Optimization of transverse alternating field electrophoresis for strain identification of Leuconostoc oenos

Genomic DNA of several strains oof oenological lactic bacteria belonging to the species Lactobacillus plantarum, Leuconostoc oenos and Pediococcus pentosaceus was digested by the rare-cutting endonucleases ApaI and SmaI. The restriction products were separated by transverse alternating field electrophoresis (TAFE). The size of the genome of L. oenos estimated by adding the molecular size of the ApaI fragments was on average 1320 kb. A strong polymorphism was observed between the strains, which could be easily differentiated except for two industrial strains of L. oenos. A simple modification of the TAFE apparatus is proposed to improve the separation of the DNA fragments. Correspondence to: J.-N. Hallet     

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P₂). Its activity is inhibited by Ca²⁺ and Zn²⁺ and is activated by Mg²⁺, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.     

Induction of autonomous endosperm in Lupinus luteus,Helleborus niger and Melandrium album by in vitro culture of unpollinated ovaries

Autonomous division of the endosperm was induced by in vitro culture of unpollinated ovaries or placenta-attached ovules in Helleborus niger, Lupinus luteus and Melandrium album. The induction frequencies for the three species were 50%, 10–20% and 0.1%, respectively. The endosperms contained up to 20 free nuclei; only a few ovules with 80–420 endosperm nuclei were found. Induction of autonomous division of the endosperm, which is unusual in amphimictic plants, was observed in three new species. No embryos appeared in the ovules. This suggests a developmental independence of the endosperm from the embryo in the culture of unpollinated ovaries or ovules.     

Time resolved spectroscopy of the tryptophyl fluorescence of the E. coli LAC repressor.

A new crystal form of a mitogenic lectin from pea seeds ($\text{Pisum sativum}$) has been obtained which is suitable for high resolution structural work. The crystals are orthorhombic, space group P2₁2₁2₁, with unit cell dimensions: $\text{a}$ = 64.2Å, $\text{b}$ = 72. 7Å, $\text{c}$ = 108. 3Å. The asymmetric unit contains one protein molecule.     

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.     

Retrograde Inhibition of Transmitter Release by ATP

Abstract: After labelling ACh tissue stores in Torpedo electric organ prisms with radioactive acetate, the release of ACh and ATP triggered by electrical stimulation or KCI depolarization was measured in the same perfusate samples. The luciferin-luciferase reaction for ATP was first counted, then the radioactive content of the sample determined. Further evidence showing that ATP release resulted from postsynaptic transmitter action was that carbachol could induce the release of ATP. A dose-response curve was obtained. Curare or α-bungarotoxin block the release of ATP elicited by carbachol. When triggered by KCI depolarization the increased efflux of ACh and ATP returned to low levels in spite of the maintained depolarization. After two successive KCI depolarizations, it was possible to dissociate the release of both substances. The efflux of ATP was exhausted while ACh release was maintained. If the second KCI depolarization was delayed ATP release recovered, but the release kinetics of ACh and ATP were sustained. The exhaustion of endogenous ATP release or the action of exogenous ATP had little or no effect on the release of ACh triggered by KCI depolarization. On the contrary, the release of ACh induced by electrical stimulation was sensitive to the action of adenine nucleotides, and a quantitative estimation of the inhibition of ACh release by ATP and adenosine could be made. At the onset of stimulation ATP release predominated, being gradually replaced by adenosine, which can be reuptaken. This would terminate the inhibitory action of the nucleotide. Carbachol inhibits evoked ACh release, while the effect of α-bungarotoxin was to increase spontaneous ACh release. These effects could be respectively mediated by an increased or a reduced release of ATP resulting from the postsynaptic action of ACh agonists or antagonists. However, a direct presynaptic effect of these substances is not excluded. It seems possible that the action of ATP on ACh release can be explained through its inhibition of the depolarization-evoked Ca²⁺ entry.