Depolarization and Neurotransmitters Increase Neuronal Protein Tyrosine Phosphorylation

Abstract: In rat hippocampal slices and in neurons in primary culture, K⁺-induced depolarization increased markedly and rapidly tyrosine phosphorylation of a 110-kDa protein (pp110) and, to a lesser degree, of a 120-kDa protein (pp120), in a calcium-dependent fashion. Qlutamate, 1-aminocyclopentane- trans -1,3-dicarboxylic acid (an agonist of metabotropic glutamate receptors), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (an agonist of ionotropic glutamate receptors) stimulated also tyrosine phosphorylation of pp110 and pp120. These effects were not observed in astrocytes in primary culture. In hippocampal slices tyrosine phosphorylation of pp110 and pp120 was stimulated by Ca²⁺-ionophores and by phorbol esters and antagonized by a chelator of intracellular Ca²⁺and by drugs that inhibit protein kinase C. Stimulation of muscarinic and α₁,-adrenergic receptors increased also tyrosine phosphorylation of pp110 and pp120. These results demonstrate that membrane depolarization and stimulation of neurotransmitter receptors activate a tyrosine phosphorylation pathway in neurons. This pathway involves an increase in intracellular Ca²⁺ concentrations and the activation of protein kinase C. It may provide a biochemical basis for some neurotrophic effects of electrical activity and neurotransmitters and may contribute to the role of tyrosine phosphorylation in long-term potentiation.     

EPR and multi-field magnetisation of reduced forms of the binuclear iron centre in ribonucleotide reductase from mouse

Mouse ribonucleotide reductase is composed of a 1?:?1 complex of two homodimeric subunits and catalyses the first unique step on the biochemical pathway to DNA synthesis. The small subunit, protein R2, contains dinuclear iron-oxygen clusters and a tyrosyl free radical required for catalytic activity. We have studied the mixed valent and fully reduced forms of the diiron oxygen cluster from mouse R2 protein by low-temperature EPR. EPR signals of the mixed-valent states of proteins R2 reconstituted with ferrous iron and oxygen in normal and deuterated water, using the same buffers, show apparent g values of 1.92, 1.73, and 1.60 for the mixed-valent state in H₂O and 1.93, 1.73, and 1.62 in D₂O. These g values are typical for diiron-oxygen proteins, while the effect of D₂O is unprecedented for this class of proteins. We estimate the coupling constant J for the Heisenberg exchange (H?=?2J*S₁*S₂) to be J?=?–7.5±1?cm^–1 for the mixed-valent form. The diferrous R2 protein shows an integer spin EPR signal in the presence of azide or 20% glycerol. Variable temperature variable field saturation magnetisation measurements show that only in the azide-complexed R2 protein does a weak ferromagnetic coupling occur (J?=?0.26±0.05?cm^–1), while R2 protein in the absence or presence of 20% glycerol contains non-coupled mononuclear ferrous iron (S?=?2) sites.     

A new approach for using cofactor dependent enzymes: example of alcohol dehydrogenase

The use of enzymes requiring a cofactor as substrate in organic synthesis is still a problem since the cofactors are expensive. This study deals with a new approach consisting of using fragments of NAD+. Three fragments of NAD(H) are examined. The activities of NMN+ and NMNH are greatly improved by the addition of adenosine in ethanol oxidation and in cyclohexanone reduction, respectively. Nicotinamide mononucleoside is not active in the ethanol oxidation but the addition of AMP promotes this reaction.     

Experimental silver bioaccumulation in the polychaetePomatoceros triqueter (L.)

Summary The tubicolous polychaetePomatoceros triqueter was exposed for 6–7 weeks to 200 or 400 g · l^–1 silver introduced as the nitrate into sea water. Survival conditions and mortality were evaluated and silver bioaccumulation analysed by atomic absorption spectrometry. Characteristic morphological lesions were recognized. Histopathologic examination was performed on paraffin or semi-thin sections and at the ultrastructural level. Histochemical examination mainly concerned the metals, reducing groups and sulfur-containing proteins. Microanalytical study involved the use of a wavelength-dispersive X-ray spectrometry microprobe and ion microanalyzer, and the use of an energy-dispersive X-ray spectrometry microprobe at the ultrastructural level. Our results emphasize the role of the branchial crown for metal penetration. Its cuticle accumulates silver as a metal, in particulate form. The internal accumulation of mainly extracellular deposits concerns the basement membranes and connective tissue present in the axis of the branchial crown filaments, or surrounding the nephridial pouches and the gut sinus. The carrier role of the closed vascular system is suggested by ultrastructural observations. The silver route from transepithelial uptake to nephridial excretion involves at least two intracellular transits, plus the vascular mesothelium. Nephridia play a role in silver storage (lysosomes) and elimination (concretions). In all parts internal to the crown cuticle, silver is at least partly associated with protein SH-groups (metallothionein-like); deposits can be enriched with silver sulfide and metallic silver.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

Occurrence of 28- and 27-Carbon ecdysteroids and sterols in developing worker pupae of the leaf-cutting ant acromyrmex octospinosus (reich) (hymenoptera,formicidae: Attini)

The major ecdysteroids in large worker pupae of the leaf-cutting ant Acromyrmex octospinosus were characterized at the peak ecdysteroid concentration by using high-performance liquid chromatography, enzyme immunoassay, and mass spectrometry. In decreasing amounts, they were determined to be makisterone A, an unidentified C₂₈ ecdysteroid bearing a molecular weight of 494, 20-hydroxyecdysone (ratio of 1 to 6 as compared to makisterone A), and putative but negligible ecdysone. The presence of both C₂₈ and C₂₇ ecdysteroids is discussed in relation to the content of 4-desmethylsterols determined by gas chromatography and mass spectrometry to be ergosta-5,7,24 (28)-trien-3β-ol, ergosterol, ergosta-5,7-dien-3β-ol and ergosta-7,24(28)-dien-3β-ol for the main sterols, and with a small amount of cholesterol.