Ligand-specific dose-response of heterologously expressed olfactory receptors.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.     

Transcription of a zebrafish gene of the hairy-Enhancer of split family delineates the midbrain anlage in the neural plate

her5 encodes a basic helix-loop-helix (bHLH) protein with all features characteristic of the Drosophila hairy-E(spl) family. her5 is expressed in a band of cells within the neural anlage from about 90% epiboly on to at least 36 h postfertilization (hpf). After completion of brain morphogenesis, her5-expressing cells are located in the caudal region of the midbrain, at the boundary with the rhombencephalon. Labelling of cells within the her5 expression domain in the neural plate by injection of fluorescein-dextran allows their labelled progeny to be localized in the 36-hpf-old embryo using an anti-fluorescein antibody. This shows that the her5 expression domain corresponds to the midbrain primordium, including both the tectum and the tegmentum, in the neural plate. A possible function for her5 in regionalization of the brain and/or control of the midbrain-hindbrain boundary is discussed.     

In vitro peptidergic neurons from the adult locust pars intercerebralis: Morphological and immunocytological studies

Primary cell cultures were prepared from a major neurosecretory center of the adult locust brain, the pars intercerebralis, in order to characterize neurosecretory cells growingin vitro. Individual pars intercerebralis could be removed free of surrounding tissue and dissociated by mechanical treatment. Mature neurosecretory neurons of different sizes regenerate new neurites during the initial three daysin vitro in serum-free medium. They show a tendency to sprout one primary neurite from which fine processes develop. By means of electron microscopy, we observed the integrity of the cellular organelles, indicating that cultured neurons are healthy, and we were able to distinguish three types of neurosecretory neurons on the basis of the ultrastructural aspects of the neurosecretory material. These three types have the same ultrastructural characteristics asin situ neuroparsin, ovary maturing parsin and locust insulin related peptide neurons. Immunogold labelling at the electron microscopic level, using the two available specific antibodies, anti-neuroparsin and anti-ovary maturing parsin, confirms the morphological characterization of neuroparsin and ovary maturing parsin cells. These results show for the first time that cultured locust neurosecretory neurons behave like thosein vivo, in terms of their ultrastructure and immunocytochemistry. Moreover, the presence of recently-formed neurosecretory material both in the Golgi zone of the perikaryon and in the neuronal processes indicates that cultured neurons have functional capacity since they are able to synthesizede novo and to transport the neurosecretory material along the neurite. Thus our well-characterized culture system provides a suitable invitro model to investigate the secretory mechanism of locust neurosecretory neurons.     

Cysteine Racemization in Peptide Synthesis: A New and Easy Detection Method

A new method has been developed for the rapid determination of D-cysteine contents in synthetic peptides. It is based on the reduction of cystine residues, when present, with tris- alkylphosphines, selective derivatization of the cysteine residues with 4-vinylpyridine, followed by acid hydrolysis of the (4-pyridylethyl)cysteine –peptides. Baseline enantiomeric resolution of theD ,L -S-β-(4-pyridylethyl)cysteine, and thus quantification ofD - enantiomer contents at levels ≤1%, is easily achieved by capillary zone electrophoresis exploiting the host–guest complexation principle with crown ethers or by gas chromatography on chiral glass capillary columns upon conventional derivatization of the hydrolysate. The acid-stability of the (4-pyridylethyl)cysteine derivative prevents racemization via thiazoline intermediates and allows for standardization of the acid hydrolysis-dependent racemization.     

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.     

Hypoxia Increases the Susceptibility to Oxidant Stress and the Permeability of the Blood-Brain Barrier Endothelial Cell Monolayer

Abstract: Using a cell culture model of the blood-brain barrier (BBB), we investigated the brain capillary endothelial cell (EC) response to hypoxia. The activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase and the GSH level of brain capillary ECs alone or in coculture with astrocytes, as well as those of pericytes, were compared with those obtained with freshly isolated microvessels. These results demonstrated that brain capillary ECs cocultured with astrocytes and used in the presence of a coculture-conditioned medium provided a relevant in vitro model for studying the effect of hypoxia-reoxygenation at the BBB level. The effect of hypoxia on antioxidant enzymes, GSH, and ATP levels was studied, as well as the modification of the permeability to small weight molecules. A decrease in all enzymes and the GSH level could explain an increase in the susceptibility of the brain capillary ECs to further oxidant injury. Second, profound rearrangements of F-actin filaments of the ECs and a decrease in the ATP level could be associated with an increase in the permeability of the monolayer. Furthermore, an apoptotic process was detected by in situ end labeling of DNA. These results indicate that hypoxia distorts the function of ECs and that these cells in culture provide a valuable tool for exploring mechanisms after hypoxia-reoxygenation.     

Easel, a gypsy LTR-retrotransposon in the Salmonidae

Despite the close similarities between retroviruses and the gypsy/Ty3 group of LTR-retrotransposons their host ranges are largely distinct: the retroviruses are found only in vertebrates, whereas the gypsy LTR-retrotransposons are almost exclusively restricted to invertebrates, plants and fungi. Here we report the amplification by PCR, and characterisation, of one of the first LTR-retrotransposons to be discovered in vertebrates - in several members of the piscine family Salmonidae. Phylogenetic analysis of this retroelement, termed easel, indicates that it is probably a phylogeneticaly basal member of the gypsy group of LTR-retrotransposons and occurs in some of the same species from which retroviruses have previously been isolated. Thus some members of the Salmonidae are the first organisms known to harbour both retroviral branch elements and the gypsy LTR-retrotransposon branch elements. This creates an overlap in the host ranges of the two retroelement families.     

Decrease of polypeptides in the PS I antenna complex with increasing growth irradiance in the red alga Porphyridium cruentum

Thylakoids isolated from cells of the red alga Porphyridium cruentum exhibit an increased PS I activity on a chlorophyll basis with increasing growth irradiance, even though the stoichiometry of Photosystems I and II in such cells shows little change (Cunningham et al. (1989) Plant Physiol 91: 1179–1187). PS I activity was 26% greater in thylakoids of cells acclimated at 280 mol photons · m^–2 · s^–1 (VHL) than in cells acclimated at 10 mol photons · m^–2 · s^–1 (LL), indicating a change in the light absorbance capacity of PS I. Upon isolating PS I holocomplexes from VHL cells it was found that they contained 132±9 Chl/P700 while those obtained from LL cells had 165±4 Chl/P700. Examination of the polypeptide composition of PS I holocomplexes on SDS-PAGE showed a notable decrease of three polypeptides (19.5, 21.0 and 22 kDa) in VHL-complexes relative to LL-complexes. These polypeptides belong to a novel LHC I complex, recently discovered in red algae (Wolfe et al. (1994a) Nature 367: 566–568), that lacks Chl b and includes at least six different polypeptides. We suggest that the decrease in PS I Chl antenna size observed with increasing irradiance is attributable to changes occurring in the LHC I-antenna complex. Evidence for a Chl-binding antenna complex associated with PS II core complexes is lacking at this point. LHC II-type polypeptides were not observed in functionally active PS II preparations (Wolfe et al. (1994b) Biochimica Biophysica Acta 1188: 357–366), nor did we detect polypeptides that showed immunocross-reactivity with LHC II specific antisera (made to Chlamydomonas and Euglena LHC II).Abbreviations Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - DCPIP 2,6-dichlorophenol indophenol - -dm dodecyl--d-maltoside - HL high light of 150 mol photons · m^–2 · s^–1 - LGB lower green band - LHC I light-harvesting complex of PS I - LHC II light-harvesting complex of PS II - LL low light of 10 mol photons · m^–2 · s^–1 - ML medium light of 50 mol photons · m^–2 · s^–1 - MES 2-(N-morpholino) ethanesulfonic acid - P700 reaction center of PS I - PFD photon flux density - Trizma tris(hydroxymethyl)aminomethane - UGB upper green band - VHL very high light of 280 mol photons · m^–2 · s^–1     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.