H-Ferritin-Regulated MicroRNAs Modulate Gene Expression in K562 Cells

In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562^shFHC) comparing it with K562 transduced with scrambled RNA (K562^shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, “Cell Death and Survival, Hematological System Development and Function, Hematopoiesis”, is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.     

New features of the cell wall of the radio-resistant bacterium Deinococcus radiodurans

We have analyzed the cell wall of the radio-resistant bacterium Deinococcus radiodurans. Unexpectedly, the bacterial envelope appears to be organized in different complexes of high molecular weight. Each complex is composed of several proteins, most of which are coded by genes of unknown function and the majority are constituents of the inner/outer membrane system. One of the most abundant complexes is constituted by the gene DR_0774. This protein is a type of secretin which is a known subunit of the homo-oligomeric channel that represents the main bulk of the type IV piliation family. Finally, a minor component of the pink envelope consists of several inner-membrane proteins. The implications of these findings are discussed.     

Synthesis, characterization and hybridization studies of an alternate nucleo-ε/γ-peptide: complexes formation with natural nucleic acids

In order to develop new oligodeoxyribonucleotide (ODN) analogs to be used in biotechnological applications, we report here the synthesis, characterization and nucleic acid binding studies of novel nucleopeptides, that we called ε-lys/γ-dabPNAs, containing a backbone of alternated l-diaminobutyric acid and l-lysine moieties. Exploring the hybridization properties of the new ODN analog, we found, by circular dichroism and UV spectroscopies, that a homothymine ε-lys/γ-dabPNA hexamer binds both DNA and RNA of complementary sequence. Furthermore, human serum stability assays on the alternate nucleopeptide evidenced a noteworthy degradation resistance. These results encourage us to deepen the knowledge of this analog, in order to evaluate its possible use in antigene/antisense or diagnostic applications.     

Spectral analysis of Delayed Luminescence from human skin as a possible non-invasive diagnostic tool

In vivo measurements of Delayed Luminescence (DL), the low-level photo-induced emission which lasts for a longer time after switching off the excitation light, have been performed on human skin, with the aim to develop a technique for optical biopsy. Preliminary tests have been performed on healthy volunteers, measuring the time decays of the spectral components (λ_emiss = 400–800 nm) starting 10 μs after switching off the excitation (λ_exc = 337 nm). Significant differences in the decay trends of DL from different subjects were revealed and quite a good reproducibility for the same subject was observed. The modeling of experimental data has been examined in detail in order to get parameters, characterizing the theoretical fit, whose changes may be correlated with age differences and seasonal variations. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Chiral tetrahydroquinoline derivatives as potent anti-hyperalgesic agents in animal models of sustained inflammation and chronic neuropathic pain

Chiral tetrahydroquinoline derivatives have been prepared by an asymmetric Mannich-type condensation reaction using commercially available vinyloxyethylsilane and a N-arylimino R-(+)-t-butyl lactate ester, in the presence of a catalytic amount of metal triflates as Lewis acids. This synthetic approach gave rise to the target aldehyde intermediate in moderate facial diastereoselectivity and in high chemical yield. This efficient route enabled to scale up the synthesis of an orally bioavailable glycine antagonist showing outstanding in vivo anti-hyperalgesic activity in different animal models of sustained inflammation and chronic neuropathic pain.     

Bent oligonucleotide duplexes as HMGB1 inhibitors: a comparative study

In this work we explore the ability of a chimeric LNA/DNA bent duplex, in which the kink is induced by 2 unpaired adenines in the middle of one strand, to bind HMGB1, a protein involved in many inflammatory processes. The LNA/DNA duplex was compared with the corresponding full DNA and PNA/DNA chimera duplexes from a thermodynamic and spectroscopic point of view.     

Electrocatalytic oxidation of salicylic acid by a cobalt hydrotalcite-like compound modified Pt electrode

In this paper a study of the electrocatalytic oxidation of salicylic acid (SA) at a Pt electrode coated with a Co/Al hydrotalcite-like compound (Co/Al HTLC coated-Pt) film is presented. The voltammetric behaviour of the modified electrode in 0.1M NaOH shows two different redox couples: Co(II)/Co(III) and Co(III)/Co(IV). The electrocatalysis occurs at the same potential of the latter couple, showing that Co(IV) centers act as the oxidant. The CV investigation demonstrates that the process is controlled both by mass and charge transfer and that the Co(IV) centers involved in the oxidation are two for each SA molecule. The estimated value of the catalytic constant is 4×10(4) M(-1) s(-1). The determination of salicylic acid was performed both by DPV and chronoamperometry. The linearity ranges and the LOD values resulted 1×10(-5) to 5×10(-4), 5×10(-7) to 1×10(-4), 6×10(-6) and 2×10(-7) M, respectively. The Co/Al HTLC electrode has been used for SA determination in BAYER Aspirina? and the obtained results are consistent with an independent HPLC analysis.     

Quantitative analysis of dynamic protein-protein interactions in planta by a floated-leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors

Dynamic protein-protein interactions are essential in all cellular and developmental processes. Protein-fragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway-compatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.