Adenosine A1 and A2A receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices.

6-Hydroxydopamine (6-OHDA) is the most used toxin in experimental Parkinson's disease (PD) models. 6-OHDA shows high affinity for the dopamine transporter and once inside the neuron, it accumulates and undergoes non-enzymatic auto-oxidation, promoting reactive oxygen species (ROS) formation and selective damage of catecholaminergic neurons. In this way, our group has established a 6-OHDA in vitro protocol with rat striatal slices as a rapid and effective model for screening of new drugs with protective effects against PD. We have shown that co-incubation with guanosine (GUO, 100 µM) prevented the 6-OHDA-induced damage in striatal slices. As the exact GUO mechanism of action remains unknown, the aim of this study was to investigate if adenosine A1 (A1R) and/or A2A receptors (A2AR) are involved on GUO protective effects on striatal slices. Pre-incubation with DPCPX, an A1R antagonist prevented guanosine effects on 6-OHDA-induced ROS formation and mitochondrial membrane potential depolarization, while CCPA, an A1R agonist, did not alter GUO effects. Regarding A2AR, the antagonist SCH58261 had similar protective effect as GUO in ROS formation and mitochondrial membrane potential. Additionally, SCH58261 did not affect GUO protective effects. The A2AR agonist CGS21680, although, completely blocked GUO effects. Finally, the A1R antagonist DPCPX, and the A2AR agonist CGS21680 also abolished the preventive guanosine effect on 6-OHDA-induced ATP levels decrease. These results reinforce previous evidence for a putative interaction of GUO with A1R-A2AR heteromer as its molecular target and clearly indicate a dependence on adenosine receptors modulation to GUO protective effect.

Involvement of adenosine A1 and A2A receptors on guanosine-mediated anti-tremor effects in reserpinized mice.

Parkinson's disease (PD) signs and symptoms regularly include tremor. Interestingly, the nucleoside guanosine (GUO) has already proven to be effective in reducing reserpine-induced tremulous jaw movements (TJMs) in rodent models, thus becoming a promising antiparkinsonian drug. Here, we aimed at revealing the mechanism behind GUO antiparkinsonian efficacy by assessing the role of adenosine A1 and A2A receptors (A1R and A2AR) on GUO-mediated anti-tremor effects in the reserpinized mouse model of PD. Reserpinized mice showed elevated reactive oxygen species (ROS) production and cellular membrane damage in striatal slices assessed ex vivo and GUO treatment reversed ROS production. Interestingly, while the simultaneous administration of sub-effective doses of GUO (5 mg/kg) and SCH58261 (0.01 mg/kg), an A2AR antagonist, precluded reserpine-induced TJMs, these were ineffective on reverting ROS production in ex vivo experiments. Importantly, GUO was able to reduce TJM and ROS production in reserpinized mouse lacking the A2AR, thus suggesting an A2AR-independent mechanism of GUO-mediated effects. Conversely, the administration of DPCPX (0.75 mg/kg), an A1R antagonist, completely abolished both GUO-mediated anti-tremor effects and blockade of ROS production. Overall, these results indicated that GUO anti-tremor and antioxidant effects in reserpinized mice were A1R dependent but A2AR independent, thus suggesting a differential participation of adenosine receptors in GUO-mediated effects.

Antibacterial potential of phytochemicals alone or in combination with antimicrobials against fish pathogenic bacteria.

AIMS: This study investigated the antibacterial activity of five phytochemicals (carvacrol, citral, eugenol, linalool and thymol) alone or in combination with florfenicol or oxytetracycline against bacteria isolated from silver catfish (Rhamdia quelen). We also analysed the potential of these compounds to inhibit biofilm formation and haemolysis caused by the bacteria. METHODS AND RESULTS: Bacteria were tested with antimicrobials to calculate the multiple antibiotic resistances. The checkerboard assay was used to evaluate a putative synergy between five phytochemicals and antimicrobials against the strains isolated. The biofilm formation inhibition assay was performed with phytochemicals and antimicrobials, and the haemolysis inhibition assay was performed with the phytochemicals. Carvacrol, eugenol and thymol were the most effective phytochemicals. Three combinations (linalool with florfenicol or oxytetracycline against Aeromonas hydrophila and citral with oxytetracycline against Citrobacter freundii) demonstrated synergy in the checkerboard assay. All phytochemicals inhibited biofilm formation and haemolysis activity. CONCLUSION: The tested phytochemicals showed satisfactory activity against fish pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The phytochemicals did not present antagonistic interactions with the antimicrobials, allowing their combined use, which may contribute to a decrease in the use of conventional drugs and their residues in aquatic environment.

Virulence markers associated with Trueperella pyogenes infections in livestock and companion animals.

Trueperella pyogenes is an opportunistic pathogen that causes diverse pyogenic infections in livestock. The genes that encode the exotoxin pyolysin (plo) and other putative factors that promote adhesion of pathogen to host cells (fimbriae fimA, fimC, fimE, fimG, neuraminidases nanH, nanP, and collagen-binding protein cbpA) have been associated with virulence, particularly in mastitis and uterus infections of dairy cows. However, the role of these virulence markers in the pathogenicity of the agent in domestic animals infections still is incompletely understood. The genes plo, fimA, fimC, fimE, fimG, nanH, nanP, and cbpA were investigated in 71 T. pyogenes strains recovered from cattle, sheep, goats, dogs, equines, and a pig, recovered from mastitis (n = 35), and non-mastitis (n = 36) cases (abscesses, reproductive tract diseases, pneumonia, lymphadenitis, encephalitis). The most common genes harboured by the isolates were: plo (71/71 = 100·0%), fimA (70/71 = 98·6%), nanP (56/71 = 78·9%), fimE (53/71 = 74·6%), fimC (46/71 = 64·8%) and nanH (45/71 = 63·4%), whereas cbpA (6/71 = 8·4%) and fimG (4/71 = 5·6%) were uncommon. The most frequent genotypes were plo/fimA/fimE/fimC/nanH/nanP (17/71 = 23·9%), plo/fimA/fimE/nanH/nanP (13/71 = 18·3%), and plo/fimA/fimE/fimC/nanP (11/71 = 15·5%). No association was observed between the presence of genes vs clinical signs or host species. To the best of our knowledge, this is the first report on aforementioned virulence factors of pathogen detected in diseased horses and dogs. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of particular virulence factors of Trueperella pyogenes that determine different pyogenic infections among domestic animals is poorly understood. Eight putative virulence genes and genotype profiles of 71 isolates were investigated among different clinical manifestations in domestic animals. The most common genes were plo (71/71 = 100·0%), fimA (70/71 = 98·6%), nanP (56/71 = 78·9%), fimE (53/71 = 74·6%), fimC (46/71 = 64·8%) and nanH (45/71 = 63·4%), whereas plo/fimA/fimE/fimC/nanH/nanP (17/71 = 23·9%), plo/fimA/fimE/nanH/nanP (13/71 = 18·3%), and plo/fimA/fimE/fimC/nanP (11/71 = 15·5%) were the most frequent genotypes. Studies involving virulence factors are critical in the investigation of molecular epidemiology, pathogenicity, and hypothetical differences in the virulence among T. pyogenes strains from different geographical areas.

Atorvastatin Prevents Glutamate Uptake Reduction Induced by Quinolinic Acid Via MAPKs Signaling.

Statins have been shown to promote neuroprotection in a wide range of neurological disorders. However, the mechanisms involved in such effects of statins are not fully understood. Quinolinic acid (QA) is a neurotoxin that induces seizures when infused in vivo and promotes glutamatergic excitotoxicity in the central nervous system. The aim of this study was to evaluate the putative glutamatergic mechanisms and the intracellular signaling pathways involved in the atorvastatin neuroprotective effects against QA toxicity. Atorvastatin (10 mg/kg) treatment for 7 days prevented the QA-induced decrease in glutamate uptake, but had no effect on increased glutamate release induced by QA. Moreover, atorvastatin treatment increased the phosphorylation of ERK1 and prevented the decrease in Akt phosphorylation induced by QA. Neither atorvastatin treatment nor QA infusion altered glutamine synthetase activity or the levels of phosphorylation of p38(MAPK) or JNK1/2 during the evaluation. Inhibition of MEK/ERK signaling pathway, but not PI3K/Akt signaling, abolished the neuroprotective effect of atorvastatin against QA-induced decrease in glutamate uptake. Our data suggest that atorvastatin protective effects against QA toxicity are related to modulation of glutamate transporters via MAPK/ERK signaling pathway.

Response to dietary-induced energy restriction in dairy sheep divergently selected for resistance or susceptibility to mastitis.

Dairy ruminants experiencing a severe postpartum negative energy balance (NEB) are considered to be more susceptible to mastitis. Although the genetic variability of mastitis resistance is well established, the biological basis of the link between energy metabolism and resistance is mostly unknown. The aim of this study was to characterize the effect of NEB on metabolism and immune response according to the genetic background for mastitis resistance or susceptibility. Forty-eight ewes from high and low somatic cell score (SCS) genetic lines were allocated to 2 homogeneous subgroups 2 wk after lambing: one group (NEB) received an energy-restricted diet to cover 60% of their energy requirements, and the other group received a control (positive energy balance: PEB) diet. Both diets met the protein requirements. After 10 d on either the NEB or PEB diet, all ewes were injected with a Pam3CSK4/MDP solution in one half-udder to induce an inflammatory response. The ewes were monitored for milk production, somatic cell count (SCC), body weight (BW), body condition score (BCS), and blood metabolites. Differential milk cell counts were determined by flow cytometry. Plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), and triiodothyronine were determined. Energy restriction resulted in an increased fat:protein ratio in milk and decreased milk yield, BW, and BCS. The NEB ewes had significantly higher NEFA and BHB and lower plasma glucose concentrations than PEB ewes, reflecting a mobilization of body reserves and ketone body synthesis. High-SCS ewes had a higher SCS than low-SCS throughout the experiment, except after the inflammatory challenge, which resulted in similar SCS in all 4 groups. A noteworthy interaction between genetic background and diet was evidenced on metabolic parameters and BW. Indeed, high-SCS ewes subjected to NEB showed greater decrease in BW and increased NEFA and BHB concentrations compared with low-SCS ewes. Thus, NEB in early lactation led to extensive mobilization of body reserves and intense ketone body synthesis in mastitis-susceptible sheep. These results reinforce the hypothesis of a genetic association between mastitis susceptibility and energy metabolism and open the way to further studies on the biological basis for this association.

Effect of acute and chronic excesses of dietary nitrogen on blood neutrophil functions in cattle.

Excess dietary nitrogen (EDN) is commonly expected in dairy herds, but no data are available regarding its consequences on cattle immunity. In this study neutrophil functions were assessed during EDN in steers. In experiment 1, 4 one-month periods, 4 diets [16% crude protein (CP; DM basis), 20% CP based on soybean meal, 20% CP based on urea, and 24% CP based on urea and soybean meal], and 4 steers were included in a crossover design to determine the effects of a chronic excess. In experiment 2, the repercussions of an acute excess were assessed with 2 periods of 10 d, the same 4 steers, and 2 diets containing 14 and 20% CP. Sampling was done during the fourth week of each period in experiment 1, and on d 0, 1, 2, 3, 7, and 9 of each period in experiment 2. Individual blood biochemistry parameters were measured and neutrophil factors, such as counts, recovery after isolation, surface expression of CD11b and CD62L, phagocytosis, diapedesis, reactive oxygen species (ROS) production, and bacteria killing, were determined. Data were analyzed by general linear models of R, with period, diet or biochemical component, and animal as explanatory variables. The outcome variables were biochemical or immune variables. The variables diet, period, and animal were forced as fixed effects. Data collected over the entire period of experiment 2 were pooled. Several multiples linear regressions or ANOVA were performed and a Bonferroni correction was applied. In experiment 2 (acute EDN), neutrophil counts were negatively associated with nitrogen intake, conversely to CD62L expression. The observed relative neutropenia may be due to neutrophil margination because CD62L-expressing neutrophils are more likely to stick to endothelium. Interestingly, ROS production was changed by EDN: chronic EDN (experiment 1) was negatively associated with opsonized zymozan (OZ)-induced ROS production and acute EDN (experiment 2) with spontaneous ROS production. For chronic EDN, ROS production upon phorbol 12-myristate 13-acetate was not modified, in contrast to OZ stimulation. Decreased ROS production during chronic EDN probably involves the early events leading to ROS production, as OZ acts through membrane receptors and phorbol 12-myristate 13-acetate directly activates protein kinase C. This is the first study to provide evidence that the modifications of neutrophil functions produced by excess nitrogen depend on the intensity and duration of the excess. Further studies, including epidemiological studies during risk periods, are needed to resolve the issues linked to EDN.

Infection of nonhost species dendritic cells in vitro with an attenuated myxoma virus induces gene expression that predicts its efficacy as a vaccine vector.

Recombinant myxoma virus (MYXV) can be produced without a loss of infectivity, and its highly specific host range makes it an ideal vaccine vector candidate, although careful examination of its interaction with the immune system is necessary. Similar to rabbit bone marrow-derived dendritic cells (BM-DCs), ovine dendritic cells can be infected by SG33, a MYXV vaccine strain, and support recombinant antigen expression. The frequency of infected cells in the nonhost was lower and the virus cycle was abortive in these cell types. Among BM-DC subpopulations, Langerhans cell-like DCs were preferentially infected at low multiplicities of infection. Interestingly, ovine BM-DCs remained susceptible to MYXV after maturation, although apoptosis occurred shortly after infection as a function of the virus titer. When gene expression was assessed in infected BM-DC cultures, type I interferon (IFN)-related and inflammatory genes were strongly upregulated. DC gene expression profiles were compared with the profiles produced by other poxviruses in interaction with DCs, but very few commonalities were found, although genes that were previously shown to predict vaccine efficacy were present. Collectively, these data support the idea that MYXV permits efficient priming of adaptive immune responses and should be considered a promising vaccine vector along with other poxviruses.

Ontology-Driven Real World Evidence Extraction from Clinical Narratives.

Unstructured clinical notes contain a huge amount of information. We investigated the possibility of harvesting such information through an NLP-based approach. A manually curated ontology is the only resource required to handle all the steps of the process leading from clinical narrative to a structured data warehouse (i2b2). We have tested our approach at the Papa Giovanni XXIII hospital in Bergamo (Italy) on pathology reports collected since 2008.

The bacterial cytolethal distending toxin (CDT) triggers a G2 cell cycle checkpoint in mammalian cells without preliminary induction of DNA strand breaks.

The bacterial cytolethal distending toxin (CDT) was previously shown to arrest the tumor-derived HeLa cell line in the G2-phase of the cell cycle through inactivation of CDK1, a cyclin-dependent kinase whose state of activation determines entry into mitosis. We have analysed the effects induced in HeLa cells by CDT, in comparison to those induced by etoposide, a prototype anti-tumoral agent that triggers a G2 cell cycle checkpoint by inducing DNA damage. Both CDT and etoposide inhibit cell proliferation and induces the formation of enlarged mononucleated cells blocked in G2. In both cases, CDK1 from arrested cells could be reactivated both in vitro by dephosphorylation by recombinant Cdc25B phosphatase and in vivo by caffeine. However, the cell cycle arrest triggered by CDT, unlike etoposide, did not originate from DNA strand breaks as demonstrated in the single cell gel electrophoresis assay and by the absence of slowing down of S phase in synchronized cells. Together with additional observations on synchronized HeLa cells, our results suggest that CDT triggers a G2 cell cycle checkpoint that is initiated during DNA replication and that is independent of DNA damage.

N-methyl-D-aspartate preconditioning prevents quinolinic acid-induced deregulation of glutamate and calcium homeostasis in mice hippocampus.

The search for new therapeutic strategies through modulation of glutamatergic transmission using effective neuroprotective agents is essential. Glutamatergic excitotoxicity is a major factor common to neurodegenerative diseases and in acute events such as cerebral ischemia, traumatic brain injury and epilepsy. We have previously demonstrated that N-methyl-D-aspartate (NMDA) preconditioning in mice showed 50 % of protection against seizures and full protection against damage to neuronal tissue induced by quinolinic acid (QA). In this study, cellular and molecular mechanisms involved on NMDA preconditioning and neuroprotection were investigated in mice treated with NMDA 24 h before QA insult. Calcium uptake and D-aspartate release from hippocampal slices obtained from mice treated with NMDA plus QA and not displaying seizures (protected mice) were similar to control (saline) or NMDA preconditioned mice. Increased calcium uptake and glutamate release is evidenced in unprotected (convulsed) mice as well as QA control, demonstrating that calcium and glutamate are involved in NMDA-induced preconditioning. Increased glutamate release evoked by QA was blocked by MK-801, whereas increased calcium uptake was abolished by voltage-dependent calcium channels inhibitors, but not MK-801. NMDA preconditioning is effective in normalizing the deregulation of glutamate transport and calcium homeostasis evoked by QA due to aberrant NMDA receptors activation that culminates in seizures and hippocampal cells damage.

Sequence of lethal events in HeLa cells exposed to the G2 blocking cytolethal distending toxin.

The bacterial cytolethal distending toxin (CDT) was previously shown to block the cell cycle of several cell lines at stage G2 through inactivation of the cyclin-dependent kinase Cdkl and without induction of DNA strand breaks. In the present study, we have analyzed, using various methods of analytical cytometry, the progressive transformation and delayed lethal events in the tumor-derived HeLa cell line temporarily exposed to CDT. The cell proliferation arrest induced by CDT was irreversible but, starting about two days after exposure, the G2 block released partially, concomitantly with a decline in the level of Cdkl phosphorylation. This partial release resulted in endoreduplication, leading to the emergence of a significant subpopulation of cells with a 8C DNA content, and by multipolar abortive mitosis which accounted for the mortality recorded 2 and 3 days after exposure. The other major lethal event was a micronucleation process which started to be significant about 3 days after exposure and amplified later on. Both multipolar abortive mitosis and micronucleation appeared topologically related to centrosomal amplification.

Intimal thickenings of human aorta contain modified reassembled lipoproteins.

The aim of this study was to determine whether in human aortas early minute changes such as minimal intimal thickenings (MIT), developed in areas known to have a predilection to atherosclerosis, contain modified reassembled lipoproteins (MRLp) such as extracellular liposomes (EL) and lipid droplets (LD). These features have been previously detected in the aortic lesion-prone areas of rabbits and hamsters fed a fat-rich diet. Tissue samples of the aortic arch and thoracic aorta from 12 young subjects who died in accidents were selectively collected from grossly normal regions. By light microscopy, some of these regions were found to contain MIT. The normal areas and the MIT were separately examined by electron microscopy or subjected to fractionation and partial biochemical characterization. The MIT (approximately 25-100 microns thick) were constituted by a pronounced proliferation of extracellular matrix, especially elastin and microfibrils, with interspersed lipid deposits appearing as EL and LD. Commonly, MIT did not contain smooth muscle cells, macrophages, foam cells or cytolytic debris. Such components were only occasionally found in specimens excised from the vicinity of fatty streaks. Saline extracts of MIT or grossly normal aortic regions were subjected to a four-step purification procedure consisting of gel filtration, affinity chromatography on anti-apo B and anti-albumin Sepharose, followed by density gradient ultracentrifugation. The entire procedure was monitored by negative staining, lipid assays, SDS PAGE and immunoblotting. From the initial MRLp mixture, two fractions were obtained: fraction 1 containing multilamellar EL and LD, and fraction 2 composed mostly of unilamellar EL. As compared with serum LDL, the cholesteryl ester/unesterified cholesterol ratio was 4-6-fold lower in fraction 1 and 15-19-fold lower in fraction 2. On SDS-PAGE the fraction 2 displayed a single protein band of 66 kDa, immunochemically identified as albumin. The MRLp isolated from human aortas with minimal intimal thickenings appeared to be similar to those purified from the prelesional stage aorta of hyperlipidemic rabbits and hamsters.

Effects of guanine nucleotides on adenosine and glutamate modulation of cAMP levels in optic tectum slices from chicks.

Glutamate and adenosine both modulate adenylyl cyclase activity through interaction of their specific receptors with stimulatory or inhibitory G-proteins. Guanine nucleotides (GN), which modulate G-protein activity intracellularly, are also involved in the inhibition of glutamate responses, acting from the outside of the cells. We had previously reported that glutamate inhibits adenosine-induced cyclic AMP (cAMP) accumulation in slices obtained from the optic tectum of chicks. In the present study we investigated the interaction of GN with these two neurotransmitters and found that GN inhibit the inhibitory effect of glutamate on adenosine-induced cAMP accumulation and potentiate adenosine-induced cAMP accumulation. These effects were observed with 5'-guanylylimidodiphosphate (GppNHp) or GMP, but not with guanosine (the nucleoside). Besides, these interactions of GN occur via a metabotropic glutamate receptor (mGluR) sensitive to (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 S,3R-ACPD) but not to L-2-amino-4-phosphonobutyrate (L-AP4). These effects were partially modulated by a mGluR antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine ((RS)M-CPG), and by an adenosine receptor antagonist, 8-phenyltheophylline. GN only potentiated the adenosine response when adenosine was acting through its receptor positively linked to adenylyl cyclase. Therefore, the data show that guanine nucleotides not only inhibit glutamate-induced responses, but also stimulate adenosine-induced responses, a fact that may contribute to the understanding of the physiological functions of guanine nucleotides.

Malnutrition increases insoluble-to-soluble tubulin ratio and in vitro incorporation of 32ATP in rat cerebral cortex.

Wistar rats were fed a normal protein (25% casein) or an isoenergetic low protein (8% casein) diet from the day of birth to weaning on day 21. Litters were killed at weaning and cerebral cortex was removed. Tubulin was prepared by centrifugation at 100,000 g, 4 degrees C, as described by Shelansky et al. [Proc. Natn. Acad. Sci. U.S.A. 70, 765-768 (1973)]. Cold-insoluble tubulin was recovered in the pellet (P1) fraction and cold-soluble tubulin in the supernatant (S1) fraction. Alpha and beta tubulin were quantified by electrophoretic and immunological methods in both fractions. Our results indicated that malnutrition enhanced the ratio of cold-insoluble-tubulin-to-cold-soluble-tubulin. Furthermore malnutrition induced an increased in vitro incorporation of 32P into both soluble and insoluble tubulins. Although tubulin phosphorylation has been related to tubulin stability properties, we cannot unequivocally ascribe the increased insoluble/soluble tubulin ratio with malnutrition to increased in vitro incorporation of 32P.

Guanine nucleotides inhibit the stimulation of GFAP phosphorylation by glutamate.

Phosphorylation of the astrocytic marker protein glial fibrillary acidic protein (GFAP) in hippocampal slices from immature rats is stimulated by glutamate agonists via a metabotropic receptor. In this study we investigated the modulation of this stimulation by guanine nucleotides. Recent work has shown that guanine nucleotides inhibit the binding of kainate to its receptors in a manner independent of G proteins. Gpp(NH)p, GDP-beta-S and GMP inhibited by approximately 50% the stimulation of GFAP phosphorylation by glutamate or 1S,3R-ACPD. In the case of glutamate and Gpp(NH)p it was shown that the inhibition was dose dependent. These results indicate that guanine nucleotides can inhibit glutamate-stimulated phosphorylation responses by interaction with a cell surface metabotropic receptor.

Chick kainate binding protein lacks GTPase activity.

Chick kainate binding protein was solubilized from cerebellar membranes and purified (x19) by use of two chromatographic steps. Measurements of [3H]kainate binding and GTPase activity in the different fractions reveal a consistent decrease of GTPase activity as the purification proceeds so that no GTPase is detectable after the final purification step. This fact, in the context of the differential involvement in nucleotide recognition of some critical amino acid residues in the p-loop motif of GTPases and in the guanine nucleotide-binding sequence of ionotropic glutamate receptors, together with significant discrepancies concerning the activity of individual nucleotides, suggests that both guanine nucleotide-recognizing sequences are unlikely to be alternative expressions of the same functional domain.

Quinolinic acid inhibits glutamate uptake into synaptic vesicles from rat brain.

Quinolinic acid (QA) is an endogenous and potent neurotoxin associated with the neurotoxicity of various common diseases. The uptake of neurotransmitters into synaptic vesicles is an important event involved in the storage and release of neurotransmitters by vesicles. The influence of QA on the uptake of glutamate, GABA and glycine into rat brain synaptic vesicles was investigated. QA (0.3-10 mM) significantly inhibited (>50%) the uptake of glutamate into synaptic vesicles, whereas QA at concentrations up to 10 mM had no significant effect on GABA or glycine uptake. Such results indicate that QA is able to selectively inhibit the vesicular uptake of glutamate, without interfering with the uptake of the inhibitory neurotransmitters GABA and glycine. These findings might be related to the neurotoxic effects of QA in the brain.

Effects of adenosine on cAMP production during early development in the optic tectum of chicks.

Accumulation of cyclic adenosine monophosphate (cAMP) elicited by adenosine was studied in slices and membrane preparations of optic tectum from chicks aged 1-13 days post-hatch. Accumulation of cAMP promoted by adenosine declined with age, the highest value being observed in three-day-old chicks and the lowest in 11-day-old chicks. However, when the slices were incubated with adenosine and the phosphodiesterase inhibitor-Ro 20-1724 the differences between the two ages were abolished, suggesting a higher phosphodiesterase activity in 11-day-old chicks. In membrane preparations, although basal adenylate cyclase activity was lower in three-day-old chicks, the guanylyl-imidodiphosphate (Gpp(NH)p) concentration curves for stimulation of adenylate cyclase activity indicated a higher sensitivity of G protein to Gpp(NH)p at this age. This hypothesis was reinforced by the observation that the binding of [3H]Gpp(NH)p to the membrane preparation was greater in three-day-old animals. In spite of these differences, the percentage of adenylate cyclase activity stimulation by 2-chloroadenosine (2CADO)+Gpp(NH)p was the same at both ages. These findings suggest that the decreased response evoked by adenosine during development is probably due to increased phosphodiesterase activity and a lower sensitivity of adenylate cyclase activity to Gpp(NH)p.