Nociceptin/orphanin FQ inhibits electrically induced contractions of the human bronchus via NOP receptor activation

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit neurogenic contractions in various tissues, including guinea pig airways. In the present study, we investigated the ability of N/OFQ to affect cholinergic contractions of human bronchi elicited by electrical field stimulation (EFS). Tissues were obtained from 23 patients undergoing surgery for lung cancer. EFS (20 Hz, 320 mA, 1.5 ms, 10 s) was applied five times every 20 min. Contractions induced by EFS were abolished by either TTX (1 microM) or atropine (1 microM) and concentration-dependently (10 nM-1 microM) inhibited by N/OFQ (Emax, 11.5+/-1.8% inhibition). The inhibitory effects of N/OFQ were mimicked by the N/OFQ receptor (NOP) ligand [Arg14, Lys15]N/OFQ which displayed however, higher significant maximal effects (17.7+/-2.9% inhibition, P<0.05). The actions of N/OFQ and [Arg14, Lys15]N/OFQ were not affected by naloxone (1 microM) while prevented by the selective NOP receptor antagonist UFP-101 (10 microM). Moreover, the inhibitory effects of NOP agonists were no longer evident in tissues treated with tertiapin (10 microM), an inhibitor of inward-rectifier potassium channels. In conclusion, the present data demonstrate that N/OFQ inhibited acetylcholine (ACh) release in the human bronchi via NOP receptor activation. This effect may involve stimulation of potassium currents.     

Molecular and functional characterisation of glutamate transporters in rat cortical astrocytes exposed to a defined combination of growth factors during in vitro differentiation

In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues and several methods have been reported for promoting their maturation into differentiated astrocytes. Hence, a combination of several nutriments/growth factors -- the G5 supplement (insulin, transferrin, selenite, biotin, hydrocortisone, fibroblast growth factor and epidermal growth factor) -- is widely used as a culture additive favouring the growth, differentiation and maturation of primary cultured astrocytes. Considering the key role played by glial cells in the clearance of glutamate in the synapses, cultured astrocytes are frequently used as a model for the study of glutamate transporters. Indeed, it has been shown that when tested separately, growth factors influence the expression and activity of the GLAST and GLT-1. The present study aimed at characterising the functional expression of these transporters during the time course of differentiation of cultured cortical astrocytes exposed to the supplement G5. After a few days, the vast majority of cells exposed to this supplement adopted a typical stellate morphology (fibrous or type II astrocytes) and showed intense expression of the glial fibrillary acidic protein. Both RT-PCR and immunoblotting studies revealed that the expression of both GLAST and GLT-1 rapidly increased in these cells. While this was correlated with a significant increase in specific uptake of radiolabelled aspartate, fluorescence monitoring of the Na+ influx associated with glutamate transporters activity revealed that the exposure to the G5 supplement considerably increased the percentage of cells participating in the uptake. Biochemical and pharmacological studies revealed that this activity did not involve GLT-1 but most likely reflected an increase in GLAST-mediated uptake. Together, these data indicate that the addition of this classical combination of growth factors and nutriments drives the rapid differentiation toward a homogenous culture of fibrous astrocytes expressing functional glutamate transporters.     

Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception

Analgesic therapies are still limited and sometimes poorly effective, therefore finding new targets for the development of innovative drugs is urgently needed. In order to validate the potential utility of blocking T-type calcium channels to reduce nociception, we explored the effects of intrathecally administered oligodeoxynucleotide antisenses, specific to the recently identified T-type calcium channel family (CaV3.1, CaV3.2, and CaV3.3), on reactions to noxious stimuli in healthy and mononeuropathic rats. Our results demonstrate that the antisense targeting CaV3.2 induced a knockdown of the CaV3.2 mRNA and protein expression as well as a large reduction of 'CaV3.2-like' T-type currents in nociceptive dorsal root ganglion neurons. Concomitantly, the antisense treatment resulted in major antinociceptive, anti-hyperalgesic, and anti-allodynic effects, suggesting that CaV3.2 plays a major pronociceptive role in acute and chronic pain states. Taken together, the results provide direct evidence linking CaV3.2 T-type channels to pain perception and suggest that CaV3.2 may offer a specific molecular target for the treatment of pain.     

Bovine herpesvirus 1 VP22 enhances the efficacy of a DNA vaccine in cattle

For this study, the intercellular trafficking ability of bovine herpesvirus 1 (BHV-1) VP22 was applied to improve the efficacy of a DNA vaccine in calves. A plasmid encoding a truncated version of glycoprotein D (tgD) fused to VP22 was constructed. The plasmid encoding tgD-VP22 elicited significantly enhanced and more balanced immune responses than those induced by a plasmid encoding tgD. Furthermore, protection against a BHV-1 challenge was obtained in calves immunized with the plasmid encoding tgD-VP22, as shown by significant reductions in viral excretion. However, less significant protection was observed for animals vaccinated with the tgD-expressing plasmid, correlating with the lower level of immunity observed prechallenge. This is the first report of the use of VP22 as a transport molecule in the context of a DNA vaccine for a large animal species.     

Optimized N-phenyl-N'-(2-chloroethyl)ureas as potential antineoplastic agents: synthesis and growth inhibition activity

In our ongoing research program aimed at the optimization of microtubule-self-assembly disrupting agents, we have prepared three series of phenylurea analogues (CEU), derived from N-(3-ω-hydroxyalkyl or 4-ω-hydroxyalkyl or 3-ω-hydroxyalkynyl)-phenyl-N′-(2-chloroethyl)ureas. Most compounds exhibit potent growth inhibitory activity on human colon carcinoma HT-29, human skin melanoma M21, and human breast carcinoma MCF-7 tumor cell lines, with a GI₅₀ ranging from 250 nM to 8 μM. Among these new molecules, three CEUs exhibit GI₅₀ in the nanomolar range. They are more potent by approximately an order of magnitude than previously described CEU analogues. As such, they are attractive hit compounds for the development of potent new alkylating antitubulin drugs.     

Adipose tissue is a regulated source of interleukin-10

White adipose tissue (WAT) is the source of pro- and anti-inflammatory cytokines and we have recently shown that this tissue is a major source of the anti-inflammatory interleukin (IL)-1 receptor antagonist (IL-1Ra). We now aimed at identifying additional adipose-derived cytokines, which might serve as regulators of IL-1Ra. We demonstrate here for the first time that the antiinflammatory cytokine IL-10 is secreted by human WAT explants and that it is up-regulated by LPS and TNF-alpha in vitro, as well as in obesity in humans (2- and 6-fold increase in subcutaneous and visceral WAT, respectively) and rodents (4-fold increase).     

Effects of interleukin-15 on antifungal responses of human polymorphonuclear leukocytes against Fusarium spp. and Scedosporium spp

Fusarium spp. and Scedosporium spp. have emerged as important fungal pathogens that are frequently resistant to antifungal compounds. We investigated the effects of human interleukin-15 (IL-15) on human polymorphonuclear leukocyte (PMNL) activity against Fusarium solani and Fusarium oxysporum as well as Scedosporium prolificans and Scedosporium apiospermum. IL-15 (100 ng/ml) significantly enhanced PMNL-induced hyphal damage of both Fusarium spp. and S. prolificans after incubation for 22 h (P < 0.01) but not S. apiospermum. In addition, IL-15 enhanced PMNL oxidative respiratory burst evaluated as superoxide anion production in response to S. prolificans but not to the other fungi after 2 h incubation. IL-15 increased interleukin-8 (IL-8) release from PMNLs challenged with hyphae of F. solani and S. prolificans (P< or = 0.04). Release of tumor necrosis factor-alpha was not affected. The species-dependent enhancement of hyphal damage and induction of IL-8 release suggest that IL-15 plays an important role in the immunomodulation of host response to these emerging fungal pathogens.     

New DNA data from a transthyretin nuclear intron suggest an Oligocene to Miocene diversification of living South America opossums (Marsupialia: Didelphidae)

Phylogenetic relationships of 19 species of didelphid marsupials were studied using two nuclear markers, the non-coding transthyretin intron 1 (TTR) and the coding interphotoreceptor retinoid binding protein exon 1 (IRBP), and two mitochondrial genes, the protein-coding cytochrome b (cyt-b) and the structural 12S ribosomal DNA (12S rDNA). Evolutionary dynamics of these four markers were compared to each other, revealing the appropriate properties presented by TTR intron 1 together with its well supported and resolved phylogenetic signal. Nuclear markers supported the monophyly of medium and large-sized opossums Metachirus+(Chironectes, Lutreolina, Didelphis, Philander), and the paraphyly of mouse-sized opossums, with the genera Gracilinanus, Thylamys, and Marmosops as a sister group to medium and large-sized didelphids. Conflicting branching patterns between mitochondrial and nuclear data involved the phylogenetic position of Marmosa-Micoureus-Monodelphis relative to other mouse-sized opossums. Nuclear phylogenetic inferences among genera were confirmed by the presence of synapomorphic indels observed in TTR intron 1. A Bayesian relaxed molecular clock dating of didelphid evolution using nuclear markers estimated their origin in the Middle Eocene (39.8 million years ago), with subsequent diversification during the Oligocene (Deseadan) and Miocene.