Changing Fatty Acid Content of Growth Cone Lipids Prior to Synaptogenesis

The developing mouse was used to assess biochemical changes in membrane lipids during the period when nerve growth cones become synapses. Growth cone particles and synaptosomes were simultaneously obtained from common brain homogenates. Incorporation of the essential fatty acid, docosahexaenoic acid (22:6 omega-3), was correlated with the developmental changes in endogenous fatty acid content of growth cones and synaptosomes. Analysis of endogenous lipid content indicated that, at all ages studied, the growth cones contained more arachidonoyl acyl chains (20:4 omega-6) than did synaptosomes. Before the onset of synaptogenesis, levels of arachidonoyl chains increased and levels of 22:6, oleoyl and linoleoyl chains decreased in synaptosomes. Although stearoyl and palmitoyl (16:0) remained stable in synaptosomes, 16:0 decreased in growth cones. With the exception of 16:0 and 20:4, endogenous fatty acyl content of growth cones and synaptosomes became similar by postnatal day 10, which coincides with the onset of synaptogenesis. When 5-day-old mouse pups were injected intraperitoneally with [3H]22:6, the incorporation into growth cone and synaptosome phospholipids was greatest in phosphatidylethanolamine, followed by phosphatidylserine and phosphatidylcholine. Nominal labeling was present in phosphatidic acid and phosphatidylinositol. Labeling in neutral lipids was less than that of phospholipids, with triacylglycerol incorporating most of the neutral lipid label, followed by diacylglycerol and free 22:6. Only the growth cone fraction contained detectable amounts of 22:6-labeled cholesterol esters. The distribution of 22:6 label in plasma 72 h after injection indicated that approximately 60% of the label was in phospholipids with approximately 40% in neutral lipids and less than 5% in free fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Analysis of the activation process of porcine procarboxypeptidase B and determination of the sequence of its activation segment

The molecular events which lead to the proteolytic transformation of porcine procarboxypeptidase B (PCPB) in carboxypeptidase B (CPB) have been determined. Among pancreatic and other tested proteinases, trypsin is the only one capable of generating carboxypeptidase B activity from the zymogen, in vitro. In the first step of this process, trypsin produces cleavage at the boundary between the activation region and the CPB region. Subsequently, a definite sequence of cleavages occurs at the C-terminal end of the released activation segment of 95 residues, giving rise to characteristic intermediates and to a proteolytically resistant activation fragment of 81 residues. In this process, the newly formed CPB participates in the quick-trimming of the released activation peptides. Only a single CPB species is formed in the activation process. This fact and the inability of the released activation peptides to inhibit CPB--and, therefore, their inability to slow down the kinetics of appearance of CPB activity--are two important characteristics differentiating between the activation processes of procarboxypeptidases A and B. The sequence of the 95 residues (MW = 12,835) of the activation region of porcine PCPB has also been deduced, largely from the information obtained by Edman degradation of its fragments and in part by considerations of homology with the rat precursor. The porcine PCPB activation region contains a high percentage of acidic residues, lacks cysteines, methionines, and side-chain posttranslational modifications, and presents a low but significant homology (31%) with the corresponding sequence of porcine procarboxypeptidase A.     

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.

     

Human glioma tumour-associated antigens

Conclusion From this brief review it appears that at least three categories of human glioma-associated antigens may exist. The first seems to be restricted and common to gliomas. The second is shared between gliomas, normal adult brain, and fetal brain. The third is present on cells from adult and fetal tissue and on cells from tumours derived from the neural crest. The expression of glioma-associated antigens is highly variable from one tumour, or tumour cell line, to another, and reflects the phenotypic heterogeneity of the glioma group. Moreover, this heterogeneity has been found in different clones of individual glioma cell lines [1]. The fact that gliomas share some antigens with normal brain is of critical importance for immunodiagnosis or immunotherapy. It is evident that active immunotherapy for gliomas should be performed with cultured cells and not with tumour extracts, because such extracts may contain MBP.The exact nature of the various glioma-associated antigens remains to be clearly defined, however. They may belong to a group of surface glycoproteins such as those described by Lloyd et al. [24] for melanoma or more recently by Lubitz et al. [25] for glial cells.