Search

Abstract: Sustained administration of opioid antagonists to rodents results in an enhanced antinociceptive response to agonists. We investigated the changes in spinal μ-opioid receptor signalling underlying this phenomenon. Rats received naltrexone (120 μg/h; 7 days) via osmotic minipumps. The antinociceptive response to the μ-agonist sufentanil was tested 24 h after naltrexone withdrawal. In spinal cord samples, we determined the interaction of μ-receptors with Gα proteins (agonist-stimulated [35S]GTPγS binding and immunoprecipitation of [35S]GTPγS-labelled Gα subunits) as well as μ-opioid receptor-dependent inhibition of the adenylyl cyclase (AC) activity. Chronic naltrexone treatment augmented DAMGO-stimulated [35S]GTPγS binding, potentiated the inhibitory effect of DAMGO on the AC/cAMP pathway, and increased the inverse agonist effect of naltrexone on cAMP accumulation. In control rats, the inhibitory effect of DAMGO on cAMP production was antagonized by pertussis toxin (PTX) whereas, after chronic naltrexone, the effect became resistant to the toxin, suggesting a coupling of μ-receptors to PTX-insensitive Gαz subunits. Immunoprecipitation assays confirmed the transduction switch from Gαi/o to Gαz proteins. The consequence was an enhancement of the antinociceptive response to sufentanil that, in consonance with the neurochemical data, was prevented by Gαz-antisense oligodeoxyribonucleotides but not by PTX. Such changes in opioid receptor signalling can be a double-edged sword. On the one hand, they may have potential applicability to the optimisation of the analgesic effects of opioid drugs for the control of pain. On the other hand, they represent an important homeostatic dysregulation of the endogenous opioid system that might account for undesirable effects in patients chronically treated with opioid antagonists. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’. [Copyright &y& Elsevier]

The laminated layer (LL) is the massive carbohydrate-rich structure that protects Echinococcus larvae, which cause cystic echinococcosis (hydatid disease) and alveolar echinococcosis. Increased understanding of the biochemistry of the LL is allowing a more informed analysis of its immunology. The LL not only protects the parasite against host attack but also shapes the overall immune response against it. Because of its dense glycosylation, it probably contains few T-cell epitopes, being important instead in T-cell independent antibody responses. Crucially, it is decoded in non-inflammatory fashion by innate immunity, surely contributing to the strong immune-regulation observed in Echinococcus infections. Defining the active LL molecular motifs and corresponding host innate receptors is a feasible and promising goal in the field of helminth-derived immune-regulatory molecules. [ABSTRACT FROM AUTHOR]

Echinococcus larvae are protected by a massive carbohydrate-rich acellular structure, called the laminated layer. In spite of being widely considered the crucial element of these host–parasite interfaces, the laminated layer has been historically poorly understood. In fact, it is still often called ‘chitinous’, ‘hyaline’ or ‘cuticular’ layer, or said to be composed of polysaccharides. However, over the past few years the laminated layer was found to be comprised of mucins bearing defined galactose-rich carbohydrates, and accompanied, in the case of Echinococcus granulosus, by calcium inositol hexakisphosphate deposits. In this review, the architecture and biosynthesis of this unusual structure is discussed at depth in terms of what is known and what needs to be discovered. [Copyright &y& Elsevier]

Abstract: The larvae of the cestodes belonging to the genus Echinococcus are outwardly protected by the laminated layer (LL), a crucial but poorly understood carbohydrate-rich acellular structure. Carbohydrate structural data strongly suggest that the main components of the LL are mucins. The most massive LL in the genus is featured by E. granulosus, agent of cystic hydatid disease. No appropriate methods existed to date for the solubilisation of the E. granulosus LL and the electrophoretic visualisation of its proposed structural mucins. We report that reduction of disulphides greatly aids LL disassembly, resulting in almost full solubilisation in combination with moderate sonication. The structural mucins can then be visualised by agarose electrophoresis and blotting with galactose-binding lectins, which also react strongly with the LL in tissue sections. A substantial portion of the material migrates as if positively charged; since the LL glycans are neutral, this may correspond to mucins with cationic peptide backbones. [ABSTRACT FROM AUTHOR]

Abstract: 5-HT1A receptor-mediated signalling in rat brain was evaluated after chronic administration (14 days; s.c.) of the selective serotonin reuptake inhibitor (SRRI) fluoxetine (10 mg/kg/day) alone, or in combination with the 5-HT1A receptor antagonist WAY100635 (0.1 mg/kg/day). The density of 5-HT1A binding sites was unchanged following fluoxetine, WAY100635, or the combination of fluoxetine and WAY100635. However, the net stimulation of [35S]GTPγS binding induced by the 5-HT1A agonist 8-OH-DPAT was significantly attenuated in dorsal raphe nucleus (DRN), but not in hippocampus, after chronic fluoxetine. Moreover, depending of the area analysed, the basal binding of [35S]GTPγS was differentially affected by this treatment: increased in DRN and decreased in hippocampal dentate gyrus. Interestingly, the changes in [35S]GTPγS basal binding and on 5-HT1A receptors functionality were prevented by the concomitant administration of WAY100635. The inhibition of dorsal raphe firing by 8-OH-DPAT was also attenuated in fluoxetine-treated rats (ED50 = 2.12 ± 0.32 μg/kg and 4.34 ± 0.09 μg/kg, for vehicle and fluoxetine respectively), an effect which was also prevented by the concomitant administration of WAY100635 (ED50 = 2.10 ± 0.58 μg/kg). Chronic administration of WAY100635 alone did not affect the 5-HT1A receptor-induced stimulation of [35S]GTPγS binding, nor the 8-OH-DPAT-induced inhibition of 5-HT neuron firing. These results demonstrate that the concomitant blockade of 5-HT1A receptors when administering fluoxetine prevents those adaptive changes of 5-HT1A receptor function associated with the chronic administration of this antidepressant. These findings could be relevant from the therapeutic point of view, and further support the potential benefit of treatments with a SSRI/5-HT1A receptor antagonist combination. [Copyright &y& Elsevier]

Morphine and related opioids are the mainstay of analgesic treatment, especially in patients suffering chronic pain. Besides their antinociceptive effects they may also exhibit anxiolytic-like properties that could contribute to pain relief. The pharmacological manipulation of the serotonergic system may not only modulate pain transmission and processing but also other behavioral effects of opioids. The present study aimed to analyze the effect of the concurrent treatment with citalopram, a selective serotonin reuptake inhibitor, on the antinociceptive, locomotor and anxiety-related effects induced by acute and subchronic administration of morphine in mice. Citalopram (15 mg/kg) enhanced the acute antinociceptive effects of morphine when concurrently administered as evidenced by a two-fold increase in the ED 50 for the antinociceptive effect of morphine in the hot-plate test. Chronic studies also revealed that concurrent citalopram treatment (15 mg/kg) delayed the development of tolerance to the thermal antinociceptive effects of morphine. Additionally, morphine-induced hyperlocomotion was potentiated by citalopram as assessed in the open-field test and in the spontaneous activity recording in the home cage, a behavioral outcome to which tolerance or desensitization was not developed. Interestingly, chronic administration of both drugs promoted an anxiolytic effect as evidenced by the increased central activity in the open field test. Future investigations on this pharmacological interaction, such as the possible translational research in clinics, might have consequences in future strategies for the therapeutic management of pain. [ABSTRACT FROM AUTHOR]

Persistent extracellular tissue-dwelling pathogens face the challenge of antibody-dependent activation of the classical complement pathway (CCP). A prime example of this situation is the larva of the cestode Echinococcus granulosus sensu lato , causing cystic echinococcosis. This tissue-dwelling, bladder-like larva is bounded by a cellular layer protected by the outermost acellular "laminated layer" (LL), to which host antibodies bind. The LL is made up of a mucin meshwork and interspersed nano-deposits of calcium inositol hexakisphosphate (calcium Ins P 6). We previously reported that calcium Ins P 6 bound C1q, apparently initiating CCP activation. The present work dissects CCP activation on the LL. Most of the C1 binding activity in the LL corresponded to calcium Ins P 6 , and this binding was enhanced by partial proteolysis of the mucin meshwork. The remaining C1 binding activity was attributable to host antibodies, which included CCP-activating IgG isotypes. Calcium Ins P 6 made only a weak contribution to early CCP activation on the LL, suggesting inefficient C1 complex activation as reported for other polyanions. CCP activation on calcium Ins P 6 gave rise to a dominant population of C3b deposited onto calcium Ins P 6 itself that appeared to be quickly inactivated. Apparently as a result of inefficient initiation plus C3b inactivation, calcium Ins P 6 made no net contribution to C5 activation. We propose that the LL protects the underlying parasite cells from CCP activation through the combined effects of inefficient permeation of C1 through the mucins and C1 retention on calcium Ins P 6. This mechanism does not result in C5 activation, which is known to drive parasite-damaging inflammation. [ABSTRACT FROM AUTHOR]

The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O -glycans in which the core Gal p β1-3 residue can initiate a chain comprising one to three additional Gal p β1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Gal p α1-4 residue, and can be ramified with GlcNAc p β1-6 residues. These, as well as the GlcNAc p β1-6 residue in core 2, can be decorated with the Gal p α1-4Gal p β1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis , showing that the non-decorated cores, together with Gal p β1-3(Gal p α1-4Gal p β1-4GlcNAc p β1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Gal p β1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc β1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis . [ABSTRACT FROM AUTHOR]

Highlights: [•] Echinococcus larvae dwell in liver and expose to the host mostly carbohydrates. [•] We observe that these carbohydrates engage selectively the Kupffer cell receptor. [•] The Kupffer cell receptor is expressed only in liver macrophages of rodents. [•] Rodents are the likely ancestral hosts for Echinococcus. [•] Liver macrophages are known to contribute to tolerogenicity typical of this organ. [ABSTRACT FROM AUTHOR]

Abstract: Progress in the biology of myo-inositol hexakisphosphate (InsP 6) has been delayed by the lack of a quantitative description of its multiple interactions with divalent cations. Our recent initial description of these [J. Torres, S. Dominguez, M.F. Cerda, G. Obal, A. Mederos, R.F. Irvine, A. Diaz, C. Kremer, J. Inorg. Biochem. 99 (2005) 828–840] predicted that under cytosolic/nuclear conditions, protein-free soluble InsP 6 occurs as Mg5(H2L), a neutral complex that exists thanks to a significant, but undefined, window of solubility displayed by solid Mg5(H2L)·22H2O (L is fully deprotonated InsP 6). Here we complete the description of the InsP 6–Mg2+–Ca2+ system, defining the solubilities of the Mg2+ and Ca2+ (Ca5(H2L)·16H2O) solids in terms of K s0 =[M2+]5[H2L10−], with pK s0 =32.93 for M=Mg and pK s0 =39.3 for M=Ca. The concentration of soluble Mg5(H2L) at 37°C and I =0.15M NaClO4 is limited to 49μM, yet InsP 6 in mammalian cells may reach 100μM. Any cytosolic/nuclear InsP 6 in excess of 49μM must be protein- or membrane-bound, or as solid Mg5(H2L)·22H2O, and any extracellular InsP 6 (e.g. in plasma) is surely protein-bound. [Copyright &y& Elsevier]

Abstract: myo-Inositol hexakisphosphate (InsP 6) is an ubiquitous and abundant molecule in the cytosol and nucleus of eukaryotic cells whose biological functions are incompletely known. A major hurdle for studying the biology of InsP 6 has been a deficiency of a full understanding of the chemistry of its interaction with divalent and trivalent cations. This deficiency has limited our appreciation of how it remains in solution within cells, and the likely degree to which it might interact in vivo with physiologically important cations such as Ca2+ and Fe3+. We report here the initial part of the description of the InsP 6-multivalent cation chemistry, including its solution equilibria studied by high resolution potentiometry and (for the Fe(III)/Fe(II) couple) cyclic voltammetry. InsP 6 forms anionic complexes of high affinities and 1:1 stoichiometry with Mg(II), Ca(II), Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II). Of particular importance is the observation that, in the exceptional case of Mg(II), InsP 6 forms the species [Mg5(H2L)] (L representing fully deprotonated InsP 6); this soluble neutral species is predicted to be the predominant form of InsP 6 under nuclear or cytosolic conditions in animal cells. Contrary to previous suggestions, InsP 6 is predicted not to interact with cytosolic calcium even when calcium is increased during signalling events. In vitro, InsP 6 also forms high affinity 1:1 complexes with Fe(III) and Al(III). However, our data predict that in the biological context of excess free Mg(II), neither Fe(III) nor Fe(II) are complexed by InsP 6. [Copyright &y& Elsevier]