Contribution of Piezo2 to endothelium-dependent pain.

BackgroundWe evaluated the role of a mechanically-gated ion channel, Piezo2, in mechanical stimulation-induced enhancement of hyperalgesia produced by the pronociceptive vasoactive mediator endothelin-1, an innocuous mechanical stimulus-induced enhancement of hyperalgesia that is vascular endothelial cell dependent. We also evaluated its role in a preclinical model of a vascular endothelial cell dependent painful peripheral neuropathy.ResultsThe local administration of oligodeoxynucleotides antisense to Piezo2 mRNA, at the site of nociceptive testing in the rat's hind paw, but not intrathecally at the central terminal of the nociceptor, prevented innocuous stimulus-induced enhancement of hyperalgesia produced by endothelin-1 (100 ng). The mechanical hyperalgesia induced by oxaliplatin (2 mg/kg. i.v.), which was inhibited by impairing endothelial cell function, was similarly attenuated by local injection of the Piezo2 antisense. Polymerase chain reaction analysis demonstrated for the first time the presence of Piezo2 mRNA in endothelial cells.ConclusionsThese results support the hypothesis that Piezo2 is a mechano-transducer in the endothelial cell where it contributes to stimulus-dependent hyperalgesia, and a model of chemotherapy-induced painful peripheral neuropathy

towards chameleon detergents for 2-D gel electrophoresis

Caged non-ionic detergents, comprised of polar oligo(ethylene glycol) and non- polar alkyl chains joined by a photocleavable ortho-nitrobenzyl sulfonate linker have been synthesized and characterized. The light-triggered transformation of such chameleon surfactant from a charge-neutral into a charged form offers great potential to improve 2-D gel electrophoretic separation of complex protein mixtures

Expression of a novel versican variant in dorsal root ganglia from spared nerve injury rats

The size and modular structure of versican and its gene suggest the existence of multiple splice variants. We have identified, cloned, and sequenced a previously unknown exon located within the noncoding gene sequence downstream of exon 8. This exon, which we have named exon 8β, specifies two stop-codons. mRNAs of the versican gene with exon 8β are predicted to be constitutively degraded by nonsense-mediated RNA decay. Here, we tested the hypothesis that these transcripts become expressed in a model of neuropathic pain

Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production

Bogen C, Al-Dilaimi A, Albersmeier A, et al. Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production. BMC Genomics. 2013;14(1): 926.BACKGROUND: Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strainof the genus Monoraphidium (SAG 48.87) was investigated in this work as apotential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated. RESULTS: Monoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category "carbohydrate metabolic process" and in "fatty acid biosynthetic process" in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana, indicating a higher metabolic diversity for applications in carbohydrate conversions of biotechnological relevance. CONCLUSIONS: The genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the diversity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the development of this strain for biotechnological applications and production concepts

Identification of a versican V2-based splicevariant as an IB4-binding marker at the surface of non-peptidergic, nociceptive C-fibers

0\. Titelblatt und Inhaltsverzeichnis 1 1\. Einleitung 10 2\. Ergebnisse 20 3\. Diskussion 55 4\. Zusammenfassung 66 5\. Material und Methoden 68 6\. Materialien und Gerätenachweis 95 7\. Literaturverzeichnis 98 8\. Eigene Veröffentlichungen 104 9\. Curriculum vitae 106 10\. Danksagung 107Eine Subpopulation von Nozizeptoren, die so genannten nicht-peptidergen C-Fasern, präsentieren ein IB4-bindendes Glykokonjugat an ihrer Plasmamembran und können deshalb durch die Verwendung von Enzym- oder auch Fluorochrom- konjugiertem IB4 spezifisch markiert und von anderen Nozizeptoren abgegrenzt werden. Die molekulare Identität des IB4-bindenden Glykokonjugates war zu Begin der vorliegenden Arbeit nicht bekannt. Die Ausbildung einer peripheren Neuropathie im experimentellen Tiermodell ist nicht nur von einer Hypersensitivierung der durch den traumatisierten Nerv innervierten Regionen, sondern außerdem auch von einer dramatischen Abnahme der IB4-Reaktivität in den von der Läsion betroffenen Hinterwurzelganglien sowie der ipsilateralen Seite des Dorsalhorns im Rückenmark begleitet. Die mit der Ausbildung der Neuropathie als auch der Abnahme der IB4-Reaktivität zugrunde liegenden plastischen Veränderungen sind nicht wirklich bekannt. Es bestand deshalb die Hoffnung, über die Identifizierung des IB4-bindenden Glykokonjugates eine Idee von den der plastischen Veränderung zugrunde liegenden molekularen Mechanismen zu erhalten. Wir haben gezeigt, dass eine V2-basierte Variante des extrazellulären Matrixproteins Versican durch die IB4-bindenden Epitope modifiziert ist. Ergebnisse verschiedener Experimente lassen außerdem vermuten, dass diese Variante auch für die IB4-Reaktivität der nicht- peptidergen C-Fasern verantwortlich ist. Wir haben im Verlauf der vorliegenden Arbeit außerdem 3 weitere Exons im Versicangen der Ratte identifiziert. Zwei der drei Exons tragen allerdings keine proteincodierenden Informationen. Aus der Kombination der drei Exons mit den bisher bekannten Exons im Versicangen lässt sich die Existenz insgesamt sechs weiterer Splicevarianten postulieren. Vier der sechs neuen Varianten sind vermutlich rattenspezifisch, die weiteren zwei dürften auch in anderen Säugetieren zu finden sein.A subpopulation of nociceptors, the so-called non-peptidergic C-fibers, is presenting one IB4-binding glycoconjugate at their plasma membrane. For that reason, the non-peptidergic C-fibers can be specifically labelled and distinguished from other nociceptors by the use of enzyme or fluorescence conjugated IB4. At the beginning of the present thesis, the molecular nature of the IB4-binding glycoconjugate was not known. The genesis of peripheral neuropathic pain in experimental animal models is not only linked to hypersensitivity of the regions that are innervated by the lesioned nerve. It is also accompanied by a dramatic decrease of IB4-reactivity in the affected DRG and the ipsilateral side of the dorsal horn in the spinal cord. The plastic changes that are responsible for the neuropathy and the decrease of IB4-reactivity are not really known. One could expect that it might be possible to get an idea of the molecular mechanisms that are responsible for the plastic changes by the identification of the IB4-binding glycoconjugate. We were able to demonstrate that one V2-based splice variant of the extracellular matrix protein versican is modified by the IB4-binding epitopes. Furthermore, results from different experiments support the idea that this variant is indeed responsible for the IB4-reactivity of the non-peptidergic C-fibers. We also were able to identify three additional exons in the versican gene of the rat. Two of these three exons do not contain any protein coding sequence information. By combination of these three exons with the exons of versican that are currently known, one can postulate the existence of six additional splice variants. Four of them are presumably rat specific, the other two should also exist in other mammals

Interleukin 6 decreases nociceptor expression of the potassium channel KV1.4 in a rat model of hand-arm vibration syndrome.

Chronic muscle pain is a prominent symptom of the hand-arm vibration syndrome (HAVS), an occupational disease induced by exposure to vibrating power tools, but the underlying mechanism remains unknown. We evaluated the hypothesis that vibration induces an interleukin 6 (IL-6)-mediated downregulation of the potassium voltage-gated channel subfamily A member 4 (KV1.4) in nociceptors leading to muscle pain. Adult male rats were submitted to a protocol of mechanical vibration of the right hind limb. Twenty-four hours after vibration, muscle hyperalgesia was observed, concomitant to increased levels of IL-6 in the gastrocnemius muscle and decreased expression of KV1.4 in the dorsal root ganglia. Local injection of neutralizing antibodies against IL-6 attenuated the muscle hyperalgesia induced by vibration, whereas antisense knockdown of this channel in the dorsal root ganglia mimicked the muscle hyperalgesia observed in the model of HAVS. Finally, knockdown of the IL-6 receptor signaling subunit glycoprotein 130 (gp130) attenuated both vibration-induced muscle hyperalgesia and downregulation of KV1.4. These results support the hypothesis that IL-6 plays a central role in the induction of muscle pain in HAVS. This likely occurs through intracellular signaling downstream to the IL-6 receptor subunit gp130, which decreases the expression of KV1.4 in nociceptors

Role of nociceptor estrogen receptor GPR30 in a rat model of endometriosis pain

Endometriosis, the most common cause of chronic pelvic pain, is an estrogen-dependent disease in which classic estrogen receptors (ERα, ERβ) play an important role. Although recent evidence suggests that the novel G protein-coupled estrogen receptor (GPR30) also plays a key role in the progression of endometriosis, whether it is also involved in endometriosis pain is still unknown. Here we tested the hypothesis that GPR30 expressed by nociceptors contributes to endometriosis pain. Intramuscular injection of the GPR30 agonists raloxifene or 17β-estradiol produced a fast-onset, persistent, mechanical hyperalgesia at the site of the injection. Intrathecal antisense (AS) oligodeoxynucleotides (ODN), but not mismatch (MM) ODN, targeting mRNA for GPR30 markedly inhibited its protein expression in nociceptors and attenuated the mechanical hyperalgesia induced by local raloxifene or 17β-estradiol. Pretreatment with the GPR30 antagonist G-36 also inhibited the hyperalgesia induced by raloxifene or 17β-estradiol in naive control rats. Surgical implant of autologous uterine tissue onto the gastrocnemius muscle, which induces endometriosis-like lesions, produced local mechanical hyperalgesia. Intrathecal AS, but not MM, ODN targeting GPR30 mRNA reversibly inhibited the mechanical hyperalgesia at the site of endometriotic lesions. Finally, intralesional injection of the GPR30 antagonist G-36 also inhibited the mechanical hyperalgesia at the site of ectopic uterine tissue. We conclude that local GPR30 agonists produce persistent mechanical hyperalgesia in naive female rats, whereas local GPR30 antagonists inhibit mechanical hyperalgesia in a model of endometriosis pain. Thus, GPR30 expressed by nociceptors innervating ectopic uterine lesions might play a major role in endometriosis pain

Nociceptor Interleukin 33 Receptor/ST2 Signaling in Vibration-Induced Muscle Pain in the Rat

Occupational exposure to mechanical vibration can produce the hand-arm vibration syndrome (HAVS), whose most disabling symptom is persistent muscle pain. Unfortunately, the pathophysiology of HAVS pain is still poorly understood, precluding the development of mechanism-based therapies. Since interleukin 33 (IL-33) is essential for inflammation and recovery that follows skeletal muscle injury, we explored its role in muscle pain in a model of HAVS, in adult male rats. Concomitant to mechanical hyperalgesia, an increase in IL-33 in the ipsilateral gastrocnemius muscle was observed 24 hours after vibration. A similar hyperalgesia was produced by intramuscular injection of recombinant rat IL-33 (rrIL-33, 10-300 ng). Intrathecal administration of an oligodeoxynucleotide antisense to IL-33R/ST2 mRNA decreased the expression of ST2 in DRG and attenuated both rrIL-33 and vibration-induced mechanical hyperalgesia. Together these data support the suggestion that IL-33 plays a central role in vibration-induced muscle pain by action, at least in part, on skeletal muscle nociceptors. PERSPECTIVE: Our findings provide evidence of the contribution of IL-33, acting on its canonical receptor, in nociceptors, to muscle pain induced by ergonomic vibration. This suggests that targeting IL-33/ST2 signaling may be a useful strategy for the treatment of muscle pain in HAVS

Role of Nociceptor αCaMKII in Transition from Acute to Chronic Pain (Hyperalgesic Priming) in Male and Female Rats

We have previously shown that activation of protein kinase Cε (PKCε) in male rats induces a chronic, long-lasting change in nociceptors such that a subsequent exposure to proinflammatory mediators produces markedly prolonged mechanical hyperalgesia. This neuroplastic change, hyperalgesic priming, is dependent on activation of cytoplasmic polyadenylation element-binding protein (CPEB), downstream of PKCε, and consequent translation of mRNAs in the peripheral terminal of the nociceptor. Since α calmodulin-dependent protein kinase II (αCaMKII), a molecule implicated in neuroplasticity, is a target of CPEB and can also affect CPEB function, we investigated its role in the transition from acute to chronic pain. Priming induced by direct activation of PKCε can be prevented by inhibition of αCaMKII. In addition, direct activation of αCaMKII induces priming, which was not prevented by pretreatment with PKCε antisense, suggesting that αCaMKII is downstream of PKCε in the induction of priming. Activation of ryanodine receptors (RyRs), which can lead to activation of αCaMKII, also induced priming, in a calcium- and αCaMKII-dependent manner. Similarly, inhibition of the RyR and a calcium buffer prevented induction of priming by PKCε. Unlike activation of PKCε, ryanodine and αCaMKII induced priming in female as well as male rats. Our results demonstrate a contribution of αCaMKII to induction of hyperalgesic priming, a phenomenon implicated in the transition from acute to chronic pain

Role of Nociceptor CaMKII in Transition from Acute to Chronic Pain (Hyperalgesic Priming) in Male and Female Rats

We have previously shown that activation of protein kinase Cε (PKCε) in male rats induces a chronic, long-lasting change in nociceptors such that a subsequent exposure to proinflammatory mediators produces markedly prolonged mechanical hyperalgesia. This neuroplastic change, hyperalgesic priming, is dependent on activation of cytoplasmic polyadenylation element-binding protein (CPEB), downstream of PKCε, and consequent translation of mRNAs in the peripheral terminal of the nociceptor. Since α calmodulin-dependent protein kinase II (αCaMKII), a molecule implicated in neuroplasticity, is a target of CPEB and can also affect CPEB function, we investigated its role in the transition from acute to chronic pain. Priming induced by direct activation of PKCε can be prevented by inhibition of αCaMKII. In addition, direct activation of αCaMKII induces priming, which was not prevented by pretreatment with PKCε antisense, suggesting that αCaMKII is downstream of PKCε in the induction of priming. Activation of ryanodine receptors (RyRs), which can lead to activation of αCaMKII, also induced priming, in a calcium- and αCaMKII-dependent manner. Similarly, inhibition of the RyR and a calcium buffer prevented induction of priming by PKCε. Unlike activation of PKCε, ryanodine and αCaMKII induced priming in female as well as male rats. Our results demonstrate a contribution of αCaMKII to induction of hyperalgesic priming, a phenomenon implicated in the transition from acute to chronic pain