents according to the different pathways activated by oxidative stress-modulated effectors. This view could also explain family differences and interethnic variations in determining risk factor appearance. This review mechanistically focused on oxidative stress events leading to individual disease factor appearance in metabolic syndrome patients and their setting for a more helpful clinical approach.     

New sulfurated derivatives of valproic acid with enhanced histone deacetylase inhibitory activity

One dithiolthione and two new methanethiosulfonate derivatives of valproic acid (VPA) were synthesized and tested in vitro as histone deacetylase (HDAC) inhibitors. The new molecules, as well as their sulfurated moieties, exhibited a much stronger inhibition of HDAC enzymatic and antiproliferative activities and histone hyperacetylation than VPA. ACS 2 is the most interesting compound among the new VPA derivatives and its sulfurated moiety, 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione, also known to be a metabolite of anethole trithione, seems to contribute significantly to its activity. This is the first time that HDAC inhibitory activity is described for dithiolethiones and thiosulfonates.     

Eradications of invasive alien species in Europe: a review

Eradication of alien species is a key conservation tool to mitigate the impacts caused by biologic invasions. The aim of the present paper is to review the eradications successfully completed in Europe and to discuss the main limits to a wider application of this management option in the region. On the basis of the available literature – including conference proceedings, national reports to the Bern Convention, etc. – a total of 37 eradication programmes have been recorded. Thirty-three eradications were carried out on islands and four on the mainland. The rat (Rattus spp.) has been the most common target (n = 25, 67%), followed by the rabbit (n = 4). In many cases, these eradications determined a significant recovery of native biodiversity. Differently to other regions of the world, no eradications of alien invertebrates and marine organisms have been recorded; regarding invasive alien plants, it appears that only some very localized removals have been completed so far in Europe. The limited number of eradications carried out in Europe so far is probably due to the limited awareness of the public and the decision makers, the inadequacy of the legal framework, and the scarcity of resources. Synthetic guidelines for improving the ability of European states to respond to aliens incursions are presented. This revised version was published online in July 2006 with corrections to the Cover Date.     

Carapichea guianensis: the correct name for Psychotria carapichea (Rubiaceae), with a correction on the authority of P. carapichea, and a new combination in Carapichea

The correct name forPsychotria carapichea isCarapichea guianensis. Also, the authority ofPsychotria carapichea was previously incorrectly cited, because of an oversight of the illegitimacy ofTapogomea carapichea Poir. The complex taxonomic history of this species is analyzed, and the authority of the binomial is correctly cited. In addition,P. ligularis, which was treated as closely related toC. guianensis in previous publications, is transferred toCarapichea and it is now known asCarapichea ligularis.     

A new synthetic approach to 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-benzimidazol-2-one(J-113397), the first non-peptide ORL-1 receptor antagonist..

An efficient approach to 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-benzimidazol-2-one (J-113397) 1, the first non-peptide ORL-1 receptor antagonist described in literature, is outlined. After construction of the piperidine framework through Dieckmann cyclization of the Michael adduct 8 of cyclooctylmethylamine to methyl acrylate, condensation with o-phenylendiamine produced the beta-enamino ester 2, which has been conveniently used to construct the benzimidazolone substituent at C-4. Catalytic hydrogenation of intermediate 11 followed by base-promoted cis--trans isomerization of the key compound 12 led to the formation of ester 13, which was converted to the racemic title compound by LiAlH(4) reduction. The pure enantiomers were obtained by chiral preparative HPLC separation using a derivatized cellulose-based stationary phase.