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1.
Magali A. Théveniau John R. Raymond Geneviève N. Rougon 《The Journal of membrane biology》1989,111(2):141-153
Summary We developed site-directed rabbit antisera against synthetic peptides selected from the deduced amino acid sequence of the hamster lung 2-adrenergic receptor (amino acids 16–31 and 174–189, respectively). All antisera directed against peptide 1 (four of four rabbits) as well as two antisera directed against peptide 2 (two of four rabbits) recognized the purified 2-adrenergic receptor in immunoblot conditions when used at a dilution of 1500. Antisera directed against peptide 1 as well as peptide 2 were able to immunoprecipitate iodinated as well as125I-cyanopindolol tabeled 2-adrenergic receptor. This last result implies that the recognized epitopes do not contain the125I-cyanopindolol binding domain of the 2-adrenergic receptor. Immunoblot experiments performed on membrane fractions from hamster lung tissue showed that immunoreactive bands at 64,000, 57,000, 47,000, 44,000 and 38,000 daltons were specifically detected. When purified 2-adrenergic receptor was iodinated and submitted to glycolytic and/or tryptic treatments, species with similar molecular weights could be recovered. Then, the immunoreactive bands probably correspond to native 2-adrenergic receptor and to degradative or nonglycosylated species of this molecule. The antisera were also able to detect immunoreactive molecules in murine and human cell lines, suggesting conservation of the probed sequences between these species. Enzymatic linked immunosorbent assay tests on intact cells and immunofluorescence studies confirmed that the amino-terminus and putative first extracellular loop are extracellularly located. Immunofluorescence studies on mouse brain primary cultures showed that cells expressing 2-adrenergic receptor-like molecules exhibited a neuronal phenotype. 相似文献
2.
In the present study, the bombesin-induced changes in cytosolic free Ca2+ ([Ca2+]i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca2+ transients. The average frequency of the Ca2+ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min−1 (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca2+ transient followed by a sustained plateau or by a decrease to basal levels. In Ca2+- free medium, bombesin caused only one or two Ca2+ transients and withdrawal of extracellular Ca2+ abolished the Ca2+ transients. The voltage-dependent Ca2+ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca2+ transients and stopped the Ca2+ transients in some cells. Thapsigargin caused a sustained rise in [Ca2+]i) in the presence of extracellular Ca2+ while in its absence the rise in [Ca2+]i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca2+], by about 50%. Depletion of intracellular Ca2+ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca2+-free medium caused an agonist-independent increase in [Ca2+]i) when extracellular Ca2+ was restored, which was larger than in control cells that had been incubated in Ca2+-free medium for the same period of time. This rise in [Ca2+]i and the thapsigargin-induced increase in [Ca2+]i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca2+ pool enhances Ca2+ influx through VDCC and voltage-independent Ca2+ channels (VICC). In conclusion, the bombesin-induced Ca2+ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca2+ transients. Intracellular Ca2+ is mobilized during each Ca2+ transient, but Ca2+ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca2+ response. Ca2+ influx in whole or part is activated by a capacitative Ca2+ entry mechanism. 相似文献
3.
Magali Basille Bruno J. Gonzalez Laurence Desrues †Myriam Demas ‡Alain Fournier Hubert Vaudry 《Journal of neurochemistry》1995,65(3):1318-1324
Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED50 = 0.12 ± 0.01 and 0.23 ± 0.07 n M , respectively), whereas vasoactive intestinal polypeptide (VIP) was ∼100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED50 = 19.1 ± 6.3 and 13.4 ± 6.0 n M , respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts. 相似文献
4.
The role of proline residues in the folding of the trypsin inhibitor derivative RCAM(14–38) has been studied by testing for slow-folding species of the unfolded protein, which could result from the introduction of wrong proline isomers after unfolding. The unfolded protein at 25 °C contains chiefly fast-folding (UF) molecules: they refold with a time constant of 40 milliseconds at pH 6.8 in 1.9 m-guanidinium chloride. At least one minor slow-folding (Us) species has been found, using fluorescence to monitor refolding. The reaction in which this Us species is formed after unfolding shows the properties expected for the cis: Irans isomerization of a proline residue. When refolding is monitored by tyrosine absorbance, two minor slow reactions are found. The faster reaction is in the same time range (15 s at 25 °C) as that studied by fluorescence, and the slower reaction is quite slow (200 s at 25 °C). It is not known whether the slower reaction results from a second Us species. There are four trans proline residues in bovine pancreatic trypsin inhibitor: the proportion of slow-folding molecules (not more than 25% at 25 °C) is smaller than expected if every proline residue can produce a Us species and if the cis to trans ratio of each residue after unfolding is at least 0.1:0.9.Criteria based on folding kinetics are given for classifying the types of folding reaction shown by unfolded molecules containing a single wrong proline isomer. Levitt (1980) has classified three types of proline residues according to the energy difference (small, intermediate or large) between the native protein and the predicted minimum energy structure containing a wrong proline isomer. He suggests that these three types of proline residues can be recognized by the types of folding reactions they produce. Only type II (intermediate) folding reactions have thus far been characterized by the criteria introduced here. We point out that the type of folding reaction depends also on the folding conditions, and a possible explanation for this effect is given. 相似文献
5.
H Jüppner G Brabant U Kapteina M Kirschner H Klein R D Hesch 《Biochemical and biophysical research communications》1986,139(3):1215-1223
A direct radioimmunoassay for the rapid and accurate detection of human ANP from unextracted plasma is described. The sensitivity was approximately 50 pg/ml, respectively 2.5 pg/tube, the intra-assay variation 4%, and the inter-assay variation less than 12%. Rat ANP (1-28, 5-25, 5-27 and 5-28), oxydized and reduced hANP as well as plasma samples from various patients run in parallel to the 1-28 hANP standard curve. These findings imply, that the antibody primarily recognizes the mid-region (amino acids 6-25) of the intact ANP, that the C-terminal portion further increases the immunoreactivity, and that circulating plasma hANP is reliably measured. Plasma hANP ranged from 50-166 pg/ml (mean +/- SD: 98.3 +/- 44.6) in healthy individuals, there was no significant difference between samples were drawn in upright or lying position, the apparent half-life of injected hANP was 5.65 minutes. Patients with liver cirrhosis revealed significantly higher hANP levels of 244.5 +/- 173.5 pg/ml. Patients with various forms of cardiac disease had hANP concentrations ranging from 50 to 1744 pg/ml, depending at least partially on the right atrial pressure. No difference was observed if the samples were drawn from either right or left intracardial locations. Our findings with this system demonstrate that hANP is reliably measured even without prior extraction. 相似文献
6.
Magali Soumillon Anamaria Necsulea Manuela Weier David Brawand Xiaolan Zhang Hongcang Gu Pauline Barthès Maria Kokkinaki Serge Nef Andreas Gnirke Martin Dym Bernard de Massy Tarjei S. Mikkelsen Henrik Kaessmann 《Cell reports》2013,3(6):2179-2190
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7.
Olfaction plays an indispensable role in human and animals in self and environmental recognition, as well as intra‐ and interspecific communication. Following the discovery of a family of olfactory receptors (ORs) by Buck and Axel in 1991, it has been established that the sense of smell begins with the molecular recognition of a chemical odorant by one or more ORs expressed in the olfactory sensory neurons. Therefore, characterization of the molecular interactions between odorant molecules and ORs is a key step in the elucidation of the general properties of the olfactory system and in the development of applications, i.e., design of new odorants, search for blockers, etc. The process putted in place at ChemCom to improve the expression of ORs at the cytoplasmic membrane of the HEK293 cell and assays enabling large‐scale deorphanization, and to characterize the interaction between chemical odorants and ORs is described. The family of human ORs includes ca. 400 putatively functional ORs which are GPCRs (G protein‐coupled receptors); to date over 100 human ORs have been deorphanized. 相似文献
8.
Magali Ferrero Frdrique Spyratos Andre Desplaces Catherine Andrieu Edelmira Phillips Jacques Rouëss 《Biology of the cell / under the auspices of the European Cell Biology Organization》1989,65(3):305-308
Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors. 相似文献
9.
Emond S André I Jaziri K Potocki-Véronèse G Mondon P Bouayadi K Kharrat H Monsan P Remaud-Simeon M 《Protein science : a publication of the Protein Society》2008,17(6):967-976
Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C. 相似文献
10.