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.     

Anthropometric and leptin changes in women following different dietary approaches to weight loss

Leptin may favorably respond to fat mass (FM) losses induced by a low-carbohydrate (LC) diet, although this is unclear. We examined serum leptin concentrations in women in midlife undergoing different dietary approaches to body weight (BW) loss. Women followed either a LC, high-protein (LCHP; n = 13) or high-carbohydrate, low-fat (HCLF; n = 12) diet for 12 weeks. Changes in anthropometric and soft-tissue mass measurements and leptin concentrations were assessed. Women in both diet groups had reductions in BW, BMI, fat-free soft-tissue mass, FM, body fat percentage, and central abdominal fat (CAF) (P < 0.001 for all variables) over the 12-week intervention. These changes were not significantly different between diet groups. Serum leptin concentrations decreased by 41.8% (P < 0.001) in the LCHP group and by 44.3% (P < 0.001) in the HCLF group from baseline to week 12, with no significant difference between groups. The association of CAF (r = 0.73) and FM (r = 0.83) change with leptin change was strong in the HCLF group. Leptin change did not relate to change in any variable in the LCHP group. Both LCHP and HCLF diets favorably lower FM, CAF, and leptin in women, suggesting that beneficial changes in leptin can be similarly achieved through different dietary approaches to BW loss.     

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC^{1, 2} methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain³ and screen printed electrodes as sensing platform.Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health⁴. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity⁵.The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.Download video file.^{(112M, mp4)}     

Investigation of de novo totally random biosequences, Part II: On the folding frequency in a totally random library of de novo proteins obtained by phage display

We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth ('Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 10(9) clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of 'Never Born Proteins' are discussed in terms of molecular evolution.     

Salivary gland-specific gene expression in the malaria vector Anopheles gambiae

Molecular studies on the tissue-specific gene expression in the salivary glands of Anopheles gambiae may provide useful tools for the development of new strategies for the control of the most efficient malaria vector in the sub-Saharan Africa. We summarize here the results of a recent investigation focused on the isolation of secreted factors and putative receptors from the salivary glands of An. gambiae. Using the Signal Sequence Trap technique we have identified the first cDNAs specifically expressed in the An. gambiae salivary glands. Among these, four are exclusively expressed in female glands and encode factors presumably involved in blood-feeding, whereas two other cDNAs seem to be expressed both in male and in female glands and are likely implicated in sugar-feeding. Homologues of genes previously identified in the yellow fever mosquito Aedes aegypti, like the apyrase and D7, as well as novel salivary gland-specific cDNAs, were identified. The isolation and characterization of promoter sequences from the corresponding genes may prove useful for the expression of anti parasitic agents in the salivary glands of transgenic mosquitoes.     

New aspects on primary aldosteronism

The adrenal cortex synthesizes and releases steroid hormones, mainly mineralocorticoids and glucocorticoids. There is a functional zonation of the adrenal cortex and steroid synthesis is thoroughly regulated. Overproduction of aldosterone, primary aldosteronism, may be much more common than previously known and may be responsible for 10% of essential hypertension. Primary aldosteronism is characterized by autonomous production of aldosterone, suppressed renin activity, hypokalemia, and hypertension. The two most common forms are unilateral adenoma and bilateral hyperplasia. In spite of thorough clinical workup and careful histopathology it is often difficult to differentiate between adenoma and hyperplasia. The gene CYP11B2 encodes the steroid synthesizing enzymes for aldosterone production, while the genes CYP17 and CYP11B1 are needed for cortisol production. Most normal controls show expression of CYP11B2 in zona glomerulosa. Expression of CYP11B1 and CYP17 is seen in zona fasciculata and reticularis, whereas the expression of CYP21 is present in all three cortical layers. Adenomas from patients with primary aldosteronism show considerable variation in the expression of CYP11B2. Adenomas from patients with Cushing's syndrome have a strong expression of CYP11B1 and CYP17. In a patient material of 29 cases of primary aldosteronism, 4 patients had small nodules detected with expression of CYP11B2 gene. These nodules were not visualized on CT, whereas adrenal masses seen on CT in these patients showed CYP11B1 and CYP17 gene expression. This suggests that these small nodules are responsible for the aldosterone production and this is characteristic of nodular hyperplasia in patients with primary aldosteronism. In conclusion, this method to visualize mRNA gene expression of steroidogenic enzymes, and especially expression of CYP11B2, has increased the knowledge of adrenal pathophysiology. The results emphasize the value to include functional studies (venous sampling and/or scintigraphy) in the preoperative work up of patients with primary aldosteronism.