Luc-Henri Tessier

Structure and expression of sunflower ubiquitin genes

Two ubiquitin genes, designatedUbB1 andUbB2, were isolated from a sunflower genomic library. They encode polyubiquitin transcripts corresponding to six repeats of the monomer. Northern blot analysis identified several different transcript size classes: bothUbB1 andUbB2 transcripts are found in the most abundant 1.6 kb class. In contrast to the previously isolatedUbF transcript which is present at high levels in flowers,UbB1 andUbB2 are expressed constitutively at low levels in different tissues. The levels of the two transcripts increase after heat stress. The two genes exhibit strong homology suggesting that they may result from duplication and conversion. Surprisingly,UbB1 gene shows structural similarities with the chicken ubiquitin heat shock gene, in particular the presence of an intron located just in front of the first ATG.

β2-adrenoceptors are critical for antidepressant treatment of neuropathic pain†

Tricyclic antidepressants (TCAs) are one of the first‐line pharmacological treatments against neuropathic pain. TCAs increase the extracellular concentrations of noradrenaline and serotonin by blocking the reuptake transporters of these amines. However, the precise downstream mechanism leading to the therapeutic action remains identified. In this work, we evaluated the role of adrenergic receptors (ARs) in the action of TCAs.

Delta-opioid receptors are critical for tricyclic antidepressant treatment of neuropathic allodynia.

BACKGROUNDnThe therapeutic effect of antidepressant drugs against depression usually necessitates a chronic treatment. A large body of clinical evidence indicates that antidepressant drugs can also be highly effective against chronic neuropathic pain. However, the mechanism by which these drugs alleviate pain is still unclear.nnnMETHODSnWe used a murine model of neuropathic pain induced by sciatic nerve constriction to study the antiallodynic properties of a chronic treatment with the tricyclic antidepressants nortriptyline and amitriptyline. Using knockout and pharmacological approaches in mice, we determined the influence of delta-opioid receptors in the therapeutic action of chronic antidepressant treatment.nnnRESULTSnIn our model, a chronic treatment by tricyclic antidepressant drugs totally suppresses the mechanical allodynia in neuropathic C57Bl/6J mice. This therapeutic effect can be acutely reversed by an injection of the delta-opioid receptor antagonist naltrindole. Moreover, the antiallodynic property of antidepressant treatment is absent in mice deficient for the delta-opioid receptor gene.nnnCONCLUSIONSnThe antiallodynic effect of chronic antidepressant treatment is mediated by a recruitment of the endogenous opioid system acting through delta-opioid receptors.

Β2-adrenoceptors are essential for desipramine, venlafaxine or reboxetine action in neuropathic pain

Neuropathic pain is a disease caused by a lesion or dysfunction of the nervous system. Antidepressants or anticonvulsants are presently the best available treatments. The mechanism by which antidepressants relieve neuropathic pain remains poorly understood. Using pharmacological and transgenic approaches in mice, we evaluated adrenergic receptor (AR) implication in the action of the tricyclic antidepressant desipramine, the noradrenaline and serotonin reuptake inhibitor venlafaxine, and the noradrenaline reuptake inhibitor reboxetine. Neuropathy was induced by cuff insertion around the sciatic nerve. We showed that chronic antidepressant treatment suppressed cuff-induced allodynia in wild-type mice but not in beta(2)-AR deficient mice, and/or that this antiallodynic action was blocked by intraperitoneal or intrathecal injection of the beta(2)-AR antagonist ICI 118,551 but not by the alpha(2)-AR antagonist yohimbine. We also showed that the anticonvulsant gabapentin was still effective in beta(2)-AR deficient mice. Our results demonstrate that beta(2)-ARs are essential for the antiallodynic action of antidepressant drugs.

Analysis of a sunflower polyubiquitin promoter by transient expression

Abstract We have recently isolated two sunflower polyubiquitin genes (UbB1 and UbB2) which are induced by heat-stress and show a highly conserved region of about 250 bp upstream of the transcription initiation site. The promoter activity of this region has been analyzed by transient expression using the β-glucoronidase (GUS) gene as reporter in tobacco protoplasts. The GUS expression in absence of any external inducer is five times higher than that observed under the control of the CaMV 35S promoter. Analysis of different deletions within the 250-bp region showed that a 65-bp sequence is responsible for most of the promoter activity in tobacco protoplasts. This activity is intron-independent and is not enhanced when protoplasts are heat-stressed. Sequence homologies strongly suggest that UbB1 plays a major role in the stress response in sunflower.

Chronic treatment with agonists of β2-adrenergic receptors in neuropathic pain

Expression of beta(2)-adrenoceptors (beta(2)-ARs) within the nociceptive system suggested their potential implication in nociception and pain. Recently, we demonstrated that these receptors are essential for neuropathic pain treatment by antidepressant drugs. The aim of the present study was to investigate whether the stimulation of beta(2)-ARs could in fact be adequate to alleviate neuropathic allodynia. Neuropathy was induced in mice by sciatic nerve cuffing. We demonstrate that chronic but not acute stimulation of beta(2)-ARs with agonists such as clenbuterol, formoterol, metaproterenol and procaterol suppressed neuropathic allodynia. By using a pharmacological approach with the beta(2)-AR antagonist ICI 118,551 or a transgenic approach with mice deficient for beta(2)-ARs, we confirmed that the antiallodynic effect of these agonists was specifically related to their action on beta(2)-ARs. We also showed that chronic treatment with the beta(1)-AR agonist xamoterol or with the beta(3)-AR agonist BRL 37344 had no effect on neuropathic allodynia. Chronic stimulation of beta(2)-ARs, but not beta(1)- or beta(3)-ARs, by specific agonists is thus able to alleviate neuropathic allodynia. This action of beta(2)-AR agonists might implicate the endogenous opioid system; indeed chronic clenbuterol effect can be acutely blocked by the delta-opioid receptor antagonist naltrindole. Present results show that beta(2)-ARs are not only essential for the antiallodynic action of antidepressant drugs on sustained neuropathic pain, but also that the stimulation of these receptors is sufficient to relieve neuropathic allodynia in a murine model. Our data suggest that beta(2)-AR agonists may potentially offer an alternative therapy to antidepressant drugs for the chronic treatment of neuropathic pain.

Glia-induced neuronal differentiation by transcriptional regulation

There is increasing evidence that different phases of brain development depend on neuron–glia interactions including postnatal key events like synaptogenesis. To address how glial cells influence synapse development, we analyzed whether and how glia‐derived factors affect gene expression in primary cultures of immunoisolated rat retinal ganglion cells (RGCs) by oligonucleotide microarrays. Our results show that the transcript pattern matched the developmental stage and characteristic properties of RGCs in vitro. Glia‐conditioned medium (GCM) and cholesterol up‐ and downregulated a limited number of genes that influence the development of dendrites and synapses and regulate cholesterol and fatty acid metabolism. The oligonucleotide microarrays detected the transcriptional regulation of neuronal cholesterol homeostasis in response to GCM and cholesterol treatment. Surprisingly, our study revealed neuronal expression and glial regulation of matrix gla protein (Mgp). Together, our results suggest that glial cells promote different aspects of neuronal differentiation by regulating transcription of distinct classes of genes.

Antidepressants suppress neuropathic pain by a peripheral β2-adrenoceptor mediated anti-TNFα mechanism.

Neuropathic pain is pain arising as a direct consequence of a lesion or disease affecting the somatosensory system. It is usually chronic and challenging to treat. Some antidepressants are first-line pharmacological treatments for neuropathic pain. The noradrenaline that is recruited by the action of the antidepressants on reuptake transporters has been proposed to act through β2-adrenoceptors (β2-ARs) to lead to the observed therapeutic effect. However, the complex downstream mechanism mediating this action remained to be identified. In this study, we demonstrate in a mouse model of neuropathic pain that an antidepressants effect on neuropathic allodynia involves the peripheral nervous system and the inhibition of cytokine tumor necrosis factor α (TNFα) production. The antiallodynic action of nortriptyline is indeed lost after peripheral sympathectomy, but not after lesion of central descending noradrenergic pathways. More particularly, we report that antidepressant-recruited noradrenaline acts, within dorsal root ganglia, on β2-ARs expressed by non-neuronal satellite cells. This stimulation of β2-ARs decreases the neuropathy-induced production of membrane-bound TNFα, resulting in relief of neuropathic allodynia. This indirect anti-TNFα action was observed with the tricyclic antidepressant nortriptyline, the selective serotonin and noradrenaline reuptake inhibitor venlafaxine and the β2-AR agonist terbutaline. Our data revealed an original therapeutic mechanism that may open novel research avenues for the management of painful peripheral neuropathies.

Isolation of flower-specific cDNA clones from sunflower

Abstract Proteins from flower, leaves and roots of sunflower (Helianthus annuus L.) have been resolved by two-dimensional polyacrylamide gel electrophoresis into distinctive patterns. A careful examination of these patterns reveals that many proteins are present in at least two of the three organs, but thateach of these organs has, in addition, a set of specific proteins. Following this observation we have used differential hybridization screening to isolate flower-specific cDNA clones from a sunflower floral cDNA library. Out of 31 clones originally isolated, 12 were subcloned and analyzed by Southern and Northern hybridization experiments. Four clones (SF1, SF2, SF6 and SF7) were shown to cross-hybridize, indicating that they carry the same (or a closely related) insert. Cross-hybridization was also found between clones SF9 and SF19. Using Northern hybridization with RNAs isolated from the individual floral parts we identified an anther-specific clone (SF18), two shown to hybridize to RNA from pistils, from anthers and, to a lesser extent, from corollae.

Structure and expression of Euglena gracilis nuclear rbcS genes encoding the small subunits of the ribulose 1,5-bisphoshate carboxylase/oxygenase: A novel splicing process for unusual intervening sequences?

In the protist Euglena gracilis, the small subunit of the chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is encoded by nuclear rbcS genes and synthesized as a polyprotein precursor containing eight mature small subunit molecules. This large precursor is encoded by at least eight different nuclear genes as ascertained by transcript analysis. The structure of three rbcS genes was established and the coding sequences were found to be interrupted by many intervening sequences (IVS). Apart from the first 5 intron involved in trans-splicing, none of these IVSs obeys the GT-AG rule characteristic of introns in higher eukaryote genes. Surprisingly, these IVSs are located at identical positions within the three genes studied. Moreover, extensive sequence homologies were found between IVSs located in the same gene as well as in different genes. The sequences of these homologous IVSs differ only by inserted and/or deleted sequences. The striking conservation of the 5 and 3 regions of these IVSs is correlated to their potential secondary structures. These structures, which bring the IVS extremities together with the exon boundaries, could be involved in a novel splicing process. The second 5 IVS is shown to be excised before the addition of the spliced leader sequence to the pre-mRNA. Similarly, two 3 IVSs are excised before the polyadenylation step. These results suggest that IVS splicing is faster than eukaryotic genomic cis-splicing and involves components other than those of the classical spliceosomes.