Modulation of Quinolinic and Kynurenic Acid Content in the Rat Brain: Effects of Endotoxins and Nicotinylalanine

Quinolinic acid, an endogenous excitotoxin, and kynurenic acid, an antagonist of excitatory amino acid receptors, are believed to be synthesized from tryptophan after the opening of the indole ring. They were measured in the rat brain and other organs using gas chromatography-mass spectrometry or HPLC. The enzyme indoleamine 2,3-dioxygenase, capable of cleaving the indole ring of tryptophan, was induced by administering bacterial endotoxins to rats, which significantly increased the brain content of both quinolinic and kynurenic acids. Nicotinylalanine, an analogue of kynurenine, inhibited this endotoxin-induced accumulation of quinolinic acid while potentiating the accumulation of kynurenic acid. The possibility of significantly increasing brain concentrations of kynurenic acid without a concomitant increase in quinolinic acid may provide a useful approach for studying the role of these electrophysiologically active tryptophan metabolites in brain function and preventing the possible toxic actions of abnormal synthesis of quinolinic acid.     

Characterization of the major allergens purified from the venom of the paper wasp Polistes gallicus

Allergic reactions to vespid stings are one of the major causes of IgE-mediated anaphylaxis. Vespa and Vespula venoms are closely related; Polistes venom is more distantly related and its allergens are less well studied. There is limited cross-reactivity between Polistes and the other vespid venoms because of differences in the epitopes on the allergen molecules.In this study, the major allergens of Polistes gallicus are isolated and characterized. P. gallicus venom contains four major allergens: phospholipase, antigen 5 (Ag5), hyaluronidase and protease that were characterized by mass spectrometry and specific binding to IgE. The complete amino acid sequence of Ag5 and the sequence of the N-terminal region of phospholipase were also determined. The alignment of Ag5 from P. gallicus (European species) and Polistes annularis (American species) shows an 85% identity that increases to 98% within the same subgenus. This could suggest the presence of specific epitopes on Ag5 molecule being the variations on the superficial loops. The features of the P. gallicus allergens could explain the partial cross-reactivity found between the American and European Polistes venoms, and suggest that the use of European Polistes venoms would improve the diagnostic specificity and the therapy of European patients and of North American patients sensitized by European Polistes.     

Structural and solution chemistry, protein binding and antiproliferative profiles of gold(I)/(III) complexes bearing the saccharinato ligand

A series of new gold(I) and gold(III) complexes based on the saccharinate (sac) ligand, namely M[Au(sac)₂] (with M being Na⁺, K⁺ or NH₄⁺), [(PTA)Au(sac)], K[Au(sac)₃Cl] and Na[Au(sac)₄], were synthesized and characterized, and some aspects of their biological profile investigated. Spectrophotometric analysis revealed that these gold compounds, upon dissolution in aqueous media, at physiological pH, manifest a rather favourable balance between stability and reactivity. Their reactions with the model proteins cytochrome c and lysozyme were monitored by mass spectrometry to predict their likely interactions with protein targets. In the case of disaccharinato gold(I) complexes, cytochrome c adducts bearing four coordinated gold(I) ions were preferentially formed in high yield. In contrast, [(PTA)Au(sac)] (PTA = 1,3,5-triaza-7-phosphaadamantane) turned out to be poorly effective, only producing a mono-metalated adduct in very low amount. In turn, the gold(III) saccharinate derivatives were less reactive than their gold(I) analogues: K[Au(sac)₃Cl] and Na[Au(sac)₄] caused moderate protein metalation, again with evidence of formation of tetragold adducts. Finally, the above mentioned gold compounds were challenged against the reference human tumor cell line A2780S and its cisplatin resistant subline A2780R and their respective cytotoxic profiles determined. [(PTA)Au(sac)] turned out to be highly cytotoxic whereas moderate cytotoxicities were observed for the gold(III) complexes and only modest activities for disaccharinato gold(I) complexes. The implications of these results are thoroughly discussed in the light of current knowledge on gold based drugs.     

The release of gamma-aminobutyric acid, glutamate, and acetylcholine from striatal slices: a mass fragmentographic study

The release processes of endogenous Acetylcholine (ACh), gamma-aminobutyric acid (GABA), glutamate (Glu) and glutamine (GLN) were studied in superfused guinea-pig caudatal slices. Basal ACh release remained constant for up to 2 h, while the basal release of GABA, Glu and GLN declined to half or less of its initial values after 1 h of superfusion. Electrical stimulation increased the ACh release by 700-800% and that of GABA by 80% whereas it decreased the output of Glu by 50% and failed to modify the GLN efflux. KCl (25 nM) increased the output of ACh by 400%, that of GABA by approximately 500% and decreased that of Glu by 40%. Substituting of CaCl(2) by MgCl(2) in the superfusion medium reduced the basal efflux of GABA, Glu and GLN. Under these conditions, no evoked release of ACh or of GABA was detected, following electrical or KCl stimulation. Tetrodotoxin 5 x 10(-7) decreased the basal ACh release by 60% and increased the GABA efflux by 40%. The toxin abolished the stimulus-evoked ACh efflux but scarcely affected that of GABA. These results are consistent with a possible neurotransmitter role of ACh and GABA in the striatum and show some differences in the ionic mechanisms underlying GABA and ACh release.     

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability

A detailed understanding of the molecular basis of protein folding and stability determinants partly relies on the study of proteins with enhanced conformational stability properties, such as those from thermophilic organisms. In this study, we set up a methodology aiming at identifying the subset of cytosolic hyperstable proteins using Sulfurispharea sp., a hyperthermophilic archaeon, able to grow between 70 and 97 degrees C, as a model organism. We have thermally and chemically perturbed the cytosolic proteome as a function of time (up to 96 h incubation at 90 degrees C), and proceeded with analysis of the remaining proteins by combining one- and two-dimensional gel electrophoresis, liquid chromatography fractionation, and protein identification by N-terminal sequencing and mass spectrometry methods. In total, 14 proteins with enhanced stabilities which are involved in key cellular processes such as detoxification, nucleic acid processing, and energy metabolism were identified including a superoxide dismutase, a peroxiredoxin, and a ferredoxin. We demonstrate that these proteins are biologically active after extensive thermal treatment of the proteome. The relevance of these and other targets is discussed in terms of the organism's ecology. This work thus illustrates an experimental approach aimed at mining a proteome for hyperstable proteins, a valuable tool for target selection in protein stability and structural studies.     

Epoetin alpha,epoetin beta and darbepoetin alfa: two-dimensional gel electrophoresis isoforms characterization and mass spectrometry analysis

Since 1989 recombinant human erythropoietin (rhEPO) has been used as a drug for the correction of anemia, but the misuse of rhEPO as an ergogenic agent among athletes is a widespread doping practice. As a consequence there is a need for developing reference methods for the detection of rhEPO in biological fluids, and to be able to differentiate the recombinant from the natural protein. Recombinant human erythropoietin differs from its natural counterpart in the glycidic part of the molecule. Three different commercial recombinant products Epoetin alpha (Eprex, Janssen Cilag), Epoetin beta (Neorecormon, Roche) and Darbepoetin alfa (Nespo, Dompè) have been used to evaluate the performance of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) for the separation of isoforms and the identification of the proteins respectively. All the compounds studied were well separated by means of 2-DE: Epoetin alpha and beta focused in the same isoelectric point region giving rise to six and eight spots respectively, whereas Darbepoetin alfa was found in a more acidic zone with two spots. Results obtained with micro high-performance liquid chromatography-electrospray ionization-time of flight (TOF) MS and matrix-assisted laser desorption/ionization-time of flight MS for the three rhEPOs are reported. These preliminary results suggest that by means of 2-DE and MS it should be possible to reveal the presence of rhEPOs for antidoping purposes.     

Use of Dufour's gland secretion in nest defence and brood nutrition by hover wasps (Hymenoptera, Stenogastrinae)

Social wasps of the subfamily Stenogastrinae produce an abdominal secretion that is used in two distinct biological contexts. First, the secretion plays an important role in larval nutrition where it serves as a substrate in which food is placed by the adults for eventual consumption by the larvae. Second, in several species, females apply the same secretion to the substrate on which their nests are constructed, where it constitutes a sticky barrier that defends the immature brood from predation by ants. This paper describes for the first time ant guard construction behaviour of three species of stenogastrine wasps belonging to the genera Eustenogaster and Liostenogaster. The identification of compounds making up these secretions was also performed by gas chromatography-mass spectrometry. Ant guards and brood secretions were similar, with saturated and unsaturated long chain hydrocarbons and alcohols as major components. We further confirm that the glandular source of abdominal secretion is the Dufour's gland. This gland contains the same hydrocarbons, and in the same proportions as ant guards and brood secretion. We discuss the fundamental importance of Dufour's gland secretion in the social life of these wasps by comparing species with and without ant guards within the subfamily.     

Nestmate recognition in Parischnogaster striatula (Hymenoptera Stenogastrinae), visual and olfactory recognition cues

The recognition of nestmates from alien individuals is a well known phenomenon in social insects. In the stenogastrine wasp Parischnogaster striatula, we investigated the ability of females to recognize nestmates and the cues on which such recognition is based. Recognition of nestmates was observed in naturally occurring interactions between wasps approaching a nest and the resident females on that nest. This recognition was confirmed in experiments in which nestmates or alien conspecifics were presented to resident females. In naturally occurring interactions, nestmates generally approach their nest with a direct flight, while aliens usually hover in front of the nest before landing. In experiments in which the presented wasps were placed close to the nest in a direct manner, antennation of the presented wasp generally occurred, indicating that chemical cues are involved. Experiments in which dead alien individuals, previously washed in hexane, and then reapplied with extracts were recognized by colonies giving further evidence that chemical cues mediate nestmate recognition. Epicuticular lipids, known to be nestmate recognition cues in social insects, were chemically analysed by GC-MS for 44 P. striatula females from two different populations (13 different colonies). Discriminant analysis was performed on the data for the lipid mixture composition. The discriminant model showed that, in the samples from these two populations, 68.2% and 81.9% of the specimens could be correctly assigned to their colony.     

In Vivo Changes in γ-Aminobutyric Acid Output from the Cerebral Cortex Induced by Inhibitors of γ-Aminobutyric Acid Uptake and Metabolism

Abstract: The effects of inhibitors of γ-aminobutyric acid (GABA) metabolism or uptake on GABA output from the cerebral cortex was studied by means of a collecting cup placed on the exposed cortex of rats anaesthetized with urethane. GABA was identified and quantified by a mass-fragmentographic method. Ethanolamine-O-sulphate (10⁻² M ) applied directly on the cerebral cortex caused a long-lasting twofold increase in GABA output, whereas dl -2, 4-diaminobutyric acid (5 × 10⁻³ M ) caused a sevenfold increase and β -alanine was inactive. The results indicate that glial uptake has little effect on GABA inactivation in the cerebral cortex. The inhibition of neuronal uptake seems a more effective tool to increase GABA concentration in the synaptic cleft, and consequently also in GABA output, than the inhibition of GABA metabolism.