Abscisic Acid Activates the Murine Microglial Cell Line N9 through the Second Messenger Cyclic ADP-ribose

Abscisic acid (ABA) is a phytohormone regulating important functions in higher plants, notably responses to abiotic stress. Recently, chemical or physical stimulation of human granulocytes was shown to induce production and release of endogenous ABA, which activates specific cell functions. Here we provide evidence that ABA stimulates several functional activities of the murine microglial cell line N9 (NO and tumor necrosis factor-α production, cell migration) through the second messenger cyclic ADP-ribose and an increase of intracellular calcium. ABA production and release occur in N9 cells stimulated with bacterial lipopolysaccharide, phorbol myristate acetate, the chemoattractant peptide f-MLP, or β-amyloid, the primary plaque component in Alzheimer disease. Finally, ABA priming stimulates N9 cell migration toward β-amyloid. These results indicate that ABA is a pro-inflammatory hormone inducing autocrine microglial activation, potentially representing a new target for anti-inflammatory therapies aimed at limiting microglia-induced tissue damage in the central nervous system.Microglial cells are the monocyte/macrophage equivalent of the central nervous system and represent the first line of defense in the brain, by removing infectious agents and damaged cells (1). Microglia can also release a variety of trophic factors and cytokines able to regulate the communication between neuronal and other glial cells and can contribute to tissue repair and neuroprotection (2–4). Pathologic microglial activation, however, confers neurotoxic properties to these cells, thereby causing neuronal degeneration (5). Excessive activation of microglia, under conditions of chronic inflammation, can contribute to the pathogenesis of neurodegenerative diseases, such as multiple sclerosis and Alzheimer and Parkinson diseases, by producing and releasing a number of potentially cytotoxic substances, including pro-inflammatory cytokines and NO (4, 6–8). Therefore, identification of the molecular mechanisms underlying microglial activation might lead to the development of new anti-inflammatory drugs for the treatment of these diseases.Abscisic acid (ABA)² is a plant hormone regulating important biological functions in higher plants, including response to abiotic stress, control of stomatal closure, regulation of seed dormancy, and germination (9). Recently, ABA was shown to behave as an endogenous pro-inflammatory hormone in human granulocytes (10), stimulating several functional activities of these cells (migration, phagocytosis, reactive oxygen species, and NO production) through a signaling cascade that involves a protein kinase A-mediated ADP-ribosyl cyclase phosphorylation and consequent overproduction of the universal Ca²⁺ mobilizer cyclic ADP-ribose (cADPR) (11). This mechanism leads to an increase of the intracellular Ca²⁺ concentration, which is ultimately responsible for granulocyte activation (10).The facts that microglial cells play a defensive role in the central nervous system similar to that of granulocytes in other tissues and that cADPR has been described as the second messenger involved in the activation of microglia induced by lipopolysaccharide (LPS) (12) prompted us to investigate the effect of ABA in these cells.Indeed, exogenous ABA, at concentrations ranging from 250 nm to 20 μm, elicits functional activation of murine N9 cells, stimulating TNF-α release and cell migration through activation of the ADP-ribosyl cyclase CD38 and overproduction of cADPR. Moreover, N9 cells produce and release ABA when stimulated with LPS, amyloid β-peptide (βA), phorbol myristate acetate (PMA), or the chemoattractant peptide f-MLP. These results indicate that ABA behaves as an endogenous, pro-inflammatory hormone in murine microglia and provide a new target for future investigations into the role of this hormone in inflammatory and degenerative diseases of the central nervous system accompanied by microglial activation.     

The vestibular cortical projection during spinal decompensation

The removal of spinal afferents in rabbits submitted to unilateral lesion of the labyrinth and fully compensated greatly modified the field potentials as well as the single unit responses recorded from the cortical vestibular area during stimulation of the intact eighth nerve. In particular, an increase of contralateral and a decrease of ipsilateral potentials were observed. These asymmetrical responses indicate that the influence of the spinal cord in compensating the effects of unilateral lesion of the labyrinth is directed to balance not only the electrical activity of the brain-stem structures, but also that of the cerebral cortex. It appears, therefore, that spinal signals intervene not only in the compensation of vestibulospinal and vestibulo-oculomotor functions but also of cortical functions, such as that related to vestibular sensation.     

Modulation of cardiac contractility by muscle metaboreflex following efforts of different intensities in humans

Accumulation of metabolic end products within skeletal muscle stimulates sensory nerves, thus evoking a pressor response termed "metaboreflex." The aim of this study was to evaluate changes in hemodynamics occurring during metaboreflex activation obtained by postexercise muscle ischemia (PEMI) after two different exercise intensities. In twelve healthy subjects, the metaboreflex was studied with the PEMI method at the start of recovery from one leg-dynamic knee extension performed at intensities of 30% (PEMI 30%) and 70% (PEMI 70%) of the maximum workload achieved in a preliminary test. Control exercise recovery tests at the same intensities were also conducted. Central hemodynamics were evaluated by means of impedance cardiography. The main findings were that 1) during metaboreflex, exercise conducted against the higher workload caused a more pronounced blood pressure increase than the strain conducted against the lower workload; and 2) during PEMI 70%, this blood pressure response was mainly achieved through enhancement of myocardial contractility that increased stroke volume and, in turn, cardiac output, whereas during PEMI 30%, the blood pressure response was reached predominantly by means of vasoconstriction. Thus a substantial enhancement of myocardial contractility was reached only in the PEMI 70% test. These results suggest that hemodynamic regulation during metaboreflex engagement caused by PEMI in humans is dependent on the intensity of the previous effort. Moreover, the cardiovascular response during metaboreflex is not merely achieved by vasoconstriction alone, but it appears that there is a complex interplay between peripheral vasoconstriction and heart contractility recruitment.     

Alterations in hepatocyte cytoskeleton caused by redox cycling and alkylating quinones

Quinones may induce toxicity by a number of mechanisms, including alkylation and oxidative stress following redox cycling. The metabolism of quinones by isolated rat hepatocytes is associated with cytoskeletal alterations, plasma membrane blebbing, and subsequent cytotoxicity. The different mechanisms underlying the effects of alkylating (p-benzoquinone), redox cycling (2,3-dimethoxy-1,4-naphthoquinone), and mixed redox cycling/alkylating (2-methyl-1,4-naphthoquinone) quinones on hepatocyte cytoskeleton have been investigated in detail in this study. Analysis of the cytoskeletal fraction extracted from quinone-treated cells revealed a concentration-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols, irrespective of the quinone employed. In the case of redox cycling quinones, these alterations were associated with an oxidation-dependent actin crosslinking (sensitive to the thiol reductant dithiothreitol). In contrast, with alkylating quinones an oxidation-independent cytoskeletal protein crosslinking (insensitive to thiol reductants) was observed. In addition to these changes, a dose-dependent increase in the relative abundance of F-actin was detected as a consequence of the metabolism of oxidizing quinones in hepatocytes. Addition of dithiothreitol solubilized a considerable amount of polypeptides from the cytoskeletal fraction isolated from hepatocytes exposed to redox cycling but not alkylating quinones. Our findings indicate that the hepatocyte cytoskeleton is an important target for the toxic effects of different quinones. However, the mechanisms underlying cytoskeletal damage differ depending on whether the quinone acts primarily by oxidative stress or alkylation.     

Pyroglutamate-modified amyloid beta-peptides--AbetaN3(pE)--strongly affect cultured neuron and astrocyte survival

N-terminally truncated amyloid-beta (Abeta) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbetaN3(pE)-40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Abeta accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Abeta1-40, Abeta1-42, AbetaN3(pE)-40 and AbetaN3(pE)-42. Our data show that fibre morphology of Abeta peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AbetaN3(pE)-40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbetaN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbetaN3(pE)-40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while full-length peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance beta-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.     

Genetic basis of Cd tolerance and hyperaccumulation in Arabidopsis halleri

The genetic basis of Cd tolerance and hyperaccumulation was investigated in Arabidopsis halleri. The study was conducted in hydroponic culture with a backcross progeny, derived from a cross between A. halleri and a non-tolerant and non-accumulating related species Arabidopsis lyrata ssp. petraea, as well as with the parents of the backcross. The backcross progeny segregates for both cadmium (Cd) tolerance and accumulation. The results support that (i) Cd tolerance may be governed by more than one major gene, (ii) Cd tolerance and Cd accumulation are independent characters, (iii) Cd and Zn tolerances co-segregate suggesting that they are under pleiotropic genetic control, at least to a certain degree, (iv) the same result was obtained for Cd and Zn accumulation.     

Structure–activity relationships of various amino-hydroxy-benzenesulfonic acids and sulfonamides as tyrosinase substrates

Background

o-Aminophenols have been long recognised as tyrosinase substrates. However their exact mode of interaction with the enzyme's active site is unclear. Properly vic-substituted o-aminophenols could help gain some insight into tyrosinase catalytic mechanism.

Methods

Eight vic-substituted o-aminophenols belonging to two isomeric series were systematically evaluated as tyrosinase substrates and/or activators and/or inhibitors, by means of spectrophotometric techniques and HPLC-MS analysis. Some relevant kinetic parameters have also been obtained.

Results

Four o-aminophenolic compounds derived from 3-hydroxyorthanilic acid (2-amino-3-hydroxybenzenesulfonic acid) and their four counterparts derived from the isomeric 2-hydroxymetanilic acid (3-amino-2-hydroxybenzenesulfonic acid) were synthesised and tested as putative substrates for mushroom tyrosinase. While the hydroxyorthanilic derivatives were quite inactive as both substrates and inhibitors, the hydroxymetanilic compounds on the contrary all acted as substrates for the enzyme, which oxidised them to the corresponding phenoxazinone derivatives.

General significance

Based on the available structures of the active sites of tyrosinases, the different affinities of the four metanilic derivatives for the enzyme, and their oxidation rates, we propose a new hypothesis regarding the interaction between o-aminophenols and the active site of tyrosinase that is in agreement with the obtained experimental results.     

CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells

NAD⁺ is mainly synthesized in human cells via the “salvage” pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the “salvage” pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD⁺ or NAD⁺ precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD⁺ precursors for NAD⁺ biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD⁺ biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.