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.     

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     

Metabolism of l-threonine and fatty acids and tylosin biosynthesis in Streptomyces fradiae

Abstract In Streptomyces fradiae l -threonine is catabolized by threonine dehydratase or threonine aldolase to 2-ketobutyrate or acetaldehyde and glycine, respectively. Threonine dehydratase synthesis is repressed and its activity is inhibited by NH₄⁺ ions. Threonine aldolase is not repressed by NH₄⁺ ions and its activity is slightly stimulated by these ions. The addition of threonine to the medium increased pronouncedly the fraction of non-branched fatty acids with an even carbon number under conditions when threonine dehydratase was repressed and inhibited. The results indicate that threonine serves as a source of propionyl-CoA and 2-methylbutyryl-CoA and also of acetyl-CoA required for tylosin and fatty acid biosynthesis.     

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.     

Fine needle aspiration cytology of spermatocytic seminoma. Report of a case

The clinical, radiologic, cytopathologic and histologic features of a case of spermatocytic seminoma in a 56-year-old man are reported and discussed. The cytologic findings in fine needle aspiration (FNA) smears suggested a diagnosis of spermatocytic seminoma, which was confirmed by subsequent histopathologic studies. The case suggests that spermatocytic seminoma can be suspected and distinguished from other types of seminoma and germ cell neoplasms by FNA cytology. The following criteria appear to be the most useful in making the cytodiagnosis: the presence of three different types of cells (large, medium and small), a clean background, an absence of a lymphoid component and "tigroid" substances and the relative paucity and clumping tendency of the neoplastic cells in the smears.     

Interaction of retinol and retinoic acid with phospholipid membranes. A differential scanning calorimetry study.

The influence of retinol and retinoic acid, two retinoids of major interest, on the main gel to liquid-crystalline phase transition of different phospholipid membranes has been studied by means of differential scanning calorimetry. Both compounds exerted perturbing effects on the phase transition of membranes composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine. At concentrations up to 42.5 mol% of retinoid in the membrane, the delta H was not much affected with respect to the pure phospholipid, indicating a rather slight interaction. As the concentration of retinol was increased the Tc transition temperature decreased. A fluid-phase immiscibility was observed for the system DPPC/retinol at concentrations between 0 and 33 mol%. Almost ideal phase diagrams were obtained for the mixture DPPE/retinol. At concentrations of 33 mol% and higher retinol was able to induce phase separations in DPPC membranes, but not in DPPE. The effect of retinoic acid was much weaker, the Tc and delta H remaining almost unaltered and equal to that of the pure phospholipid up to concentrations of 30 mol%, at neutral pH. Retinoic acid exerted a pH-dependent effect. As the pH decreased, and therefore increased the extent of protonation of retinoic acid, the pertubation of the membrane induced by this compound was less. A strong effect, both on Tc and delta H, was observed at pH 10, where the retinoic acid moiety will be mainly unprotonated and the negative charge will generate repulsive forces thus destabilizing the membrane. The mixture DPPC/retinoic acid presents a region of fluid-phase immiscibility. At low pH, when the retinoic acid moiety was fully protonated, this fluid-immiscibility region extended from 0 to 36 mol% of retinoic acid, but its size decreased with increasing pH, and at pH 10 it was only found from 0 to 3 mol%. These results are discussed in terms of the possible retinoid/phospholipid interactions and the disposition of the retinoid moiety in the bilayer.     

Effect of beta-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk.

The interaction of bovine beta-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine beta-lactoglobulin displays only one high affinity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 x 10(6) and 2.3 x 10(6) M-1, respectively. However, other binding sites with low affinity are also present. The existence of one high affinity binding site is in accordance with the amount of fatty acids naturally bound to beta-lactoglobulin isolated from milk. The effect of beta-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of beta-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that beta-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.