257 research outputs found
Molecular mechanisms of sensitization of pain transducing P2X3 receptors by the migraine mediators CGRP and NGF
No full textMigraine headache originates from the stimulation of nerve terminals of trigeminal ganglion neurons that innervate meninges. Characteristic features of migraine pain are not only its delayed onset but also its persistent duration. Current theories propose that endogenous substances released during a migraine attack (the neuropeptide calcitonin gene-related peptide [CGRP] and the neurotrophin nerve growth factor [NGF]) sensitize trigeminal neurons to transmit nociceptive signals to the brainstem, though the mechanisms remain poorly understood. Recent studies indicate that acute, long-lasting sensitization of trigeminal nociceptive neurons occurs via distinct processes involving enhanced expression and function of adenosine triphosphate (ATP)-gated P2X(3) receptors known to play a role in chronic pain. In particular, on cultured trigeminal neurons, CGRP (via protein kinase A-dependent signaling) induces a slowly developing upregulation of the ionic currents mediated by P2X(3) receptors by enhancing receptor trafficking to the neuronal membrane and activating their gene transcription. Such upregulated receptors acquire the ability to respond repeatedly to extracellular ATP, thus enabling long-lasting signaling of painful stimuli. In contrast, NGF induces rapid, reversible upregulation of P2X(3) receptor function via protein kinase C phosphorylation, an effect counteracted by anti-NGF antibodies. The diverse intracellular signaling pathways used by CGRP and NGF show that the sensitization of P2X(3) receptor function persists if the action of only one of these migraine mediators is blocked. These findings imply that inhibiting a migraine attack might be most efficient by a combinatorial approach. The different time domains of P2X(3) receptor modulation by NGF and CGRP suggest that the therapeutic efficacy of novel antimigraine drugs depends on the time of administration
Nanostructure-Based Fluorescent Biosensors
No full textNanostructure-Based Fluorescent Biosensors
Shivaram K. a, Gunnella R. a, Giuliodori A. M.b, Spurio R.b, Fabbretti A. b, Perrozzi F.c
Ottaviano L.c
aSchool of Science and Technology, Physics Division, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino; e-mail: [email protected]
bSchool of Biosciences and Veterinary Medicine, Biology Division, University of Camerino, Via Gentile III da Varano, 62032 Camerino
cDipartimento di Scienze Fisiche e Chimiche, Universita' dell'Aquila Via Vetoio, 67100 L'Aquila
We have investigated a fluorescent biosensor based on graphene oxide (GO) for the measurement of interaction between a fluorophore FAM (Carboxyfluorescein)-labeled single-stranded DNA with its complementary single-stranded DNA oligonucleotide (target). The graphene oxide adsorbs the FAM-labeled single stranded DNA (probe) and quenches its fluorescence. Upon addition of the complementary single stranded DNA oligonucleotide, the probe hybridizes to its target [1] thus producing a double- stranded DNA, which detaches from the GO. The release of the double helix leads to the recovery of dye fluorescence that can be monitored by fluorimetric techniques. Pristine GO [2,3] flakes were prepared using a modified Hummers method and dispersed in water with a concentration of 0.5 mg/mL. The samples were prepared by drop casting, the GO and DNA with buffered solution [1] on 300 nm SiO2 /Si(100) at room temperature. AFM image of the GO flakes,) shows the typical AFM image of the DNA-GO complex, where the bright areas on the GO surface might be due to the adsorption of DNA. In this complex the thickness is about 3 nm.This observation indicates that GO can strongly adsorb ssDNA and can efficiently quench its fluorescence. The fluorescently labeled ssDNA-GO complex displayed significant fluorescence enhancement upon addition of complementary target DNA oligonucleotide (Figure 1b). This recovery of fluorescence increases with increasing concentration of the target DNA added to the mixture.
References
1. Lu, C.-H., Angew. Chem. Int. Ed. 48: 4785–4787 (2009). doi: 10.1002/anie.200901479.
2. Nan-Fu Chiu et al. M. Aliofkhazraei (Ed.), ISBN: 978-953-51-1182-5, InTech (2013). DOI: 10.5772/56221.
3. F. Perrozzi et al., J. Phys.: Condens. Matter 27, 013002 (2015) doi:10.1088/0953-8984/27/1/013002 Graphene oxide: from fundamentals to applications
Morphological features P2X3 expression in sensory neurons of rodent trigeminal ganglia under different culture conditions
No full textFunctional differences between ATP-gated Human and Rat P2X3 Receptors are caused by critical residues of the Intracellular C-Terminal Domain
No full textATP-activated P2X3 receptors of sensory ganglion neurons contribute to pain transduction and are involved in chronic pain signaling. Although highly homologous (97%) in rat and human species, it is unclear whether P2X3 receptors have identical function. Studying human and rat P2X3 receptors expressed in patch-clamped human embryonic kidney (HEK) cells, we investigated the role of non-conserved tyrosine residues in the C-terminal domain (rat tyrosine-393 and human tyrosine-376) as key determinants of receptor function. In comparison with rat P2X3 receptors, human P2X3 receptors were more expressed and produced larger responses with slower desensitization and faster recovery. In general, desensitization was closely related to peak current amplitude for rat and human receptors. Downsizing human receptor expression to the same level of the rat one still yielded larger responses retaining slower desensitization and faster recovery. Mutating phenylalanine-376 into tyrosine in the rat receptor did not change current amplitude; yet, it retarded desensitization onset, demonstrating how this residue was important to functionally link these two receptor states. Conversely, removing tyrosine from position 376 strongly down-regulated human receptor function. The different topology of tyrosine residues in the C-terminal domain has contrasting functional consequences and is sufficient to account for species-specific properties of this pain-transducing channel. 'Of Mice and Men'? A single amino acid in the pain-transducing P2X3 receptor structure can determine human or rat function. The reason why the human P2X3 receptor generates much larger responses than the homologous rat one was investigated. Changing the position of one tyrosine in the intracellular C-terminus converted the function of the human receptor to the rat one, and vice versa. Species-specific differences in P2X3 responses to harmful stimuli may be attributed to the location of a single tyrosine residue, which is modulated by intracellular kinases to determine the strength of pain signaling to the brain. © 2012 International Society for Neurochemistry
Neutralization of Nerve Growth Factor induces plasticity of ATP-sensitive P2X3 receptors of nociceptive trigeminal ganglion neurons
No full textThe antibiotic Furvina® targets the P-site of 30S ribosomal subunits and inhibits translation initiation displaying start codon bias.
No full textFurvina®, also denominated G1 (MW 297), is a synthetic nitrovinylfuran [2-bromo-5-(2-bromo-2-nitrovinyl)-furan] antibiotic with a broad antimicrobial spectrum. An ointment (Dermofural®) containing G1 as the only active principle is currently marketed in Cuba and successfully used to treat dermatological infections. Here we describe the molecular target and mechanism of action of G1 in bacteria and demonstrate that in vivo G1 preferentially inhibits protein synthesis over RNA, DNA and cell wall synthesis. Furthermore, we demonstrate that G1 targets the small ribosomal subunit, binds at or near the P-decoding site and inhibits its function interfering with the ribosomal binding of fMet-tRNA during 30S initiation complex (IC) formation ultimately inhibiting translation. Notably, this G1 inhibition displays a bias for the nature (purine vs. pyrimidine) of the 3'-base of the codon, occurring efficiently only when the mRNA directing 30S IC formation and translation contains the canonical AUG initiation triplet or the rarely found AUA triplet, but hardly occurs when the mRNA start codon is either one of the non-canonical triplets AUU or AUC. This codon discrimination by G1 is reminiscent, though of opposite type of that displayed by IF3 in its fidelity function, and remarkably does not occur in the absence of this factor
Characterization of the regulatory genes for the production of the glycopeptide antibiotic A40926 by Nonomuraea ATCC 39727
No full textTime-resolved assembly of a nucleoprotein complex between Shigella flexneri virF promoter and its transcriptional repressor H-NS.
No full textThe virF gene of Shigella, responsible for triggering the virulence cascade in this pathogenic bacterium, is transcriptionally repressed by the nucleoid-associated protein H-NS. The primary binding sites of H-NS within the promoter region of virF have been detected here by footprinting experiments in the presence of H-NS or its monomeric DNA-binding domain (H-NSctd), which displays the same specificity as intact H-NS. Of the 14 short DNA fragments identified, 10 overlap sequences similar to the H-NS binding motif. The 'fast', 'intermediate' and 'slow' H-NS binding events leading to the formation of the nucleoprotein complex responsible for transcription repression have been determined by time-resolved hydroxyl radical footprinting experiments in the presence of full-length H-NS. We demonstrate that this process is completed in ≤1 s and H-NS protections occur simultaneously on site I and site II of the virF promoter. Furthermore, all 'fast' protections have been identified in regions containing predicted H-NS binding motifs, in agreement with the hypothesis that H-NS nucleoprotein complex assembles from a few nucleation sites containing high-affinity binding sequences. Finally, data are presented showing that the 22-bp fragment corresponding to one of the HNS binding sites deviates from canonical B-DNA structure at three TpA steps
Identification of negative residues in the P2X3 ATP receptor ectodomain as structural determinants for desensitization and the Ca2+ sensing modulatory sites
No full textAgonist-dependence of recovery from desensitization of P2X3 receptors provides a novel and sensitive approach for their rapid up or down regulation
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