Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin

We have analyzed the anti- or pro-oxidant effects of the flavonoid quercetin (QU) by evaluating, in U937 cell line, hydrogen peroxide (H(2)O(2)), superoxide anion reduced glutathione (GSH) content, mitochondrial membrane potential, DNA content, phosphatidylserine exposure on the outer face of the plasma membrane and cell viability. Polychromatic flow cytometry was used to evaluate in the same cells several functional parameters. For short periods of treatment QU exerted an anti-oxidant effect (decrease in H(2)O(2) levels), whereas for long periods it showed a pro-oxidant activity (increase in ). In these conditions, GSH content was reduced, and this correlated with a lack of anti-oxidant activity of QU, which in turn could be correlated with proapoptotic activity of this molecule. Thus, QU can exert different effects (anti-/prooxidant) depending on exposure times and oxidative balance, and in particular on stores of GSH.     

Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells

Neisseria meningitidis is a human pathogen, which is a major cause of sepsis and meningitis. The bacterium colonizes the upper respiratory tract of approximately 10% of humans where it lives as a commensal. On rare occasions, it crosses the epithelium and reaches the bloodstream causing sepsis. From the bloodstream it translocates the blood-brain barrier, causing meningitis. Although all strains have the potential to cause disease, a subset of them, which belongs to hypervirulent lineages, causes disease more frequently than others. Recently, we described NadA, a novel antigen of N. meningitidis, present in three of the four known hypervirulent lineages. Here we show that NadA is a novel bacterial invasin which, when expressed on the surface of Escherichia coli, promotes adhesion to and invasion into Chang epithelial cells. Deletion of the N-terminal globular domain of recombinant NadA or pronase treatment of human cells abrogated the adhesive phenotype. A hypervirulent strain of N. meningitidis where the nad A gene was inactivated had a reduced ability to adhere to and invade into epithelial cells in vitro. NadA is likely to improve the fitness of N. meningitidis contributing to the increased virulence of strains that belong to the hypervirulent lineages.     

Changes in the tobacco leaf apoplast proteome in response to salt stress

The apoplast of plant cells is a dynamic compartment involved in many processes, including maintenance of tissue shape, development, nutrition, signalling, detoxification and defence. In this work we used Nicotiana tabacum plants as a model to investigate changes in the soluble apoplast composition induced in response to salt stress. Apoplastic fluid was extracted from leaves of control plants and plants exposed to salt stress, using a vacuum infiltration procedure. Two-dimension electrophoretic analyses revealed about 150 polypeptide spots in the pH range of 3.0 to 10.0, in independent protein extracts, with a high level of reproducibility between the two sample sets. Quantitative evaluation and statistical analyses of the resolved spots in treated and untreated samples revealed 20 polypeptides whose abundance changed in response to salt stress. Mass spectroscopic peptide separation and sequencing was used to identify polypeptides affected by salt stress. While the levels of some proteins were reduced by salt-treatment, an enhanced accumulation of protein species known to be induced by biotic and abiotic stresses was observed. In particular, two chitinases and a germin-like protein increased significantly and two lipid transfer proteins were expressed entirely de novo. Some apoplastic polypeptides, involved in cell wall modifications during plant development, remained largely unchanged. The significance of these components is discussed in the context of stress responses in plants.     

Adaptor protein ARH is recruited to the plasma membrane by low density lipoprotein (LDL) binding and modulates endocytosis of the LDL/LDL receptor complex in hepatocytes

ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 degrees C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.     

Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus

The proton-pumping and the ATP hydrolysis activities of the ATP synthase of Rhodobacter capsulatus have been compared as a function of the ADP and P(i) concentrations. The proton pumping was measured either with the transmembrane pH difference probe, 9-amino-6-chloro-2-methoxyacridine, or with the transmembrane electric potential difference probe, bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol, obtaining consistent results. The comparison indicates that an intrinsic uncoupling of ATP synthase is induced when the concentration of either ligand is decreased. The half-maximal effect was found in the submicromolar range for ADP and at about 70 microM for P(i). It is proposed that a switch from a partially uncoupled state of ATP synthase to the coupled state is induced by the simultaneous binding of ADP and P(i).     

Differential phosphorylation of c-Jun and JunD in response to the epidermal growth factor is determined by the structure of MAPK targeting sequences

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation.     

Opposite modulatory roles for adenosine A1 and A2A receptors on glutamate and dopamine release in the shell of the nucleus accumbens. Effects of chronic caffeine exposure

Previous studies have demonstrated opposing roles for adenosine A1 and A2A receptors in the modulation of extracellular levels of glutamate and dopamine in the striatum. In the present study, acute systemic administration of motor-activating doses of the A2A receptor antagonist MSX-3 significantly decreased extracellular levels of dopamine and glutamate in the shell of the rat nucleus accumbens (NAc) and counteracted both dopamine and glutamate release induced by systemic administration of motor-activating doses of either the A1 receptor antagonist CPT or caffeine. Furthermore, exposure to caffeine in the drinking water (1 mg/mL, 14 days) resulted in tolerance to the effects of systemic injection of CPT or caffeine, but not MSX-3, on extracellular levels of dopamine and glutamate in the NAc shell. The present results show: first, the existence of opposite tonic effects of adenosine on extracellular levels of dopamine and glutamate in the shell of the NAc mediated by A1 and A2A receptors; second, that complete tolerance to caffeine's dopamine- and glutamate-releasing effects which develops after chronic caffeine exposure is attributable to an A1 receptor-mediated mechanism. Development of tolerance to the dopamine-releasing effects of caffeine in the shell of the NAc may explain its weak addictive properties and atypical psychostimulant profile.     

Acetylcholinesterase inhibitors increase ADAM10 activity by promoting its trafficking in neuroblastoma cell lines

Acetylcholinesterase inhibitors (AChEIs) are the only currently available drugs for treating Alzheimer's Disease (AD). Some authors have suggested a function of AChEIs not only in the induction of AChE overproduction and alternative splicing shifts but also a possible role of these drugs in amyloid metabolism beyond their well-known symptomatic effect. Here, we investigate the mechanisms of action of the AChEI donepezil on APP (amyloid precursor protein) metabolism and on the activity/trafficking of the alpha-secretase candidate ADAM 10, in differentiated human neuroblastoma cells (SH-SY5Y). In these cells, the activity of AChE is significantly decreased after 2 h of donepezil treatment. Further, SH-SY5Y cells released significantly more sAPPalpha into the medium, whereas total APP levels in cell lysates were unchanged. Interestingly, treated cells showed increased ADAM 10 levels in membrane compartments. This effect was prevented by pretreatment with tunicamycin or brefeldin, suggesting that donepezil affects trafficking and/or maturation of ADAM 10; additionally, this pretreatment significantly decreased sAPPalpha levels. Pre-incubation with atropine decreased release of sAPPalpha significantly but did not revert ADAM 10 activity to control levels further suggesting that donepezil acts not solely through a purely receptor mediated pathway. These findings indicate that donepezil exerts multiple mechanisms involving processing and trafficking of key proteins involved in AD pathogenesis.     

Using drug-discrimination techniques to study the abuse-related effects of psychoactive drugs in rats

Drug-discrimination (DD) techniques can be used to study abuse-related effects by establishing the interoceptive effects of a training drug (e.g., cocaine) as a cue for performing a specific operant response (e.g., lever pressing reinforced by food). During training with this protocol, pressing one lever is reinforced when the training drug is injected before the start of the session, and responding on a second lever is reinforced when vehicle is injected before the session. Lever choice during test sessions can then be used as an indication of whether a novel drug has effects similar to the training drug, or whether a potential therapeutic alters the effects of the training drug. Although training can be lengthy (up to several months), the pharmacological specificity of DD procedures make them a perfect complement to other techniques used to study drug-abuse phenomena, such as intravenous self-administration and conditioned place-preference procedures.