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.     

Response to cytosolic nickel of Slow Vacuolar channels in the hyperaccumulator plant <Emphasis Type="Italic">Alyssum bertolonii</Emphasis>

We applied the patch-clamp technique to investigate the transport properties of the Slow Vacuolar (SV) channel identified in leaf vacuoles of Alyssum bertolonii Desv., a nickel hyperaccumulator plant growing in serpentine soil of the northern Apennines (Italy). SV currents recorded in vacuoles from adult plants collected in their natural habitat showed high sensitivity towards cytosolic nickel. Dose-response analyses indicated half-maximal current inhibition at submicromolar concentrations, i.e. up to three orders of magnitude lower than previously reported values from other plant species. The voltage-dependent increase of residual currents at saturating nickel concentrations could be interpreted as relief of channel block by nickel permeation at high positive membrane potentials. Including young plants of A. bertolonii into the study, we found that SV channels from these plants did not display elevated nickel sensitivity. This difference may be related to age-dependent changes in nickel hyperaccumulation of A. bertolonii leaf cells.     

The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4

The JNK‐interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP‐binding protein ARF6. The interaction of ARF6–GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin‐1 and dynactin. Here, we report the crystal structure of ARF6–GTP bound to the JIP4‐LZII at 1.9 Å resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6–(JIP4)₂–ARF6 configuration. Comparison of the ARF6–JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site‐directed mutagenesis and surface plasmon resonance, we further show that non‐conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure‐derived model of the association of the ARF6–JIP3/JIP4 complex with membranes shows that the JIP4‐LZII coiled‐coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6‐mediated motor switch regulatory function.     

γ-Glutamyltransferase-dependent resistance to arsenic trioxide in melanoma cells and cellular sensitization by ascorbic acid

The cell ability of tumor cells to tolerate stress conditions is a typical feature of solid tumors. In particular, the resistance to oxidative stress of melanoma cells likely contributes to their intrinsic drug resistance. In an attempt to develop novel strategies for overcoming the mechanisms of cellular protection against oxidative stress, in this study we have explored the efficacy of the combination of two prooxidant agents in two human melanoma cell clones. The selected clones are characterized by a marked difference in expression of γ-glutamyltransferase, which is known to produce a persistent low level of oxidative stress resulting in the stimulation of protective systems. The γ-glutamyltransferase-overexpressing clone exhibited a low susceptibility to arsenic trioxide-induced apoptosis, associated with low reactive oxygen species induction and increased catalase activity. The combination of arsenic trioxide with subtoxic concentrations of ascorbic acid resulted in a sensitization to apoptotic cell death. The expression of protective mechanisms, in particular catalase activity, accounted for the behavior of the resistant clone. The sensitization achieved by the combination was associated with a cellular response involving the ASK1/p38 axis, which is implicated in the regulation of catalase expression and the activation of apoptotic signals. In conclusion, the results of our study provide evidence that a rational combination of prooxidant agents may be effective in overcoming cellular tolerance to oxidative stress.     

Enantioselective catalytic reduction of ketoimines with trichlorosilane promoted by readily available chiral Lewis bases

The straightforward synthesis of a series of enantiomerically pure Lewis basic amides by simple condensation of commercially available enantiopure diamines with picolinic acid is reported. These compounds were shown to promote the enantioselective reduction of ketoimines with trichlorosilane. Working with the model substrate N-phenyl benzophenone imine, the new organocatalysts led to the formation of the corresponding amine, with excellent chemical efficiency (up to 99% chemical yield) and good stereoselectivity (up to 73% ee). Up to 83% of enantioselectivity was reached in the reduction of differently substituted imines.     

Molecular dynamics simulations of mouse ferrochelatase variants: what distorts and orientates the porphyrin?

Molecular dynamics simulations of the wild-type and variant forms of the mouse ferrochelatase in complex with the product (haem) have been performed using the GROMOS96 force field, in the NpT ensemble. Ferrochelatase, the last enzyme in the catalytic pathway of the haem biosynthesis, catalyses the reaction of insertion of a ferrous ion into protoporphyrin IX by distorting the planar geometry of the latter reactant. The simulations presented aim at understanding the role of active-site residues in this catalytic process. Analysis of the simulation trajectories explains the consequences of the mutations introduced and sheds more light on the role of the His209 residue in porphyrin macrocycle distortion. The function of residues coordinating propionate groups of the haem molecule is discussed in terms of stability of the substrate and product complexes.     

Polyazetidine-based immobilization of redox proteins for electron-transfer-based biosensors

A highly stable functional composite film was prepared using polyazetidine prepolymer (PAP) with peroxidase from horseradish (HRP) and/or glucose oxidase (GOx). The good permeability of the PAP layer to classical electrochemical mediators, as evaluated by the determination of the diffusion coefficient of different redox molecules, is of great importance in view of the use of PAP as an immobilizing agent in second-generation biosensor development. Cyclic voltammetry of the HRP-PAP layer on a glassy carbon electrode (GCE) showed a pair of stable and quasi-reversible peaks for the HRP-Fe((III))/Fe((II)) redox couple at about -370 mV vs. Ag/AgCl electrode in pH 6.5 phosphate buffer. The electrochemical reaction of HRP entrapped in the PAP film exhibited a surface-controlled electrode process. This film and the successive modifications (HRP-PAP self-assembled monolayer (SAM) modified Au electrode) were used as a biological catalyst (hydrogen peroxide transducers) for glucose biosensors, after coupling to GOx. Both HRP/GOx-PAP and HRP/GOx-PAP SAM third generation biosensors were prepared and characterized. The use of PAP as immobilizing agent offers a biocompatible micro-environment for confining the enzyme and foreshadows the great potentiality of this immobilizing agent not only in theoretical studies on protein direct electron transfer but also from an applications point of view in the development of second- and third-generation biosensors.     

Potential of Ginkgo biloba L. leaves in the management of hyperglycemia and hypertension using in vitro models

Leaves from four different Ginkgo biloba L. trees (1 and 2 – females; 3 and 4 – males), grown at the same conditions, were collected during a period of 5 months (from June to October, 2007). Water and 12% ethanol extracts were analyzed for total phenolics content, antioxidant activity, phenolic profile, and the potential in vitro inhibitory effects on α-amylase, α-glucosidase, and Angiotensin I-Converting Enzyme (ACE) enzymes related to the management of diabetes and hypertension. The results indicated a significant difference among the trees in all functional benefits evaluated in the leaf extracts and also found important seasonal variation related to the same functional parameters. In general, the aqueous extracts had higher total phenolic content than the ethanolic extracts. Also, no correlation was found between total phenolics and antioxidant activity. In relation to the ACE inhibition, only ethanolic extracts had inhibitory activity.     

Ozonation strategies to reduce sludge production of a seafood industry WWTP

In this work, several alternatives related to the application of ozone in different streams of a seafood industry WWTP were evaluated to minimize the production of waste sludge. The WWTP was composed of two coagulation-flocculation units and a biological unit and generated around of 6550 kg/d of sludge. Ozone was applied to sludge coming from flotation units (110 g TSS/L) at doses up to 0.03 g O(3)/g TSS during batch tests, no solids solubilization being observed. Ozone doses ranging from 0.007 to 0.02 g O(3)/g TSS were also applied to the raw wastewater in a bubble column reaching a 6.8% of TSS removal for the highest ozone dose. Finally, the effect of the pre-ozonation (0.05 g O(3)/g TSS) of wastewater coming from the first flotation unit was tested in two activated sludge systems during 70 days. Ozonation caused a reduction of the observed yield coefficient of biomass from 0.14 to 0.07g TSS/g COD(Tremoved) and a slight improvement of COD removal efficiencies. On the basis of the capacity for ozone production available in the industry, a maximum reduction of sludge generated by the WWTP of 7.5% could be expected.