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  • 1
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We have modelled the conformation of the III–IV loop of the Cav2.1 subunit of P/Q calcium channels, a loop that is implicated in fast voltage-dependent inactivation. Change in channel inactivation requires its direct interaction with the I–II loop. This interaction occurs with an affinity in the order of 70 nm. Intracellular injection of a 40-mer III–IV loop-derived peptide produces an increase in the rate of fast inactivation. This alteration in channel kinetic is also accompanied by a hyperpolarizing shift in the steady-state voltage-dependence of inactivation. None of these effects are observed in the presence of a β subunit, suggesting the existence of a competitive mechanism of action between the β subunit and the III–IV loop. Amino acid sequence comparison using BLAST reveals that the III–IV loop shares 53% identity with the γ subunit of G proteins. Because of the pivotal contribution of the III–IV loop to inactivation, an atomic model of the III–IV loop was generated by both homology modelling and molecular mechanics calculations. Using the X-ray structures of the βγ dimer of the heterotrimeric G-proteins as templates, the III–IV loop is predicted to contain a well-structured α-helix at the amino-terminus with both the N- and C-termini having the same orientation in the plane of the inner lipid bilayer. We provide a hypothetical working model in which we propose that the III–IV loop interacts with the I–II loop via its Gβγ binding domain.
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  • 2
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The serotonin transporter (5-HTT) is the target of most antidepressant drugs, whose therapeutic action is related to their facilitatory influence on 5-HT neurotransmission. In this study, we investigated the functional adaptive properties of 5-HT1A autoreceptors, which regulate serotonergic neuronal firing, in knockout mice deficient in 5-HTT. Neurons of the dorsal raphe nucleus (DRN) were recorded extracellularly under chloral hydrate anaesthesia in male and female knockout 5-HTT mice and their wild-type counterparts. The inhibitory response of DRN neurons to intravenous injection of the 5-HT1A agonist 8-OH-DPAT was dramatically reduced in knockout 5-HTT compared with wild-type mice, especially in females. Changes in 8-OH-DPAT-induced hypothermia and autoradiographic labelling of 5-HT1A sites in the DRN confirmed a greater level of desensitization/down-regulation of 5-HT1A autoreceptors in female than in male knockout 5-HTT mice. After gonadectomy, the functional status of 5-HT1A autoreceptors was unchanged in wild-type mice, whereas in knockout 5-HTT, castrated males exhibited a down-regulation, and ovariectomized females an up-regulation of these receptors, as shown by electrophysiological recording and autoradiographic labelling in the DRN, as well as by changes in 8-OH-DPAT-induced hypothermia. Finally, in gonadectomized knockout 5-HTT mice, treatment with testosterone or estradiol restored the DRN neuronal firing sensitivity to 8-OH-DPAT back to sham control level in males or females, respectively. These data indicate that sexual hormones participate in the mechanisms responsible for the desensitization of 5-HT1A autoreceptors in knockout 5-HTT mice. The differential effects of testosterone and estradiol on 5-HT1A-mediated control of 5-HT neurotransmission might be related to the well-established gender differences in the vulnerability to depression.
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  • 3
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Serotonin (5-HT) plays an important role in the regulation of the time-keeping system in rodents. In the present study, we have investigated the interplay between the rhythms of 5-HT synthesis and release in the suprachiasmatic nuclei (SCN) of the rat. The quantitative distribution of tryptophan hydroxylase (TpH) protein was used as an index of 5-HT synthesis, in perikarya and terminals areas. In the raphe medianus, the maximal levels of TpH was reached in the early daytime period, followed by a decrease before the onset of darkness. Conversely, in the axon terminals of the SCN the highest levels of TpH were found before the onset of the dark-period. Furthermore, TpH amount in SCN displays variations depending on the anatomical area of the SCN. Extracellular 5-HT peaked at the beginning of the night, as evidenced by in vivo microdialysis in the SCN. The 5-HT metabolite, 5-HIAA, presented a similar pattern, but the acrophase occurred in the middle of the dark period. These results suggest that TpH is transported from the soma to the nerve terminals in which 5-HT is synthesized during daytime. This would fill the intracellular stores of 5-HT to provide for its nocturnal release.
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  • 4
    ISSN: 1546-170X
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Azodicarbonamide was recently identified as a new anti-HIV agent that targets the zinc finger domains of the HIV-1 NCp7 nucleocapsid protein. Here, we demonstrate that azodicarbonamide inhibits in a dose-dependent manner the responses of purified human CD4+ T lymphocytes stimulated either by ...
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Examination of 55 clinical isolates of uropathogenic Escherichia coli producing the CNF1 toxin demonstrated that the cnf1 gene is systematically associated with a hly operon via a highly conserved hlyD-cnf1 intergenic region (igs, 943 bp) as shown in the J96 UPEC strain. We examined if this association could reflect a co-regulation of the production of these toxins. Translation of cnf1 from an immediately upstream promoter has been shown to be controlled by means of an anti-Shine–Dalgarno sequence present in the cnf1 coding sequence [fold-back inhibition (cnf1 fbi)]. The cnf1 fbi was not regulated by elements present in the igs. An RNA covering the full hlyD sequence, the igs and extending on the cnf1 gene, was then detected in the J96 strain. This RNA could be part of a HlyCABD mRNA. Transcription of the haemolysin operon requires RfaH antitermination activity. Inactivation of rfaH in J96 resulted in a 100-fold reduction of the CNF1 content of bacteria. The production of CNF1 from a plasmidic igscnf1 DNA was not sensitive to RfaH, indicating that this factor acted on cnf1 transcription via the hly promoter. This way the cnf1 fbi mechanism might be overcome by transcription of cnf1 from the haemolysin promoter and antitermination by RfaH. This constitutes a novel system of bacterial virulence factors co-regulation.
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  • 6
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Surface expression level of voltage-dependent calcium channels is tightly controlled in neurons to avoid the resulting cell toxicity generally associated with excessive calcium entry. Cell surface expression of high voltage-activated calcium channels requires the association of the pore-forming subunit, Cavα, with the auxiliary subunit, Cavβ. In the absence of this auxiliary subunit, Cavα is retained in the endoplasmic reticulum (ER) through mechanisms that are still poorly understood. Here, we have investigated, by a quantitative method based on the use of CD8α chimeras, the molecular determinants of Cavα2.1 that are responsible for the retention, in the absence of auxiliary subunits, of P/Q calcium channels in the ER (referred to here as ‘ER retention’). This study demonstrates that the I–II loop of Cavα2.1 contains multiple ER-retention determinants beside the β subunit association domain. In addition, the I–II loop is not the sole domain of calcium channel retention as two regions identified for their ability to interact with the I–II loop, the N- and C-termini of Cavα2.1, also produce ER retention. It is also not an obligatory determinant as, similarly to low-threshold calcium channels, the I–II loop of Cavα1.1 does not produce ER retention in COS7 cells. The data presented here suggests that ER retention is suppressed by sequential molecular events that include: (i) a correct folding of Cavα in order to mask several internal ER-retention determinants and (ii) the association of other proteins, including the Cavβ subunit, to suppress the remaining ER-retention determinants.
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  • 7
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Glucocorticoid hormones exert strong influences on central neurotransmitter systems. In the present work, we examined the functional consequences of corticosterone suppression on the dopaminergic transmission in the dorsolateral striatum by studying the expression of Fos-like proteins and extracellular dopamine levels. Glucocorticoid hormones were suppressed by adrenalectomy, and the specificity of the effects assessed by restoring physiological plasmatic corticosterone concentrations. We show that, in the dorsolateral striatum, glucocorticoids modify postsynaptic dopaminergic transmission. Suppression of glucocorticoids decreased the induction of Fos proteins in response to a direct agonist of dopamine D1 receptors (SKF 82958, 1.5 mg/kg, i.p.), but not the release of dopamine induced by morphine (2 mg/kg, s.c.) or the density of the limiting enzyme of dopamine synthesis, tyrosine hydroxylase. In contrast to the dopaminergic response to morphine, the response to cocaine (15 mg/kg, i.p.) was modified by the suppression of corticosterone. In this case, adrenalectomy increased cocaine-induced changes in extracellular dopamine but did not modify the expression of Fos-like proteins. This absence of changes in cocaine-induced Fos-like proteins might result from a compensatory mechanism between the increase in the dopaminergic response and the decrease in the functional activity of dopamine D1 receptors. The increased dopaminergic response to cocaine also contrasts with the decreased response previously observed in the shell of the nucleus accumbens [Barrot et al. (2000) Eur. J. Neurosci., 12, 973–979]. The present data highlight the profound heterogeneous influence of glucocorticoids within dopaminergic projections.
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  • 8
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamine neurotransmitters, is expressed in a restricted number of areas, and subject to numerous regulations during development and in adulthood. Two transcription factor binding sites present in the proximal region of the TH gene, the TPA-responsive element (TRE) and the c-AMP responsive element (CRE), have been shown to play important roles in TH gene regulation in vitro. In order to elucidate in vivo the role of these two sites, we produced transgenic mice bearing a 5.3-kb fragment from the 5′ flanking sequence of the TH gene with mutations in either the CRE- or TRE-sites. Using the intact 5.3-kb fragment fused to two different reporter genes (HSV1-tk and lacZ), we show that this promoter fragment is able to specifically direct expression in catecholaminergic tissues both in adult mice and embryos. Interestingly, the CRE- and TRE-mutated transgenes were not expressed in adult mice, contrary to the situation in embryos where they were specifically expressed in catecholaminergic regions. These results demonstrate that the CRE and TRE play an essential role in basal TH expression in adult tissues in vivo. Moreover, they suggest that distinct transcription factors are involved in TH regulation in developing and adult tissues. In support of this, gel mobility shift experiments revealed a complex present only in embryonic tissues. Taken together, these data highlight the diversity of the mechanisms underlying the establishment and maintenance of the catecholaminergic phenotype.
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  • 9
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: We have investigated the contribution of the Cavβ subunits to the process of inactivation dependent of the I–II loop of Cavα2.1. Two amino acid residues located in the alpha1 interaction domain (AID) of the I–II loop of Cavα2.1 (Arg387 and Glu388) have been directly implicated in voltage-dependent inactivation of this channel. Various point mutations of these residues disrupt the interaction between the I–II loop and the III–IV loop, and thereby modify the inactivation properties of the channel by accelerating its kinetics and shifting the steady-state inactivation curve towards hyperpolarized potentials. A similar disruption is produced by Cavβ4 subunit association with the I–II loop. Moreover, in the presence of Cavβ4 subunit, introducing negatively charged residues at positions 387 or 388 slows inactivation kinetics down, whereas introducing positive charges has the opposite effect. The shift of the steady-state inactivation curve is also amino acid charge-dependent. In contrast, mutation of Arg387 or Glu388 does not alter the differential regulation of the different Cavβ isoforms on inactivation. These results suggest that the expression of Cavβ4 alters the contribution of charged residues at positions 387 and 388 to inactivation. We discuss these results with regard to the actual hypotheses on the mechanisms of calcium channel inactivation. We introduce the working concept that Cavβ-subunits produce a conformational repositioning of charged AID residues within the electric field.
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  • 10
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs) are emerging as important modulators of brain physiopathology. Dramatic changes in the expression of MMPs and TIMPs occur during excitotoxic/neuroinflammatory processes. However, only the measurement of net protease activity is relevant physiologically, and the functional consequences of MMP/TIMP ratio modifications in the brain remain elusive. In order to assess MMP activity and effects in brain tissue, we combined in vivo and organotypic culture models of kainate (KA)-induced excitotoxicity to provoke selective neuronal death and neuroinflammation in the hippocampus. Using in situ zymography, we show that KA-induced excitotoxic seizures in rats increase net MMP activity in hippocampal neurons 8 h after seizures, before their death, and that this increase is neuronal activity-dependent. Three days after KA, proteolytic activity increases in blood vessels and reactive glial cells of vulnerable areas, in relation with neuroinflammation. At 7 and 15 days, proteolysis remains high in blood vessels whereas it is reduced in glia. In organotypic hippocampal cultures, which lack blood cell-mediated inflammation and extrinsic connections, a broad-spectrum inhibitor of MMPs (MMPI), but also a selective MMP-9 inhibitor, protect hippocampal neurons against KA-induced excitotoxicity. Moreover, recombinant MMP-9, but not MMP-2, induces selective pyramidal cell death in these cultures and KA-induced neuronal activity exacerbates the neuronal death promoting effects of MMP-9. These data strongly implicate MMPs, and MMP-9 in particular, in both excitotoxic neuronal damage and subsequent neuroinflammatory processes, and suggest that selective MMPIs could be therapeutically relevant in related neurological disorders.
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