Biological properties of a new fluorescent biphalin fragment analogue

Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH<--X, where X=Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X=DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both mu and delta opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation.     

Synthesis,antiprotozoal and antibacterial activity of nitro- and halogeno-substituted benzimidazole derivatives

Two series of benzimidazole derivatives were sythesised. The first one was based on 5,6-dinitrobenzimidazole, the second one comprises 2-thioalkyl- and thioaryl-substituted modified benzimidazoles. Antibacterial and antiprotozoal activity of the newly obtained compounds was studied. Some thioalkyl derivatives showed remarkable activity against nosocomial strains of Stenotrophomonas malthophilia, and an activity comparable to that of metronidazole against gram-positive and gram-negative bacteria. Of the tested compounds, 5,6-dichloro-2-(4-nitrobenzylthio)-benzimidazole showed the most distinct antiprotozoal activity.     

Utilisation of glycogen,ATP and arginine phosphate in exercise and recovery in terrestrial red crabs,Gecarcoidea natalis

Intermittent locomotion by terrestrial crustaceans may under specific circumstances increase walking distance and may allow partial re-oxidization of anaerobic products, and replenishment of ATP and arginine phosphate. The Christmas Island red crab G. natalis undertakes a substantial breeding migration each year. The leg muscles of G. natalis subjected to bouts of 2.5 min walking and 2.5 min rest were severely anaerobic. Adenylate energy charge and the large arginine phosphate stores were greatly reduced. Walking for 4 min with pauses of only 1 min exacerbated the anaerobiosis and utilised 50% of the endogenous muscle glycogen. Post-exercise, the adenylate energy charge recovered before the arginine phosphate charge and a large and persistent hyperglycaemia accompanied the restoration of glycogen. Arginine phosphate functioned as a large, longer term, energy reservoir-almost as part of the adenylate pool. Gluconeogenesis is yet to be generally substantiated in decapod crustaceans but G. natalis appears to remove lactate slowly and to reincorporate exogenous glucose into muscle glycogen in the same time frame as lactate removal from the haemolymph. The 4:1 exercise/pause regimen facilitated access to energy stores and increased walking distance, and it allowed L-lactate and H(+) efflux from the muscle during pausing. These responses are similar to those of G. natalis in the field, except during the migration when walking was entirely aerobic. Determinations of adenylate, fuel and arginine phosphate reserves and usage during the migration are required together with more detailed behavioral analysis to resolve the dichotomy in metabolic response.     

The mechanism of the hemolytic activity of polyene antibiotics

The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca2+ or Ba2+ but not by Mg2+, Na+ or SO42-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO4(2-) to permeate.     

Arabidopsis genes encoding mitochondrial type II NAD(P)H dehydrogenases have different evolutionary origin and show distinct responses to light

In addition to proton-pumping complex I, plant mitochondria contain several type II NAD(P)H dehydrogenases in the electron transport chain. The extra enzymes allow the nonenergy-conserving electron transfer from cytoplasmic and matrix NAD(P)H to ubiquinone. We have investigated the type II NAD(P)H dehydrogenase gene families in Arabidopsis. This model plant contains two and four genes closely related to potato (Solanum tuberosum) genes nda1 and ndb1, respectively. A novel homolog, termed ndc1, with a lower but significant similarity to potato nda1 and ndb1, is also present. All genes are expressed in several organs of the plant. Among the nda genes, expression of nda1, but not nda2, is dependent on light and circadian regulation, suggesting separate roles in photosynthesis-associated and other respiratory NADH oxidation. Genes from all three gene families encode proteins exclusively targeted to mitochondria, as revealed by expression of green fluorescent fusion proteins and by western blotting of fractionated cells. Phylogenetic analysis indicates that ndc1 affiliates with cyanobacterial type II NADH dehydrogenase genes, suggesting that this gene entered the eukaryotic cell via the chloroplast progenitor. The ndc1 should then have been transferred to the nucleus and acquired a signal for mitochondrial targeting of the protein product. Although they are of different origin, the nda, ndb, and ndc genes carry an identical intron position.     

A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in aplysia

Synapse-specific facilitation requires rapamycin-dependent local protein synthesis at the activated synapse. In Aplysia, rapamycin-dependent local protein synthesis serves two functions: (1) it provides a component of the mark at the activated synapse and thereby confers synapse specificity and (2) it stabilizes the synaptic growth associated with long-term facilitation. Here we report that a neuron-specific isoform of cytoplasmic polyadenylation element binding protein (CPEB) regulates this synaptic protein synthesis in an activity-dependent manner. Aplysia CPEB protein is upregulated locally at activated synapses, and it is needed not for the initiation but for the stable maintenance of long-term facilitation. We suggest that Aplysia CPEB is one of the stabilizing components of the synaptic mark.     

Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other.     

Interactions of trimeric purine nucleoside phosphorylases with ground state analogues--calorimetric and fluorimetric studies

Binding enthalpies, dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen and Cellulomonas sp. purine nucleoside phosphorylases with their ground state analogues (substrates and inhibitors) were studied by calorimetric and spectrofluorimetric methods. Data for all ligands, with possible exception of hypoxanthine, are consistent with three identical non-interacting binding sites.