Joana M. Marques

Saccharomyces cerevisiae Hog1 Protein Phosphorylation upon Exposure to Bacterial Endotoxin

The yeast Hog1 protein is both functionally and structurally similar to the mammalian p38, belonging to the same family of mitogen-activated protein (MAP) kinases and responding to extracellular changes in osmolarity. Since p38 mediates lipopolysaccharide (LPS) effects in mammalian cells, we now tested the responsiveness of Hog1 upon exposure of the yeast Saccharomyces cerevisiae to bacterial LPS. In the presence of Escherichia coli LPS (100 ng/ml) and an endotoxically active, hexaacylated, synthetic lipid A (compound 506; 100 ng/ml), Hog1 becomes phosphorylated with a maximum of phosphorylation between 3 and 6 h, whereas a tetraacylated, inactive form of lipid A (compound 406) did not cause any modification in the phosphorylation state of Hog1. A triple labeling immunocytochemical study showed that phosphorylated Hog1 translocates into the nucleus after a 90-min incubation and becomes sparsely located in the cytoplasm. The translocation of the phospho-Hog1 is preceded by an increased expression of the HOG1 gene and concomitant with the expression of the Hog1 target gene, GPD1. We also observed that cells unable to synthesize Hog1 do not resist LPS as efficiently as wild-type cells. We conclude that the yeast S. cerevisiae is able to respond to the presence of Gram-negative bacteria endotoxin and that Hog1 is involved in this response.

Presynaptic adenosine A2A receptors dampen cannabinoid CB1 receptor-mediated inhibition of corticostriatal glutamatergic transmission.

Both cannabinoid CB1 and adenosine A2A receptors (CB1 receptors and A2A receptors) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A postsynaptic CB1−A2A receptor interaction has already been elucidated, but the presynaptic A2A receptor‐mediated control of presynaptic neuromodulation by CB1 receptors remains to be defined. Because the corticostriatal terminals provide the major input to the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions.

Presynaptic A2A adenosine receptors dampen CB1 cannabinoid receptor-mediated inhibition of corticostriatal glutamatergic transmission

Both cannabinoid CB1 and adenosine A2A receptors (CB1 receptors and A2A receptors) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A postsynaptic CB1−A2A receptor interaction has already been elucidated, but the presynaptic A2A receptor‐mediated control of presynaptic neuromodulation by CB1 receptors remains to be defined. Because the corticostriatal terminals provide the major input to the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions.

Adenosine A2b receptors control A1 receptor‐mediated inhibition of synaptic transmission in the mouse hippocampus

Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A2BR) are Gs/q‐protein‐coupled receptors with low expression in the brain. As A2BR function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A2BR‐knockout (A2BR‐KO) and C57/BL6 mice were submitted to a behavioural test battery (open field, elevated plus‐maze, Y‐maze). The A2BR agonist BAY60‐6583 (300 nm) decreased the paired‐pulse stimulation ratio, an effect prevented by the A2BR antagonist MRS 1754 (200 nM) and abrogated in A2BR‐KO mice. Accordingly, A2BR immunoreactivity was present in 73 ± 5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60‐6583 attenuated the A1R control of synaptic transmission, both the A1R inhibition caused by 2‐chloroadenosine (0.1–1 μm) and the disinhibition caused by the A1R antagonist DPCPX (100 nm), both effects prevented by MRS 1754 and abrogated in A2BR‐KO mice. BAY 60‐6583 decreased glutamate release in slices and also attenuated the A1R inhibition (CPA 100 nm). A2BR‐KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus‐maze and the Y‐maze and no alteration of locomotion, anxiety or working memory. We conclude that A2BR are present in hippocampal glutamatergic terminals where they counteract the predominant A1R‐mediated inhibition of synaptic transmission, impacting on exploratory behaviour.

Retrospective analysis of clinical yeast isolates in a hospital in the centre of Portugal: spectrum and revision of the identification procedures.

Abstract We conducted a four-year (2003-2006) retrospective study of yeasts recovered in a hospital laboratory in the centre of Portugal to evaluate the epidemiology of yeast infections. Clinical isolates and data were gathered from 751 patients corresponding to 906 episodes of yeast infection. The isolates were first identified using classical and commercial methods, routinely employed at the hospital laboratory. We then re-identified the same isolates using RFLP of the ITS 5.8S rRNA gene and sequence of the D1/D2 domain of the 26S rRNA gene. Candida parapsilosis isolates were re-identified using the Ban I digestion of the SADH gene. C. albicans was the most frequently isolated of the yeasts found in the analysed specimens, with an overall incidence of 69.6% and then in decreasing order, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei. C. parapsilosis was most frequently recovered from younger patients, decreasing with age, while C. glabrata occurrence increased with age. We found an increased number of cases of fungemia per 100,000 people per year, reaching a maximum of 4.4 during 2006.

Hierarchical glucocorticoid-endocannabinoid interplay regulates the activation of the nucleus accumbens by insulin.

Here we asked if insulin activation of the nucleus accumbens in vitro is reflected by an increase in (3)H-deoxyglucose ([(3)H]DG) uptake, thus subserving a new model to study molecular mechanisms of central insulin actions. Additionally, we investigated the dependence of this insulin effect on endocannabinoids and corticosteroids, two major culprits in insulin resistance. We found that in acute accumbal slices, insulin (3 and 300nM but not at 0.3nM) produced an increase in [(3)H]DG uptake. The synthetic cannabinoid agonist, WIN55212-2 (500nM) and the glucocorticoid dexamethasone (10μM), impaired insulin (300nM) action on [(3)H]DG uptake. The glucocorticoid receptor (GcR) antagonist, mifepristone (10μM) prevented dexamethasone from inhibiting insulins action. Strikingly, this anti-insulin action of dexamethasone was also blocked by two CB1 cannabinoid receptor (CB1R) antagonists, O-2050 (500nM) and SR141716A (500nM), as well as by tetrahydrolipstatin (10μM), an inhibitor of diacylglycerol lipases-the enzymes responsible for the synthesis of the endocannabinoid, 2-arachidonoyl-glycerol (2-AG). On the other hand, the blockade of the post-synaptic 2-AG metabolizing enzymes, α,β-serine hydrolase domain 6/12 by WWL70 (1μM) also prevented the action of insulin, probably via increasing endogenous 2-AG tone. Additionally, an anti-insulin receptor (InsR) antibody immunoprecipitated CB1Rs from accumbal homogenates, indicating a physical complexing of CB1Rs with InsRs that supports their functional interaction. Altogether, insulin stimulates glucose uptake in the nucleus accumbens. Accumbal GcR activation triggers the synthesis of 2-AG that in turn binds to the known CB1R-InsR heteromer, thus impeding insulin signaling.

Presynaptic P2X1-3 and α3-containing nicotinic receptors assemble into functionally interacting ion channels in the rat hippocampus.

Previous studies documented a cross-talk between purinergic P2X (P2XR) and nicotinic acetylcholine receptors (nAChR) in heterologous expression systems and peripheral preparations. We now investigated if this occurred in native brain preparations and probed its physiological function. We found that P2XR and nAChR were enriched in hippocampal terminals, where both P2X1-3R and α3, but not α4, nAChR subunits were located in the active zone and in dopamine-β-hydroxylase-positive hippocampal terminals. Notably, P2XR ligands displaced nAChR binding and nAChR ligands displaced P2XR binding to hippocampal synaptosomes. In addition, a negative P2XR/nAChR cross-talk was observed in the control of the evoked release of noradrenaline from rat hippocampal synaptosomes, characterized by a less-than-additive facilitatory effect upon co-activation of both receptors. This activity-dependent cross-inhibition was confirmed in Xenopus oocytes transfected with P2X1-3Rs and α3β2 (but not α4β2) nAChR. Besides, P2X2 co-immunoprecipitated α3β2 (but not α4β2) nAChR, both in HEK cells and rat hippocampal membranes indicating that this functional interaction is supported by a physical association between P2XR and nAChR. Moreover, eliminating extracellular ATP with apyrase in hippocampal slices promoted the inhibitory effect of the nAChR antagonist tubocurarine on noradrenaline release induced by high- but not low-frequency stimulation. Overall, these results provide integrated biochemical, pharmacological and functional evidence showing that P2X1-3R and α3β2 nAChR are physically and functionally interconnected at the presynaptic level to control excessive noradrenergic terminal activation upon intense synaptic firing in the hippocampus.

Glutamate-induced and NMDA receptor-mediated neurodegeneration entails P2Y1 receptor activation

Despite the characteristic etiologies and phenotypes, different brain disorders rely on common pathogenic events. Glutamate-induced neurotoxicity is a pathogenic event shared by different brain disorders. Another event occurring in different brain pathological conditions is the increase of the extracellular ATP levels, which is now recognized as a danger and harmful signal in the brain, as heralded by the ability of P2 receptors (P2Rs) to affect a wide range of brain disorders. Yet, how ATP and P2R contribute to neurodegeneration remains poorly defined. For that purpose, we now examined the contribution of extracellular ATP and P2Rs to glutamate-induced neurodegeneration. We found both in vitro and in vivo that ATP/ADP through the activation of P2Y1R contributes to glutamate-induced neuronal death in the rat hippocampus. We found in cultured rat hippocampal neurons that the exposure to glutamate (100 µM) for 30 min triggers a sustained increase of extracellular ATP levels, which contributes to NMDA receptor (NMDAR)-mediated hippocampal neuronal death through the activation of P2Y1R. We also determined that P2Y1R is involved in excitotoxicity in vivo as the blockade of P2Y1R significantly attenuated rat hippocampal neuronal death upon the systemic administration of kainic acid or upon the intrahippocampal injection of quinolinic acid. This contribution of P2Y1R fades with increasing intensity of excitotoxic conditions, which indicates that P2Y1R is not contributing directly to neurodegeneration, rather behaving as a catalyst decreasing the threshold from which glutamate becomes neurotoxic. Moreover, we unraveled that such excitotoxicity process began with an early synaptotoxicity that was also prevented/attenuated by the antagonism of P2Y1R, both in vitro and in vivo. This should rely on the observed glutamate-induced calpain-mediated axonal cytoskeleton damage, most likely favored by a P2Y1R-driven increase of NMDAR-mediated Ca2+ entry selectively in axons. This may constitute a degenerative mechanism shared by different brain diseases, particularly relevant at initial pathogenic stages.

Towards improved adsorption of phenolic compounds by surface chemistry tailoring of silica aerogels

The high toxicity/volatility and low biodegradation of phenolic compounds are serious concerns in terms of environmental and health impact—their recommended max. value for drinking water is 0.005 mg/L. They are usually removed from effluents by adsorption, but they show a complex interaction behavior with adsorbents, because the hydroxyl group and the hydrophobic aromatic ring are very close. In this work, the versatility of Si chemistry was explored to tailor the surface chemistry of silica aerogels and improve their adsorption performance towards phenolic compounds. Methyltrimethoxysilane and tetramethylorthosilicate were combined to adjust the hydrophobicity of the obtained aerogels. In the next stage, β-cyclodextrin, with its highly hydrophobic cavity, was grafted into the gels to improve the capturing of aromatic rings. For a sustainable linkage of β-cyclodextrin to silica, the methyltrimethoxysilane/tetramethylorthosilicate precursor system was modified by adding an epoxy functionalized silane. A first screening of the adsorption performance shows a 1.5–2-fold increase of the adsorption capacity and removal efficiencies of the epoxy-cyclodextrin-modified aerogel toward phenol and p-cresol when compared to aerogel counterpart without modification. Freundlich isotherm model was the most suitable to describe the equilibrium data of aerogels with or without β-cyclodextrin, with the curves showing favorable profiles, more evident in the case of aerogels with β-cyclodextrin. Apart from the improving of the sorption capacity for phenolic compounds (achieving a maximum of 60 mg g−1 in the case of p-cresol), the utilization of the biodegradable β-cyclodextrin moiety obtained from natural and sustainable resources is a further asset of the epoxy-cyclodextrin-modified aerogel.Graphical Abstract