Glutamate Exocytosis and MARCKS Phosphorylation Are Enhanced by a Metabotropic Glutamate Receptor Coupled to a Protein Kinase C Synergistically Activated by Diacylglycerol and Arachidonic Acid

Abstract: 4-Aminopyridine evokes repetitive firing of synaptosomes and exocytosis of glutamate by inhibiting a dendrotoxin-sensitive K⁺ channel responsible for stabilizing the membrane potential. We have shown previously that activation of protein kinase C (PKC) by high concentrations of phorbol ester (4β-phorbol dibutyrate) can increase release by inhibiting a dendrotoxin-insensitive ion channel, whereas the metabotropic glutamate receptor (mGluR) agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate [(1 S ,3 R )-ACPD] mimics the action of 4β-phorbol dibutyrate, but only in the presence of 2 µ M arachidonic acid (AA). In this article, we investigate the role of AA. AA plus (1 S ,3 R )-ACPD is without effect on KCl-induced glutamate exocytosis, indicating that the regulatory pathway acts upstream of the release-coupled Ca²⁺ channel or Ca²⁺-secretion coupling. Diacylglycerol concentrations are greatly enhanced by (1 S ,3 R )-ACPD alone, independently of AA, indicating that AA acts downstream of phospholipase C. Myristoylated alanine-rich C kinase substrate (MARCKS) is the major presynaptic substrate for PKC. mGluR activation by (1 S ,3 R )-ACPD enhances phosphorylation of MARCKS, but only in the presence of AA. These results strongly suggest that AA acts on presynaptic PKC synergistically with diacylglycerol generated by the phospholipase-coupled mGluR, consistent with the known behaviour of certain purified PKC isoforms. The magnitude of the effects observed in a population of rat cerebrocortical synaptosomes suggests that this is a major mechanism regulating the release of the brain's dominant excitatory neurotransmitter and supports the concept that AA, or a related compound with a similar locus of action, may in certain circumstances play a role in synaptic plasticity.     

Cationic and Anionic Requirements for the Binding of 2β-Carbomethoxy-3β-(4-Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier

Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [³H]CFT binding to synaptosomal P₂ membranes requires low concentrations of Na⁺ (peak binding between 20 and 50 m M Na⁺), is stimulated by phosphate anion or l^-, but is unaffected or only slightly affected by F^-, Cl^-, Br^-, NO₃^-, or SO₄^2-, Concentrations of Na⁺ of >50 m M become inhibitory except in the presence of l^-, which shifts peak binding levels toward higher Na⁺ concentrations and also elevates the peak binding level. K⁺ strongly decreased [³H]CFT binding with a shallow inhibition curve, and Na⁺ could not overcome this effect. Saturation analysis of [³H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 m M ; 1 mM plus 49 mM NaCIversus 10 m M plus 40 m M NaCI; or 1 mM plus 129 m M Nal versus 10 m M plus 120 m M Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae

Abstract Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 μg benzylpenicillin ml⁻¹. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.     

Mesodern-inducing factors and mesodermal patterning

Identification of the signalling molecules involved in mesoderm formation in amphibian embryos still presents problems. None of the original candidates, such as activin, have been definitively ruled out, and the new factors, such as the nodal-related genes, have come on to the scene. Of the original candidates, activin has been definitively shown to act as a morphogen, whereas bone morphogenetic protein (BMP)-4 has emerged as a ventral inducer and an inhibitor of neural differentiation. The effects of BMP-4 are antagonized by chordin, a molecule related to the product of the Drosophila gene short gastrulation.     

Analogs of arachidonic acid methylated at C-7 and C-10 as inhibitors of leukotriene biosynthesis

The syntheses and biological activity of (all )-7,7-dimethyl-5-8,- 11,14-eicosatetraenoic acid, (all )-7,7,-dimethyl-5,8,11-eicosatrienoic acid, ( , -7,7-dimethyl-5,8-eicosadienoic acid, (all )-10,10-dimetyl- 5,8,11,14-eicosatetraenoic acid, (all -10,10-dimethyl-5,8,11-eicosatrienoic acid, and .-( , -15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid are described. These arachidonic acid analogs are all inhibitors of ionophore-induced SRS-A biosynthesis in rat peritoneal cells. Their mode of action may involve inhibition of phospholipase A₂ rather than Δ⁵-lipoxygenase. These compounds failed to exhibit significant activity in an model designed to detect inhibitors of antigen-induced, leukotriene-mediated bronchoconstriction is sensitized guinea pigs.