A neural-specific hypomethylated domain in the 5' flanking region of the glial fibrillary acidic protein gene

In the present study we examined the methylation status of the dial fibrillary acidic protein (GFAP) gene promoter, analyzing various CG sites in both the human and rat gene in GFAP-expressing and nonexpressing tissues, Moreover, we studied the methylation of specific CG sites in different rat brain areas during postnatal development. in cell cultures highly enriched in specific neural-or non-neural-cell types (fibroblasts), and in human gliomas. The obtained results do not support a simple correlation between demethylation and expression of the GFAP gene but help to identify a cluster of CG sites in the 5' flanking region (from -1176 to -1471 in the rat) that are hypomethylated in neural cell types and localized in a region highly conserved between rat, mouse and human GFAP promoters, Neural-specific hypomethylation of this conserved zone can be observed also in the human GFAP gene both in normal brain tissue and neoplastic dial cells, A higher demethylation of the -1176 site at early stage of postnatal life was observed in specific rat brain areas, such as hippocampus and cerebellum. The most dramatic differences were observed in the cerebellum where a peak of demethylation of the -1176 site was detected at 15 days of postnatal life, followed by an intense remethylation of this site, Results of experiments in the CG4 dial progenitor cell line showed that demethylation of the -1176 site is already established before transcriptional activation of the GFAP gene, Moreover, results of experiments in primary cell cultures show that in neuronal cell types, such as cerebellar granule cells and embryonic cerebral hemisphere neurons, the level of demethylation of the -1176 site is comparable to that observed in cultured astrocytes, In contrast a high level of methylation can be observed in cultured non-neural cell types (fibroblasts), Such neural-specific hypomethylation could be established in a very early stage in the progression along the neural cell lineage and could play a role in maintaining a local open chromatin conformation which is then necessary to allow the interaction with specific regulatory factors present in astroglial cells

Molecular beacon as oligonucleotide nanosensors for intracellular mRNA

Molecular beacons, molecules capable to turn on or to modify their light emission upon the interaction with well- defined molecular targets, are among the most promising optical intracellular nanosensors proposed in the recent years. They are oligonucleotidic sequences labeled at the two side-ends, with a fluorophore (F1) at one side and a quencher Q (or a second fluorophore ) at the other side, characterized by an absorption band overlapping with the F1 emission band. In the absence of the target, these structures have a closed conformation with F1 and Q so closed that in the presence of F1 excitation fluorescence resonance energy transfer (FRET) occurs and no emission is observed. In the presence of the target, these structures open and fluorescence emission is observed since F1 and Q are sufficiently distant that FRET does not take place. Their intracellular internalization must be performed using suitable carriers capable to allow these nanosensors to penetrate the cell membrane and enter the cell. We describe here the design, implementation and characterization of structured polymethylmethacrylate (PMMA) nanoparticles (NPs) and carbon nanotubes (CNTs) for intracellular mRNA monitoring. PMMA NPs and CNTs were characterized as potential intracellular nanocarriers of the molecular beacon (MB) for the detection of mRNA encoding surviving, a protein member of the inhibitor of apoptosis family, highly expressed in most types of cancer. Atto647N and Blackberry 650 are the MB fluorophore/quencher pair. Hybridization studies with a target sequence analogous to survivin specific mRNA were conducted in vitro, and the MB functionalities, in solution and once anchored to NPs and CNTs, were successfully demonstrated. An increase of fluorescence signal was observed with the increase of the target concentration from 10 to 500 nM, confirming the dependence of the MB fluorescence on the presence of the target also after its immobilization onto the NPs. MB functionality and specificity were also tested in living cells by transfection with a classical lipid agent, lipofectamine, and by confocal microscopy imaging. A fluorescence increase was observed in the cytoplasm after 1 h from the transfection without fluorescent aggregates or fluorescence in the extracellular environment. On the contrary, no fluorescence was observed in transfected cells not expressing surviving

Physico-chemical characterization and synthesis of neuronally active alpha-conotoxins

The high speciFIcity of alpha-conotoxins for different neuronal nicotinic acetylcholine receptors makes them important probes for dissecting receptor subtype selectivity. New sequences continue to expand the diversity and utility of the pool of available alpha-conotoxins. Their identification and characterization depend on a suite of techniques with increasing emphasis on mass spectrometry and microscale chromatography, which have benefited from recent advances in resolution and capability. Rigorous physicochemical analysis together with synthetic peptide chemistry is a prerequisite for detailed conformational analysis and to provide sufficient quantities of alpha-conotoxins for activity assessment and structure-activity relationship studies

alpha-conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes

Cone snails comprise approximate to 500 species of venomous molluscs, which have evolved the ability to generate multiple toxins with varied and often exquisite selectivity. One class, the alpha-conotoxins, is proving to be a powerful tool for the differentiation of nicotinic acetylcholine receptors (nAChRs). These comprise a large family of complex subtypes, whose significance in physiological functions and pathological conditions is increasingly becoming apparent. After a short introduction into the structure and diversity of nAChRs, this overview summarizes the identification and characterization of alpha-conotoxins with selectivity for neuronal nAChR subtypes and provides examples of their use in defining the compositions and function of neuronal nAChR subtypes in native vertebrate tissues