Antiviral and cytotoxic activities of aminoarylazo compounds and aryltriazene derivatives

Twelve aminoarylazocompounds (A–C) and 46 aryltriazene 7 derivatives (D–G) have been synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses.Eight aminoazocompounds and 27 aryltriazene derivatives exhibited antiviral activity, sometimes of high level, against one or more viruses. A marked activity against BVDV and YFV was prevailing among the former compounds, while the latter type of compounds affected mainly CVB-2 and RSV. None of the active compounds inhibited the multiplication of HIV-1, VSV and VV.Arranged in order of decreasing potency and selectivity versus the host cell lines, the best compounds are the following; BVDV: 1 > 7 > 8 > 4; YFV: 7 > 5; CVB-2: 25 > 56 > 18; RSV: 14 > 20 > 55 > 38 > 18 > 19; HSV-1: 2. For these compounds the EC₅₀ ranged from 1.6 μM (1) to 12 μM (18), and the S. I. from 19.4 (1) to 4.2 (2).Thus the aminoarylazo and aryltriazene substructures appear as interesting molecular component for developing antiviral agents against ss RNA viruses, particularly against RSV and BVDV, which are important human and veterinary pathogens.Finally, molecular modeling investigations indicated that compounds of structure A–C, active against BVDV, could work targeting the viral RNA-dependent RNA-polymerase (RdRp), having been observed a good agreement between the trends of the estimated IC₅₀ and the experimental EC₅₀ values.     

Expression of the PsMT A1 gene in white poplar engineered with the MAT system is associated with heavy metal tolerance and protection against 8-hydroxy-2′-deoxyguanosine mediated-DNA damage

Marker-free transgenic white poplar (Populus alba L., cv ‘Villafranca’) plants, expressing the PsMT _A1 gene from Pisum sativum for a metallothionein-like protein, were produced by Agrobacterium tumefaciens-mediated transformation. The 35SCaMV-PsMT _A1 -NosT cassette was inserted into the ipt-type vector pMAT22. The occurrence of the abnormal ipt-shooty phenotype allowed the visual selection of transformants, while the yeast site-specific recombination R/RS system was responsible for the excision of the undesired vector sequences with the consequent recovery of normal marker-free transgenic plants. Molecular analyses confirmed the presence of the 35SCaMV-PsMT _A1 -NosT cassette and transgene expression. Five selected lines were further characterized, revealing the ability to withstand heavy metal toxicity. They survived 0.1 mM CuCl₂, a concentration which strongly affected the nontransgenic plants. Moreover, root development was only slightly affected by the ectopic expression of the transgene. Reactive oxygen species were accumulated to a lower extent in leaf tissues of multi-auto-transformation (MAT)-PsMT_A1 plants exposed to copper and zinc, compared to control plants. Tolerance to photo-oxidative stress induced by paraquat was another distinctive feature of the MAT-PsMT_A1 lines. Finally, low levels of DNA damage were detected by quantifying the amounts of 8-hydroxy-2′-deoxyguanosine in leaf tissues of the transgenic plants exposed to copper.     

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.     

Human Neuroserpin: Structure and Time-Dependent Inhibition

Human neuroserpin (hNS) is a protein serine protease inhibitor expressed mainly in the nervous system, where it plays key roles in neural development and plasticity by primarily targeting tissue plasminogen activator (tPA). Four hNS mutations are associated to a form of autosomal dominant dementia, known as familial encephalopathy with neuroserpin inclusion bodies. The medical interest in and the lack of structural information on hNS prompted us to study the crystal structure of native and cleaved hNS, reported here at 3.15 and 1.85 Å resolution, respectively. In the light of the three-dimensional structures, we focus on the hNS reactive centre loop in its intact and cleaved conformations relative to the current serpin polymerization models and discuss the protein sites hosting neurodegenerative mutations. On the basis of homologous serpin structures, we suggest the location of a protein surface site that may stabilize the hNS native (metastable) form. In parallel, we present the results of kinetic studies on hNS inhibition of tPA. Our data analysis stresses the instability of the hNS-tPA complex with a dissociation half-life of minutes compared to a half-life of weeks observed for other serpin-cognate protease complexes.     

Biological control of great brome (<Emphasis Type="Italic">Bromus diandrus</Emphasis>) in durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis>): specificity,physiological traits and impact on plant growth and root architecture of the fluorescent pseudomonad strain X33d

The rhizobacterial strain X33d was previously shown to suppress the growth of the weed great brome (Bromus diandrus Roth.). The aim of this work was to identify X33d, characterize its physiological activities, assess its specificity on different non-target crops, and its impact on the growth and the root architecture of great brome and durum wheat (Triticum durum Desf.) grown alone and together. Based on 16S rDNA sequencing, X33d was identified as Pseudomonas trivialis. The specificity assay, performed on a mixture of soil/sand/peat, highlighted the suppressive activity of P. trivialis X33d against great brome and the promoting effect on most of the considered crops, especially durum wheat. Although the growth of durum wheat on quartz sand was unaffected, P. trivialis X33d suppressed the growth and affected the root architecture of great brome, especially when co-seeded with durum wheat. Great brome plants inoculated with X33d and co-seeded with durum wheat showed low root biomass, short root systems and low surface area, volume and number of tips. Moreover, P. trivialis X33d synthesized indole-acetic acid (IAA) that could be involved both in great brome growth suppression and durum wheat growth promotion. Our results indicate that P. trivialis X33d could be exploited as a potential biocontrol agent against great brome without affecting the durum wheat growth. These results are discussed in relation to the competitive capability of great brome towards durum wheat.     

Proteomic analysis of Pteris vittata fronds: two arbuscular mycorrhizal fungi differentially modulate protein expression under arsenic contamination

Arbuscular mycorrhizae (AM) are the most widespread mutualistic symbioses between the roots of most land plants and a phylum of soil fungi. AM are known to influence plant performance by improving mineral nutrition, protecting against pathogens and enhancing resistance or tolerance to biotic and abiotic stresses. The aim of this study was to investigate the frond proteome of the arsenic hyperaccumulator fern Pteris vittata in plants that had been inoculated with one of the two AM fungi (Glomus mosseae or Gigaspora margarita) with and without arsenic treatment. A protective role for AM fungi colonisation in the absence of arsenic was indicated by the down-regulation of oxidative damage-related proteins. Arsenic treatment of mycorrhizal ferns induced the differential expression of 130 leaf proteins with specific responses in G. mosseae- and Gi. margarita-colonised plants. Up-regulation of multiple forms of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase, primarily in G. mosseae-inoculated plants, suggests a central role for glycolytic enzymes in arsenic metabolism. Moreover, a putative arsenic transporter, PgPOR29, has been identified as an up-regulated protein by arsenic treatment.     

Morpho-functional analysis of lung tissue in mild interstitial edema

Mild pulmonary interstitial edema was shown to cause fragmentation of interstitial matrix proteoglycans. We therefore studied compartmental fluid accumulation by light and electron microscopy on lungs of anesthetized rabbits fixed in situ by vascular perfusion after 0.5 ml.kg(-1).min(-1) iv saline infusion for 180 min causing approximately 6% increase in lung weight. Morphometry showed that a relevant portion (44%) of extravascular fluid is detected early in the alveolar septa, 85% of this fluid accumulating in the thick portion of the air-blood barrier. The arithmetic mean thickness of the barrier increased in interstitial edema from 1.06 +/- 0.05 (SE) to 1.33 +/- 0.06 microm. The harmonic mean thickness increased from 0.6 +/- 0.03 to 0.86 +/- 0.07 microm, mostly due to thickening of the thin portion causing an increase in gas diffusion resistance. Despite some structural damage, the air-blood barrier displays a relatively high structural resistance providing a safety factor against the development of severe edema. It is suggested that the increase in extra-alveolar perivascular space occurs as a consequence of fluid accumulation in the air-blood barrier.