Separate Intramolecular Targets for Protein Kinase A Control N-Methyl-d-aspartate Receptor Gating and Ca2+ Permeability

Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca²⁺ flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca²⁺ permeability (P_Ca/P_Na) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca²⁺ conductance, because neither Na⁺ conductance nor Ca²⁺-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca²⁺ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca²⁺ permeability.     

Possible role of cytokinins in cereals with regard to the resistance to obligate fungus parasites

The cultivars of barley and wheat resistant to mildew had a higher level of free zeatin and its derivatives during the whole ontogeny than the susceptible cultivars. The effect of exogenous application of cytokinins on the growth ofErysiphe graminis DC. was different. Kinetin and benzylaminopurine showed only a slight inhibitory effect. Zeatin and its derivatives completely inhibited growth of this fungus. Presented at the International Symposium “Plant Growth Regulators” held on June 18-22 1984 at Liblice, Czechoslovakia.     

Fragile sites,chromosome evolution,and human neoplasia

Summary In a study of the possible relationship between human fragile sites, chromosomal rearrangements related to neoplasia, and chromosome regions involved in evolutionary changes, we have found that 17 fragile sites related to cancer, 15 fragile sites not related to cancer, and 17 non-fragile regions also related to human malignancy correspond or are close to bands involved in rearrangements that have taken place during chromosomal evolution in primates.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

The molecular organization of nerve membranes

Summary The lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 226 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.The preceding papers of this series were published inArchives of Biochemistry and Biophysics.     

Multiple forms of lactate dehydrogenase in Staphylococcus aureus

Activities for nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent forms of lactate dehydrogenase (LDH) were measured in cell-free extracts of Staphylococcus aureus strain PS 6 for the d and l isomers of lactate. Data obtained for the NAD-dependent lactate dehydrogenases indicate that oxidation of both isomers of lactate is due to both an l-lactate-specific LDH and a lactate racemase. After acrylamide gel electrophoresis, two bands exhibiting LDH activity were detected in crude or in partially purified cell-free extracts. The fast band exhibited LDH activity that was not NAD-dependent for both isomers of lactate, whereas, the slow band had very high NAD-dependent LDH activity for the l isomer but just detectable activity or the d isomer. Both bands appeared when d-lactate was used as the substrate, but only the slow band was formed when l-lactate was the substrate. NAD-dependent LDH, in apparent association with a nonspecific tetrazolium-reducing protein, is responsible for the production of the slow band.     

Instrumental assay of microbial lipase at constant pH

A rapid, accurate method with high sensitivity and reproducibility, and having the advantage of a short incubation period under constant pH, has been developed for routine measurement of microbial lipase. Assembled from readily available and economical instrumental components, the apparatus includes a pH meter, a thermoelectric heating and stirring device, a motor-driven burette, and an automatic recorder. The reaction mixture, consisting of 5 ml of a 10% olive oil-gum arabic emulsion, 2 ml of 3 m NaCl, 2 ml of sodium taurocholate (15 mg/ml) of 0.075 m CaCl(2), 5 ml of water, and 1 ml of enzyme solution, was adjusted to pH 8.0 and 37 C. The pH was maintained at a constant value by automatic addition of 0.01 n NaOH during the incubation period, which usually lasted 5 min. A lipase unit, derived from the use of this technique, may be defined as the number of microequivalents of acid liberated per minute under the specified conditions. The method was sensitive to 0.01 units. Various organisms tested produced 0.17 to 1.32 units per ml of the cell filtrate. An Arrhenius plot for staphylococcal lipase yielded 14,500 cal for function A (energy of activation).     

Nephelometric Assay of Bovine Antistaphylocoagulase Serum

A nephelometric assay of staphylococcal coagulase has been utilized to measure coagulase inhibition by bovine anticoagulase serum. Suitably diluted antisera produced maximal inhibition when incubated with purified coagulase at pH 7.3 in phosphate-buffered saline for 15 min at 22 C or 1 hr at 4 C. Neutralization of coagulase activity was measured as the reduction in the clotting rate of a fibrinogen-plasma substrate, and was directly proportional to the concentration of antiserum over a wide range of coagulase activity. A unit of anticoagulase was defined as the amount of inhibitor that neutralized one unit of coagulase. In addition to the heat-stable (56 C, 30 min) antibody contained in the crude gamma-globulin fraction, a heat-labile, nondialyzable coagulase inhibitor was also detected in the sera from 15 of 16 randomly selected dairy cows.