Search

Background and purpose Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT1 receptor and in the inactivation of the closely related orphan receptor GPR50. Experimental approach Chimeric MT1 -GPR50 receptors were generated and functionally analysed in terms of 2-[125 I]iodomelatonin binding, Gi /cAMP signalling and β-arrestin2 recruitment. We also used computational molecular dynamics (MD) simulations. Key results MD simulations of 300 ns revealed (i) the tight hairpin structure of the E2 loop of the MT1 receptor (ii) the most suitable features for melatonin binding in MT1 receptors and (iii) major predicted rearrangements upon MT1 receptor activation, stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions, because reciprocal replacement of MT1 and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT1 receptors and GPR50 respectively. Conclusions and implications Our work demonstrated the crucial role of the E2 loop for MT1 receptor and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT1 receptor conformations and by showing how evolutionary processes appear to have selected for modifications in the E2 loop in order to make GPR50 unresponsive to melatonin. Linked articles This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

The human thioredoxin (TRX)-interacting protein is found in multiple subcellular compartments and plays a major role in redox homeostasis, particularly in the context of metabolism (e.g., lipidemia and glycemia) and apoptosis. A molecular approach to the protein's modus operandi is still needed because some aspects of the TRX-interacting protein-mediated regulation of TRX are not clearly understood. To this end, His-tagged TRX-interacting proteins were over-expressed in Escherichia coli. Because the protein is expressed mainly in inclusion bodies, it was denatured in high concentrations of guanidium hydrochloride, centrifuged, and purified by Ni-NTA affinity chromatography. His-TRX-interacting protein was then refolded by dialysis and its restructuring monitored by circular dichroism spectrometry. This preparation resulted in the formation of a covalent complex with recombinant human TRX, demonstrating that association occurs without the intervention of other partner proteins. Multiple cysteine-to-serine mutants of TRX-interacting protein were produced and purified. These mutations were efficient in limiting the formation of disulfide-linked homo-oligomers in an oxidizing environment. The mutants were also used to gain functional insight into the formation of the TRX-interacting protein-TRX complexes. These complexes were able to form in the absence of internal disulfide bridges. A mutant with all but one cysteine changed to serine (Cys247) also showed an enhanced capacity to form complexes with TRX demonstrating, in a pure molecular system, that this particular cysteine is likely responsible for the disulfide bridge between TRX-interacting protein and TRX.

Background and purpose For many years, it was suspected that sheep expressed only one melatonin receptor (closely resembling MT(1) from other mammal species). Here we report the cloning of another melatonin receptor, MT(2), from sheep. Experimental approach Using a thermo-resistant reverse transcriptase and polymerase chain reaction primer set homologous to the bovine MT(2) mRNA sequence, we have cloned and characterized MT(2) receptors from sheep retina. Key results The ovine MT(2) receptor presents 96%, 72% and 67% identity with cattle, human and rat respectively. This MT(2) receptor stably expressed in CHO-K1 cells showed high-affinity 2[(125)I]-iodomelatonin binding (K(D)= 0.04 nM). The rank order of inhibition of 2[(125)I]-iodomelatonin binding by melatonin, 4-phenyl-2-propionamidotetralin and luzindole was similar to that exhibited by MT(2) receptors of other species (melatonin > 4-phenyl-2-propionamidotetralin > luzindole). However, its pharmacological profile was closer to that of rat, rather than human MT(2) receptors. Functionally, the ovine MT(2) receptors were coupled to G(i) proteins leading to inhibition of adenylyl cyclase, as the other melatonin receptors. In sheep brain, MT(2) mRNA was expressed in pars tuberalis, choroid plexus and retina, and moderately in mammillary bodies. Real-time polymerase chain reaction showed that in sheep pars tuberalis, premammillary hypothalamus and mammillary bodies, the temporal pattern of expression of MT(1) and MT(2) mRNA was not parallel in the three tissues. Conclusion and implications Co-expression of MT(1) and MT(2) receptors in all analysed sheep brain tissues suggests that MT(2) receptors may participate in melatonin regulation of seasonal anovulatory activity in ewes by modulating MT(1) receptor action.

Melatonin is a neurohormone implicated in both biorhythm synchronization and neuroprotection from oxidative stress. Its functions are mediated by two G-protein-coupled-receptors (MT1 and MT2) and MT3, which corresponds to quinone oxidoreductase 2 (QR2). To determine the binding site of QR2 for melatonin, point mutations of residues crucial for the enzymatic activity of hQR2 were performed. The substitution of the hydrophobic residues Phe126, Ile128 and Phe178 by tyrosines at the active site significantly increased enzymatic activity and decreased the affinity of a structural analog of melatonin, the 2[ 125 I]iodo-MCANAT. The mutation of residues implicated in zinc chelating (His 173 ; His 177 ) had no effect on radioligand binding. Destabilisation of the cofactor FAD by mutation N18E showed that 2[ 125 I]iodo-MCANAT binding was closely linked to the conformational integrity of human QR2. Surprisingly, the mutations C222F and N161A, which are distant from the determined binding site of the ligand, increased the affinity of 2[ 125 I]iodo-MCANAT for hQR2. What seems to better explain the binding variations among the mutants are the activity recorded with BNAH and coenzyme Q1. Various hypotheses are discussed based on the various parameters used in the study: nature of the substrates and co-substrates and nature of the amino acid changes. This study, which constitutes the first structural analysis of hQR2, should enable to better understand the biological role of melatonin on this enzyme and particularly, the discrepancies between the pharmacologies of the melatonin binding site (MT3) and the QR2 catalytic activity.

Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called alpha, beta, and gamma) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the alpha isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform gamma and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, K(m) and V(max) values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.

NRH:quinone oxidoreductase 2 (QR2) is a long forgotten oxidoreductive enzyme that metabolizes quinones and binds melatonin. We used the potency of the RNA interference (RNAi)-mediated gene silencing to build a cellular model in which the role of QR2 could be studied. Because standard approaches were poorly successful, we successively used: (1) two chemically synthesized fluorescent small interfering RNA (siRNA) duplexes designed and tested for their gene silencing capacity leading to a maximal 40% QR2 gene silencing 48 h post-transfection; (2) double transfection and cell-sorting of high fluorescent siRNA-transfected HT22 cells further enhancing QR2 RNAi silencing to 88%; (3) stable QR2 knock-down HT22 cell lines established with H1and U6 promoter driven QR2 short hairpin RNA (shRNA) encoding vectors, resulting in a 71–80% reduction of QR2 enzymatic activity in both QR2 shRNA HT22 cells. Finally, as a first step in the study of this cellular model, we observed a 42–48% reduction of menadione/BNAH-mediated toxicity in QR2 shRNA cells compared to the wild-type HT22 cells. Although becoming widespread and in some cases effective, siRNA-mediated cellular knock-down proves in the present work to be of marginal efficiency. Much development is required for this technique to be of general application.

Background and purpose: Ivabradine, a specific and use-dependent If inhibitor, exerts anti-ischaemic activity purely by reducing heart rate. The aim of this work was to characterize its effect on the predominant HCN channel isoform expressed in human sino-atrial nodes (hSAN), to determine its kinetics in HCN channels from multicellular preparations and rate-dependency of its action. Experimental approach: RT-PCR analysis of the four HCN channel isoforms was carried out on RNAs from hSAN. Patch-clamp and intracellular recordings were obtained from CHO cells stably expressing hHCN4 and isolated SAN, respectively. Beating rate of rat isolated atria was followed using a transducer. Key results: hHCN4 mRNAs were predominant in hSAN. Ivabradine induced a time-dependent inhibition of hHCN4 with an IC50 of 0.5 μM. In rabbit SAN, ivabradine progressively reduced the frequency of action potentials: by 10% after 3 h at 0.1 μM, by 14% after 2 h at 0.3 μM and by 17% after 1.5 h at 1 μM. After 3h, ivabradine reduced the beating rate of rat right atria with an IC30 of 0.2 μM. The onset of action of ivabradine was use-dependent rather than time-dependent with slower effects than caesium, an extracellular If blocker. Ivabradine 3 μM decreased the frequency of action potentials in SAN from guinea-pig, rabbit and pig by 33%, 21% and 15% at 40 min, respectively. Conclusions and implications: The use-dependent inhibition of hHCN4 current by ivabradine probably contributes to its slow developing effect in isolated SAN and right atria and to its increased effectiveness in species with rapid SAN activity. British Journal of Pharmacology (2007) 150, 37–46. doi:10.1038/sj.bjp.0706940

The catabolism of melatonin, whether naturally occurring or ingested, takes place via two pathways: approximately 70% can be accounted for by conjugation (sulpho- and glucurono-conjugation), and approximately 30% by oxidation. It is commonly thought that the interferon-induced enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42), which oxidizes tryptophan, is also responsible for the oxidation of 5-hydroxytryptamine (serotonin) and its derivative, melatonin. Using the recombinant enzyme expressed in Escherichia coli, we show in the present work that indoleamine 2,3-dioxygenase indeed cleaves tryptophan; however, under the same conditions, it is incapable of cleaving the two other indoleamines. By contrast, myeloperoxidase (EC 1.11.1.7) is capable of cleaving the indole moiety of melatonin. However, when using the peroxidase conditions of assay -- with H2O2 as co-substrate -- indoleamine 2,3-dioxygenase is able to cleave melatonin into its main metabolite, a kynurenine derivative. The present work establishes that the oxidative metabolism of melatonin is due, in the presence of H2O2, to the activities of both myeloperoxidase and indoleamine 2,3-dioxygenase (with lower potency), since both enzymes have Km values for melatonin in the micromolar range. Under these conditions, several indolic compounds can be cleaved by both enzymes, such as tryptamine and 5-hydroxytryptamine. Furthermore, melatonin metabolism results in a kynurenine derivative, the pharmacological action of which remains to be studied, and could amplify the mechanisms of action of melatonin.

α 1 -Adrenoceptors are G-protein-coupled receptors that bind catecholamines. Sixteen distinct human α 1A -adrenoceptor isoforms have been identified from human tissues, including five full-length and 11 truncated versions. An updated scheme for the identification of α 1A -adrenoceptor splice variants is proposed. Given the established roles of α 1 -adrenoceptors in benign prostatic hyperplasia, myocardial hypertrophy and other cardiovascular disorders, elucidation of the biological significance of the signaling diversity and potential pharmacological roles of α 1A -adrenoceptor splice variants are important areas of future research.

1. Endothelin-1 (ET-1) and tumor necrosis factor alpha (TNFalpha) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNFalpha induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNFalpha-induced iNOS expression and to determine whether or not ET-1 could influence TNFalpha-induced iNOS expression and NO production. 2. In differentiated 3T3-F442A, treatment with TNFalpha (20 ng ml(-1)) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription-polymerase chain reaction and Northern blot analysis. TNFalpha-induced iNOS expression requires nuclear factor kappaB activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38-MAP kinase pathways. 3. ET-1, but not ET-3, inhibited the TNFalpha-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA(2) 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. 4. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. 5. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNFalpha, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. 6. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFalpha-induced iNOS expression.

Chymase mediates a major alternative way of angiotensin II production from angiotensin I beside angiotensin converting enzyme in the final step of the renin-angiotensin system. This enzyme is also involved in other physio-pathological processes such as angiogenesis, atherosclerosis and inflammation. Several purification attempts of natural or recombinant chymase were reported in the literature. Most of these reports were not successful in obtaining the recombinant enzyme in a highly active form and in large quantity. In the present study, we describe a facile route for the purification of the human recombinant chymase. Chymase being produced as inactive prochymase, to be cathepsin C-activated, newly raised anti-chymase Ig were used to follow the purification. In order to complete the available tools for the search of chymase inhibitors, we developed and assessed a new 96-well plate based assay for the measurement of enzyme activity, as well as a low throughput, HPLC-based one. The assays used an original derivative of angiotensin I, or the native hormone. Chymase was produced in CHO cells and appropriately matured. The amount of enzyme obtained at the end of the process is compatible with the medium-throughput screening (up to 10,000 points per day), about 800 microg x L(-1) of culture medium with a specific activity of 6.16 mmol of angiotensin I cleaved per minute per mg of protein. All the biological and technical tools are now available for the discovery of new classes of chymase inhibitors.

We report the characterization by genomics-based approach of the human H4-receptor gene structure. The H4-receptor gene have been mapped by radiation hybrid experiments (Gene Bridge 4) on chromosome 18q11.2, between the AFMBB11WH5 and CHLC.GATA85D10 markers. The H4-receptor gene spans more than 21 kbp and contains three exons separated by two large introns (>7 kbp). RT-PCR analysis showed that the H4-receptor gene encoded a 3.7 kb mRNA which did not seem to be alternatively spliced within its coding region. The H4-receptor transcripts were found to be highly expressed in peripheral tissues implicated in inflammatory responses such as leukocytes, spleen, lung, and liver. In addition, low expression level of the H4-receptor mRNA was also detected in several human brain regions. Analysis of the 5'-flanking region of the H4-receptor gene did not reveal the existence of canonical TATA or CAAT-box. However, several putative regulatory elements mediating TNFalpha or IL-6-stimulated transcriptional activation were detected. The uteroglobin promoter binding factor, known to mediate anti-inflammatory response of uteroglobin, in the lung, was also found in this region. Thus, the description of the H4-receptor gene promoter region will facilitate the elucidation of its transcriptional control by factors secreted during inflammatory responses.

We have identified from human liver eight alpha(1A)-adrenoceptor (alpha(1A)-AR) splice variants that were also expressed in human heart, prostate and hippocampus. Three of these alpha(1A)-AR isoforms (alpha(1A-1)-AR, alpha(1A-2a)-AR and alpha(1A-3a)-AR) gave rise to receptors with seven transmembrane domains (7TMalpha(1A)-AR). The other five (alpha(1A-2b)-AR, alpha(1A-2c)-AR, alpha(1A-3c)-AR, alpha(1A-5)-AR and alpha(1A-6)-AR) led to truncated receptors lacking transmembrane domain VII (6TMalpha(1A)-AR). The 7TMalpha(1A)-AR isoforms transiently expressed in COS-7 cells bound [(3)H]prazosin with high affinity (K(d) 0.2 nM) and mediated a noradrenaline (norepinephrine)-induced increase in cytoplasmic free Ca(2+) concentration, whereas the 6TMalpha(1A)-AR isoforms were incapable of ligand binding and signal transduction. Immunocytochemical studies with N-terminal epitope-tagged alpha(1A)-AR isoforms showed that the 7TMalpha(1A)-AR isoforms were present both at the cell surface and in intracellular compartments, whereas the 6TMalpha(1A)-AR isoforms were exclusively localized within the cell. Interestingly, in co-transfected cells, each truncated alpha(1A)-AR isoform inhibited [(3)H]prazosin binding and cell-surface trafficking of the co-expressed 'original' 7TMalpha(1A-1)-AR. However, there was no modification of either the [(3)H]prazosin-binding affinity or the pharmacological properties of alpha(1A-1)-AR. Immunoblotting experiments revealed that co-expression of the alpha(1A-1)-AR with 6TMalpha(1A)-AR isoforms did not impair alpha(1A-1)-AR expression. Therefore the expression in human tissues of many truncated isoforms constitutes a new regulation pathway of biological properties of alpha(1A)-AR.

The main melatonin receptors are two G-protein coupled receptors named MT(1) and MT(2). Having described the molecular pharmacology of the human versions of these receptors, we turned to two of the three species most useful in studying melatonin physiology: rat and sheep (a diurnal species used to understand the relationship between circadian rhythm and depression). We also employed previously used compounds to describe the mouse melatonin receptors; despite the early cloning of mouse receptors, few molecular pharmacology studies on these receptors exist. To our surprise, we detected no major differences between the data obtained from mice and those from other species.

International audience; c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS. (Journal of Biomolecular Screening 2010:956-967) Key w ords: meganuclease, homologous gene targeting, protein expression, cell-based assay, high-throughput screening

The L-DOPA decarboxylase mRNA levels were determined by a sensitive S1 nuclease method in four organs and one tumor of adult rat. S1 mapping analysis, with probes corresponding to the mRNA coding region, whowed that this region is conserved in all L-DOPA decarboxylase mRNA of neuronal and non-neuronal tissues. The mRNA was not very abundant; its representation varies approximately from 0.00035% of total RNA in the mid brain to 0.013% of total mRNA in the pheochromocytoma. A strong correlation between mRNA level and enzyme amount was observed (correlation coefficient = 0.99). The results indicate that the level of mRNA is a primary factor determining the L-DOPA decarboxylase level.

Autotaxin is a member of the phosphodiesterase family of enzymes, (NPP2). It is an important secreted protein found in conditioned medium from adipocytes. It also has a putative role in the metastatic process. Based on these observation, further validation of this potential target was necessary, apart from the classical biochemical ones. The construction of a knock out mouse strain for ATX was started. In this paper, we report the generation of a mouse line displaying an inactivated ATX gene product. The KO line was designed in order to generate a functional inactivation of the protein. In this respect, the threonine residue T210 was replaced by an alanine (T210A) leading to a catalytically inactive enzyme. If the experimental work was straight forward, we disappointedly discovered at the final stage that the breeding of heterozygous animals, ATX −/+, led to the generation of a Mendelian repartition of wild-type and heterozygous, but no homozygous were found, strongly suggesting that the ATX deletion is lethal at an early stage of the development. This was confirmed by statistical analysis. Although other reported the same lethality for attempted ATX−/− mice generation [van Meeteren, L.A., Ruurs, P., Stortelers, C., Bouwman, P., van Rooijen, M.A., Pradère, J.P., Pettit, T.R., Wakelam, M.J.O., Saulnier-Blache, J.S., Mummery, C.L., Moolenar, W.H. and Jonkers, J. (2006) Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development, Mol. Cell. Biol. 26, 5015–5022; Tanaka, M., Okudaira, S., Kishi, Y., Ohkawa, R., Isei, S., Ota, M., Noji, S., Yatomi, Y., Aoki, J., and Arai, H. (2006) Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid, J. Biol. Chem. 281, 25822–25830], they used more drastic multiple exon deletions in the ATX gene, while we chose a single point mutation. To our knowledge, the present work is the first showing such a lethality in any gene after a point mutation in an enzyme catalytic site.

Stable expression of G protein coupled receptors in cell lines is a crucial tool for the characterization of the molecular pharmacology of receptors and the screening for new antagonists. However, in some instances, many difficulties have been encountered to obtain stable cell lines expressing functional receptors. Here, we addressed the question of vector optimization to establish cell lines expressing the human neuropeptide Y receptor 5 (NPY5-R) or histamine receptor 4 (HH4R). We have compared bicistronic vectors containing viral or cellular internal ribosome entry sites (IRES), co-expressing the receptor and the neomycine resistance gene from a single mRNA, to a bigenic vector containing two distinct promoters upstream each different genes. This study is the first one to validate the use of three cellular IRESs for long-term transgene expression. Our results demonstrate for both NPY5-R and HH4R that the bicistronic vectors with EMCV, VEGF, FGF1A or FGF2 IRES provide clones expressing functional receptors with yields between 25% and 100%. In contrast, the bigenic vector provided no functional clones, related to a low expression of NPY5R mRNA. The cell lines expressing active receptor were stable after more than 50 passages. These data indicate that IRES-based bicistronic vectors are particularly appropriate to establish cell clones expressing active G-coupled protein receptors with a high yield. In the case of NPY5, it was a new way to produce such a stable cell line. Furthermore, the characteristics-presented herein-of this receptor pharmacological property are perfectly in line with those reported in the literature.

The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt(1) and MT(2), the existence of a novel putative melatonin receptor, MT(3), was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt(1) and MT(2). In this report, we present the purification of MT(3) from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR(2), EC ). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT(3) to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR(2). After transfection of CHO-K1 cells with the human QR(2) gene, not only did the QR(2) enzymatic activity appear, but also the melatonin-binding sites with MT(3) characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT(3) binding and QR(2) enzymatic activity obtained from samples of Syrian hamster kidney or QR(2)-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT(3) as the quinone reductase QR(2) and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.

A cDNA clone for dopa decarboxylase (EC 4.1.1.28) has been isolated from a rat pheochromocytoma cDNA library and the cDNA sequence has been determined. It corresponds to an mRNA of 2094 nucleotides. The length of the mRNA was measured by primer-extension of rat pheochromocytoma RNA and the 5' end of the sequence of the mRNA was confirmed by the PCR. A probe spanning the translation initiation site of the mRNA was used to hybridize with mRNAs from various organs of the rat. S1 nuclease digestion of the mRNAs annealed with this probe revealed two classes of mRNAs. The comparison of the cDNA sequence and published sequences for rat liver, human pheochromocytoma, and Drosophila dopa decarboxylase supported the conclusion that two mRNAs are produced: one is specific for tissue of neuronal origin and the other is specific for tissues of nonneuronal (mesodermal or endodermal) origin. The neuronal mRNA contains a 5' untranslated sequence that is highly conserved between human and rat pheochromocytoma including a GA stretch. The coding sequence and the 3' untranslated sequence of mRNAs from rat liver and pheochromocytoma are identical. The rat mRNA differs only in the 5' untranslated region. Thus a unique gene codes for dopa decarboxylase and this gene gives rise to at least two transcripts presumably in response to different signals during development.

International audience; Total poly (A +) RNA has been extracted from rat pheochromocytoma and translated in vitro by means of a reticulocyte lysate system. We show that two antisera, prepared against pig kidney DOPA decarboxylase (DDC) or rat pheochromocytoma DDC, immunoprecipitate an in vitro synthetized 50 kDa polypeptide identified as DDC by competition experiments with pure DDC. The proportion of specific mRNA has been calculated and represents 0.05% of total poly A + mRNA. Its size has been established by electrophoresis in methylmercuric hydroxide containing agarose gel, corresponding to a 2.2 kb length mRNA.

Zhang et al . (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein–coupled receptor GPR39. However, we found that I 125 -obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.

The discovery of novel drugs for neurodegenerative diseases has been a real challenge over the last decades. The development of patient- and/or disease-specificin vitromodels represents a powerful strategy for the development and validation of lead candidates in preclinical settings. The implementation of a reliable platform modeling dopaminergic neurons will be an asset in the study of dopamine-associated pathologies such as Parkinson’s disease. Disease models based on cell reprogramming strategies, using either human-induced pluripotent stem cells or transcription factor-mediated transdifferentiation, are among the most investigated strategies. However, multipotent adult stem cells remain of high interest to devise direct conversion protocols and establishin vitromodels that could bypass certain limitations associated with reprogramming strategies. Here, we report the development of a six-step chemically defined protocol that drives the transdifferentiation of human nasal olfactory stem cells into dopaminergic neurons. Morphological changes were progressively accompanied by modifications matching transcript and protein dopaminergic signatures such as LIM homeobox transcription factor 1 alpha (LMX1A), LMX1B, and tyrosine hydroxylase (TH) expression, within 42 days of differentiation. Phenotypic changes were confirmed by the production of dopamine from differentiated neurons. This new strategy paves the way to develop more disease-relevant models by establishing reprogramming-free patient-specific dopaminergic cell models for drug screening and/or target validation for neurodegenerative diseases.

Nitric oxide (NO) is a potent vasodilator, but it can also modulate contractile responses of the airway smooth muscle. Whether or not endothelial (e) NO synthase (NOS) contributes to the regulation of bronchial tone is unknown at present. Experiments were designed to investigate the isoforms of NOS that are expressed in murine airways and to determine whether or not the endogenous release of NO modulates bronchial tone in wild-type mice and in mice with targeted deletion of eNOS [eNOS(−/−)]. The presence of neuronal NOS (nNOS), inducible NOS (iNOS), and eNOS in murine trachea and lung parenchyma was assessed by RT-PCR, immunoblotting, and immunohistochemistry. Airway resistance was measured in conscious unrestrained mice by means of a whole body plethysmography chamber. The three isoforms of NOS were constitutively present in lungs of wild-type mice, whereas only iNOS and nNOS were present in eNOS(−/−) mice. Labeling of nNOS was localized in submucosal airway nerves but was not consistently detected, and iNOS immunoreactivity was observed in tracheal and bronchiolar epithelial cells, whereas eNOS was expressed in endothelial cells. In wild-type mice, treatment with N-nitro-l-arginine methyl ester, but not with aminoguanidine, potentiated the increase in airway resistance produced by inhalation of methacholine. eNOS(−/−) mice were hyperresponsive to inhaled methacholine and markedly less sensitive to N-nitro-l-arginine methyl ester. These results demonstrate that the three NOS isoforms are expressed constitutively in murine lung and that NO derived from eNOS plays a physiological role in controlling bronchial airway reactivity.