Biomedical subjects

Paola Zacchi

Publications and source records attributed to Paola Zacchi.

5 recordsLinked to original sources

PubMed2004

Clustering of extrasynaptic GABA(A) receptors modulates tonic inhibition in cultured hippocampal neurons.

Tonic inhibition plays a crucial role in regulating neuronal excitability because it sets the threshold for action potential generation and integrates excitatory signals. Tonic currents are known to be largely mediated by extrasynaptic gamma-aminobutyric acid type A (GABA(A)) receptors that are persistently activated by submicromolar concentrations of ambient GABA. We recently reported that, in cultured hippocampal neurons, the clustering of synaptic GABA(A) receptors significantly affects synaptic transmission. In this work, we demonstrated that the clustering of extrasynaptic GABA(A) receptors modulated tonic inhibition. Depolymerization of the cytoskeleton with nocodazole promoted the disassembly of extrasynaptic clusters of delta and gamma(2) subunit-containing GABA(A) receptors. This effect was associated with a reduction in the amplitude of tonic currents and diminished shunting inhibition. Moreover, diffuse GABA(A) receptors were less sensitive to the GAT-1 inhibitor NO-711 and to flurazepam. Quantitative analysis of GABA-evoked currents after prolonged exposure to submicromolar concentrations of GABA and model simulations suggest that clustering affects the gating properties of extrasynaptic GABA(A) receptors. In particular, a larger occupancy of the singly and doubly bound desensitized states can account for the modulation of tonic inhibition recorded after nocodazole treatment. Moreover, comparison of tonic currents recorded during spontaneous activity and those elicited by exogenously applied low agonist concentrations allows estimation of the concentration of ambient GABA. In conclusion, receptor clustering appears to be an additional regulating factor for tonic inhibition.

Animals↗

PubMed2004

Pin1 links the activities of c-Abl and p300 in regulating p73 function.

Activation of p73 upon genotoxic treatment triggers apoptosis of tumor cells lacking functional p53 and involves the activities of c-Abl and p300. Here, we demonstrate that conformational changes of p73 catalyzed by the prolyl isomerase Pin1 are crucial in this pathway. Lack of Pin1 reduces p73 stability, hampering its accumulation upon genotoxic stress. Indeed, we show that upon treatment with chemotherapeutic drugs c-Abl enhances the phosphorylation-dependent interaction between Pin1 and p73, and this in turn promotes p73 acetylation by p300. Consistently, the ability of c-Abl and p300 to increase p73 stability and transcriptional activity requires Pin1. As a consequence, Pin1 appears to be essential for activation of the apoptotic response by endogenous p73.

Acetyltransferases↗

PubMed2003

Declusterization of GABAA receptors affects the kinetic properties of GABAergic currents in cultured hippocampal neurons.

Speed and reliability of synaptic transmission are essential for information coding in neuronal networks and require the presence of clustered neurotransmitter receptors at the plasma membrane in precise apposition to presynaptic terminals. Receptor clusterization is the result of highly regulated processes involving functional and structural proteins. Among the structural elements, microtubules are known to play a crucial role in anchoring of gamma-aminobutyric acid, type A (GABA(A)) receptors. Here we show that microtubule depolymerization with nocodazole induces the declusterization of GABA(A) receptors and modifies the kinetic properties of GABAergic currents in cultured hippocampal neurons. In particular, this drug, applied either in the bath or via the patch pipette, induced the acceleration of the onset kinetics of miniature inhibitory postsynaptic currents (mIPSCs) without significantly affecting their frequency, thus suggesting a main postsynaptic site of action. After nocodazole treatment, current responses to ultrafast applications of GABA exhibited a faster rise time and an accelerated onset of desensitization. A quantitative analysis of GABA-evoked currents and model simulations suggest that declusterization affects the gating properties of GABA(A) receptors. In particular, a faster entry into the desensitized state of declustered GABA(A) receptors may account for the changes in the kinetic properties of mIPSCs after nocodazole treatment. Hence it appears that the clustered condition of GABA(A) receptors contributes in shaping GABAergic currents.

Animals↗

PubMed2003

Selecting open reading frames from DNA.

We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector. Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used. By cloning DNA fragments upstream of a fusion gene, consisting of the beta-lactamase gene flanked by lox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive. After selection, the beta-lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3. This vector has been tested on a plasmid containing tissue transglutaminase. All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides. Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope. This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems.

Antibodies, Monoclonal↗

PubMed2002

The prolyl isomerase Pin1 reveals a mechanism to control p53 functions after genotoxic insults.

The tumour suppressor p53 is important in the cell decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 post-translational modifications and association with other proteins have been implicated in the regulation of its stability and transcriptional activities. Here we report that, on DNA damage, p53 interacts with Pin1, a peptidyl-prolyl isomerase, which regulates the function of many proteins involved in cell cycle control and apoptosis. The interaction is strictly dependent on p53 phosphorylation, and requires Ser 33, Thr 81 and Ser 315. On binding, Pin1 generates conformational changes in p53, enhancing its transactivation activity. Stabilization of p53 is impaired in UV-treated Pin1(-/-) cells owing to its inability to efficiently dissociate from Mdm2. As a consequence, a reduced p53-dependent response was detected in Pin1(-/-) cells, and this correlates with a diminished transcriptional activation of some p53-regulated genes. Our results suggest that, following stress-induced phosphorylation, p53 needs to form a complex with Pin1 and to undergo a conformational change to fulfil its biological roles.

Amino Acid Motifs↗