Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.     

Recombination between similar but not identical DNA sequences during yeast transformation occurs within short stretches of identity.

Interactions between similar but not identical (homeologous) DNA sequences play an important biological role in the evolution of genes and genomes. To gain insight into the underlying molecular mechanism(s) of genetic recombination, we have studied inter- and intramolecular homeologous recombination in S. cerevisiae during transformation. We found that homeologous DNAs recombine efficiently. Hybrid sequences were obtained between two mammalian cytochrome P450 cDNAs, sharing 73% identity, and between the yeast ARG4 gene and its human homeologous cDNA, sharing 52% identity. Sequencing data showed that the preferred recombination events are those corresponding to the overall alignment of the DNA sequences and that the junctions are within stretches of identity of variable length (2-21 nt). We suggest that these events occur by a conventional homologous recombination mechanism.     

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.     

Low-serum medium development for human diploid fibroblast microcarrier cultures

A new medium supplement mixture, PPRF92, has been developed to enable the serial subculture of human diploid fibroblasts (MRC-5 cells) on microcarriers. Furthermore, the PPRF92 supplements enable cell growth at serum levels as low as 1%. Through an optimization programme, the PPRF92 supplements have evolved into a simple mixture with the concentrations of key components at a level that makes the overall cost very competitive with medium containing 10% foetal bovine serum (FBS). Furthermore, the PPRF92 supplement mixture is most efficacious when FBS is replaced with the cheaper, and more widely available, adult bovine serum (ABS). Although medium exchange with serum is necessary in order to achieve confluence on microcarriers, the PPRF92 mixture is only necessary at the initiation of each passage. Using the medium replinishment protocol that has been developed in our laboratory, MRC-5 cells were successfully serially passaged through 13 bead-to-bead transfers on microcarriers in DMEM/F12 medium enriched with the PPRF92 supplement mixture reported here, and 1% ABS.Correspondence to: L. A. Behie     

Dermenkephalin and deltorphin I reveal similarities within ligand-binding domains of mu- and delta-opioid receptors and an additional address subsite on the delta-receptor.

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to the mu- and delta-opioid receptors, respectively. The amino-terminal domains Tyr-D-X-Phe (where X is either Ala or Met) of these peptides behave as selective and potent mu-receptor ligands. Routing of Tyr-D-X-Phe to the delta- or the mu- receptor is associated with the presence or the absence at the C-terminus of an additional hydrophobic and negatively charged tetrapeptide by-passing the mu-addressing ability of the amino-terminal moiety. A study of 20 Tyr-D-X-Phe-Y-NH2 analogs with substitution of X and Y by neutral, hydrophobic, aromatic amino acids as well as by charged amino acid residues shows that tetrapeptides maintain high binding affinity and selectivity for the mu-opioid receptor. Although residue in position 4 serves a delta-address function, the tripeptide motif at the C-terminus of dermenkephalin and deltorphin I are critical components for high selectivity at delta-opioid receptor. Results demonstrate that mu- and delta-opioid receptors share topologically equivalent ligand-binding domains, or ligand-binding sequences similarities, that recognized Tyr-D-X-Phe as a consensus message-binding sequence. The delta-receptor additionally contains a unique address subsite at or near the conserved binding domain that accommodates the C-terminal tetrapeptide motif of dermenkephalin and deltorphin I.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad.

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied. 2. CA secretion was induced by ACh or nicotine, but not by muscarine. 3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response. 4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine. 5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M2, which may play a regulatory function.     

Comparative value of four arcelin variants in the development of dry bean lines resistant to the Mexican bean weevil

Wild Phaseolus vulgaris L. accessions containing arcelin codominant alleles 1 through 5 were reconfirmed and characterized for resistance to the Mexican bean weevil, Zabrotes subfasciatus (Boheman) (Coleoptera: Bruchidae). Accession G 02771 (arcelin 5) had the highest level of antibiosis resistance, followed by G 12952 (arcelin 4), G 12882 (arcelin 1) and G 12866 (arcelin 2). Arcelin 3 accessions conferred the lowest levels of resistance. As the presence of arcelin is inherited as a single dominant gene, a backcross breeding program has been used to transfer resistance to the Mexican bean weevil from wild beans to bean cultivars using serological techniques to detect the presence of arcelin and replicated insect feeding tests to measure resistance levels. Progeny containing arcelin 1 showed resistance equal or superior to that of the resistant check. Arcelin 2-deerived lines had intermediate levels of resistance while no resistant progenies were obtained from crosses with arcelin 3 and 4 sources. Results are discussed in relation to the deployment of arcelin alleles in bean cultivars.

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Valeurs comparées de 5 types d'arcéline dans l'obtention de lignées de Phaseolus vulgaris résistantes à Zabrotes subfasciatus

Résumé La résistance à Zabrotes subfasciatus est associée à la présence d'arcéline, une nouvelle protéine des graines, découverte chez quelques populations de Phaseolus vulgaris. 5 types d'arcéline, hérités comme allèles codominants ont été décrits dans la littérature. Nous avons reprécisé les différentes populations contenant différents types d'arcéline et caractérisé leurs résistances à Z. subfasciatus. La population G 02771, correspondant à l'arcéline 5, présente la résistance la plus élevée par antibiose, suivie de G 12952 (arcéline 4), G 12882 (arcéline 1) et G 12866 (arcéline 2). Les populations contenant l'arcéline 3 présentent le moins de résistance à Z. subfasciatus.Un programme de croisements en retour associé à des tests sérologiques pour déceler la présence d'arcéline chez les descendants jeunes et des expériences répétées d'alimentation par les insectes vec BC2F₃ a été réalisé pour transférer la résistance de populations naturelles à des cultivars de haricots. Les lignées, provenant de croisements avec des populations sauvages avec de l'arcéline 1, ont été fortement résistantes à Z. subfasciatus. Les lignées contenant de l'arcéline 2 ont été considérées comme ayant une résistance intermédiaire. Les lignées avec arcélines 3 et 4 étaient sensibles. Les raisons de l'échec du transfert de la résistance élevée des parents contenant de l'arcéline 4, sont inconnues. On a constaté que la concentration de l'arcéline dans les lignées contenant cet allèle était très faible, tandis que la concentration en arcéline 1 restait remarquablement élevée. Les recherches sont poursuivies pour déterminer les raisons de l'absence de transfert de l'arcéline 4 chez les descendants contenant cet allèle. Quoi qu'il en soit, les caractéristiques agronomiques et les qualités des lignées résistantes (codées RAZ) ont été évaluées en vue d'une diffusion pour les programmes nationaux de recherche des pays de basses altitudes intertropicaux ou Zabrotes subfasciatus fait des dégâts importants.

     

Ultrastructure of scales in a teleost (Carassius auratus L.) after use of rapid freeze-fixation and freeze-substitution

Summary New data on the ultrastructural features of the elasmoid scales ofCarassius auratus have been obtained by use of rapid freezing with subsequent freeze-substitution in anhydrous solvents. These are compared with the results obtained using conventional aqueous fixatives.The external layer of the scales is composed of randomly oriented collagen fibres. In the first stages of mineralization, mineral deposits are located in the interfibrillary substance where dense granules appear to be active sites of mineralization. Spheritic mineralization occurs in this layer.The fibrillary plate is composed of two kinds of collagen fibres. Most of them are organized in lamellae forming the plywood-like structure. They are thicker than the so-called TC fibres, which are oriented from the basal part towards the superficial layer. These TC fibres are involved in the first stages of mineral deposition in the fibrillary plate where inotropic mineralization occurs.The mineral phase is almost always located in the interfibrillary matrix in both layers of the elasmoid scale. In this respect, teleost scales differ from those described so far in other lower vertebrates.

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Resumé Des précisions concernant les aspects ultrastructuraux des dépôts minéraux dans les écailles deCarassius auratus ont été obtenues grâce à l'utilisation de la congélation ultra-rapide suivie d'une cryosubstitution en milieu anhydre. Ces données sont comparées à celles fournies par les méthodes usuelles utilisant des fixateurs aqueux.La couche externe des écailles comprend des fibres collagènes disposées sans ordre apparent. Les dépôts minéraux se produisent surtout dans la substance interfibrillaire où des granules denses semblent représenter des sites actifs au cours de la minéralisation apparentée au type sphéritique.La plaque basale comporte deux catégories de fibres collagènes. Les unes, les plus nombreuses, de plus fort diamètre, sont organisées en lamelles formant une structure en contre-plaqué; les autres appelées fibres TC, orientées de la base de l'écaille vers la zone superficielle, jouent un rôle important dans les premières phases de la minéralisation de type inotropique dans cette partie de l'écaille.Dans les deux couches de l'écaille, la phase minérale est surtout trouvée dans la substance interfibrillaire. De ce fait, les écailles élasmoides des Téleostéens peuvent être distinguées des autres écailles dermiques connues de Vertébrés inférieurs.