Tagging genes with cassette-exchange sites

In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5′ and 3′ ‘gene trap’ vectors containing heterospecific Flp recognition target sites and the corresponding ‘exchange’ vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE–PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.     

Antiviral 6-amino-quinolones: molecular basis for potency and selectivity

Structural modifications introduced in 6-amino-quinolones to increase antiviral activity can strongly affect cytotoxicity to host cells. By competition to Tat-TAR complex and binding experiments to viral and cellular DNA and RNA structures, we show that the nature of the substituent at position 7 modifies drug affinity and specificity for the nucleic acid. Interestingly, the basicity of the above substituent modulates chelation of the quinolone template to magnesium ions, which, in turn, critically affects the potency and target selectivity in the antiviral quinolone family.     

Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site

Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/c)(A/t)A*GNNCt(T/a)N(C/a) and GN4G(G/c)(A/c)G*GNNCtTN(C/a) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.     

Molecular characterization of cytotoxic and resistance mechanisms induced by NCX 4040, a novel NO-NSAID, in pancreatic cancer cell lines*

Although non steroidal antiinflammatory drugs (NSAIDs) have been shown to be effective as chemopreventive agents, important side-effects limit their clinical use. A promising novel class of drugs, nitric oxide-donating NSAIDs (NO-NSAIDs), has been found to be more active than classical NSAIDs. This study explored the effect of the NO-donating aspirin derivative, NCX 4040, on three human pancreatic adenocarcinoma cell lines (Capan-2, MIA PaCa-2 and T3M4). NCX 4040 activity was compared with that of NCX 4016 (an NO₂-positional isomer of NCX 4040), SNAP (a standard NO-releasing molecule), NCX 4042 (denitrated analog of NCX 4040), and aspirin. NCX 4040 showed a striking cytocidal activity in all cell lines, already inducing significant percentages of apoptotic cells at 10 μM in Capan-2 cell lines. This study focused on the biological mechanisms of sensitivity and resistance to NCX 4040, highlighting that the cytotoxic action of this drug may be due to the hyperexpression of Bax, its translocation to the mitochondria, the release of Cytochrome C, and the activation of caspases-9 and -3, overall in a p53-independent manner. Moreover, the use of a specific COX-2 inhibitor (NS 398) in the experimental models showed that COX-2 hyperexpression could partially explain the resistance mechanisms to NCX 4040. This study was supported by Istituto Oncologico Romagnolo, Forlì, and by the Italian, Ministry of Health, 2004.     

Cell surface protein detection with immunogold labelling in ESEM: optimisation of the method and semi-quantitative analysis

In this work we used a combination of immunogold labelling (IGL) and environmental scanning electron microscopy (ESEM) to detect the presence of a protein on the cell surface. To achieve this purpose we chose as experimental system 3T3 Swiss Albino Mouse Fibroblasts and galectin-3. This protein, whose sub-cellular distribution is still under discussion, is involved in a large number of cell physiological and pathological processes. IGL technique has been utilised by many authors in combination with SEM and TEM to obtain the identification/localisation of receptors and antigens, both in cells and tissues. ESEM represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artefacts and interfere with IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labelling detection is based on the atomic number difference between Nanogold spheres and the biological material. Using the gaseous secondary electron detector (GSED) compositional contrast is easily revealed by the backscattered electrons component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimised to improve the intensity and the specificity of the labelling signal, in order to obtain a semi-quantitative evaluation of the labelling signal.     

Emergence of Assortative Mixing between Clusters of Cultured Neurons

The analysis of the activity of neuronal cultures is considered to be a good proxy of the functional connectivity of in vivo neuronal tissues. Thus, the functional complex network inferred from activity patterns is a promising way to unravel the interplay between structure and functionality of neuronal systems. Here, we monitor the spontaneous self-sustained dynamics in neuronal cultures formed by interconnected aggregates of neurons (clusters). Dynamics is characterized by the fast activation of groups of clusters in sequences termed bursts. The analysis of the time delays between clusters'' activations within the bursts allows the reconstruction of the directed functional connectivity of the network. We propose a method to statistically infer this connectivity and analyze the resulting properties of the associated complex networks. Surprisingly enough, in contrast to what has been reported for many biological networks, the clustered neuronal cultures present assortative mixing connectivity values, meaning that there is a preference for clusters to link to other clusters that share similar functional connectivity, as well as a rich-club core, which shapes a ‘connectivity backbone’ in the network. These results point out that the grouping of neurons and the assortative connectivity between clusters are intrinsic survival mechanisms of the culture.