Simultaneous detection of the release of glutamate and nitric oxide from adherently growing cells using an array of glutamate and nitric oxide selective electrodes

The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which the individual electrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redox hydrogel-based glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positively charged Ni porphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working electrode by a bienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adherently growing C6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current responses recorded at each electrode after stimulation of glutamate and NO release by means of K+ and bradykinin clearly demonstrate the ability of the individual electrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the neighbouring electrode or other analytes present in the test mixture.     

Cell-compatible array of three-dimensional tip electrodes for the detection of nitric oxide release

An array of electrodes on which cells could be grown directly was fabricated using silicon anisotropic etching and a thick-photoresist process and employed for the detection of nitric oxide (NO) released from a population of adherently growing human umbilical vein endothelial cells (HUVEC). The electrodes are tip-shaped and are 40 microm high of which only the top 15 microm are exposed Pt-tips. After electrochemical induced modification of the exposed Pt tips using Ni phthalocyanine the individual addressable electrode tips were sensitive and selective for the detection of NO at an applied constant potential of 750 mV. The silicon nitride insulation of the lower part of the tip electrodes prevented the death of the cells upon the application of the working potential at which NO was detected. It also helped to avoid the perturbation of the integrity of the sensing chemistry imparted on the electrode surface that could have resulted from the contact of the adherently growing cells with the active electrode surface. The release of nitric oxide from HUVEC was successfully monitored with different numbers of tip electrodes simultaneously connected as combined working electrode.     

Integrin activation is required for VEGF and FGF receptor protein presence on human microvascular endothelial cells

Endothelial cell proliferation and migration is initiated by growth factors including FGF and VEGF that bind to specific transmembrane receptor tyrosine kinases. Mechanisms that regulate in vivo expression of fibroblast growth factor receptors (FGFR) and vascular endothelial growth factor receptors (VEGFR) are not well understood. Since it is well known that different matrices influence the proliferation and migration of endothelial cells in culture, we hypothesized that changes in the extracellular matrix environment can regulate growth factor receptors on endothelial cells. We cultured human microvascular endothelial cells on different matrices (vitronectin, laminin, fibronectin, fibrin, and collagen IV) and examined for the presence of growth factor receptors (FGFR-1, FGFR-2, VEGFR-1, and VEGFR-2). We show that vitronectin increased the presence of all four growth factor receptors and most notably, VEGFR-1. In contrast, fibrin decreased all four receptors, especially FGFR-1 and FGFR-2. Inhibiting phosphotyrosine signaling abolished immunostaining for all four receptors, regardless of the matrix, but was not dependent on activating the Fyn-Shc pathway. Cells plated on vitronectin in the presence of blocking antibodies to integrins _v3 and _v5 similarly decreased presence of these growth factor receptors. Our data suggests a possible mechanism of how matrix-integrin interactions regulate endothelial cell responsiveness to growth factors and anchorage-dependent cell growth.     

Genetic variation in resistance to Phytophthora cinnamomi in seedlings of two Turkish Abies species

Genetic variation in resistance to Phytophthora cinnamomi was investigated for two fir species endemic to the Republic of Turkey. Open-pollinated families of seedlings of Trojan fir (Abies equi-trojani) and Turkish fir (Abies bornmuelleriana) were grown from seed in a greenhouse for approximately 15 months, inoculated with rice grains colonized with P. cinnamomi, and subsequent mortality assessed biweekly for 16 weeks. Final seedling mortality was higher in Trojan fir (56.4 %) compared to Turkish fir (32.9 %). Mortality in both species varied by geographic origin, decreasing from west (59.8 %, ) to east (21.4 %, Karabük). As mortality increased following inoculation, both narrow-sense individual-tree $ \left( {h_i^2} \right) $ and family mean $ ( {h_f^2} ) $ heritabilities increased, plateauing at 0.62?±?0.162 and 0.97?±?0.011 for Trojan fir and 0.50?±?0.102 and 0.96?±?0.01 for Turkish fir, respectively. Terminal and lateral branch bud break assessed under greenhouse conditions were also under strong genetic control. For terminal bud break, individual-tree heritabilities for Trojan and Turkish fir were 0.49?±?0.146 and 0.45?±?0.099, respectively, while family mean heritabilities were 0.88?±?0.035 and 0.88?±?0.027, respectively. The family mean correlation between bud break and final disease mortality was not significant for lateral buds but positive and significant for terminal buds (r?=?0.32) suggesting that selection for resistance would either not alter, or slightly reduce, early bud break. These are encouraging results for ongoing tree improvement efforts in North America and Europe to develop planting stock for the Christmas tree industry.     

Evalution of toxicity of abcisic acid and gibberellic acid in rats: 50 days drinking water study

In the present study, the influence of subchronic effects of two plant growth regulators (PGRs) [Abcisic acid (ABA) and Gibberellic acid (GA₃)] on antioxidant defense systems [reduced glutathione (GSH), glutathione reductase (GR), superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT)] and lipid peroxidation level (malondialdehyde = MDA) in various tissues of the rat were investigated during treatment as a drinking water model. 75 ppm of ABA and GA₃ in drinking water were continuously administered orally to rats (Sprague-Dawley albino) ad libitum for 50 days. The PGRs treatments caused different effects on the antioxidant defense systems and MDA content of dosed rats compared to controls. The lipid peroxidation end product MDA significantly increased in the lungs, heart and kidney of rats treated with GA₃ without significant change in the spleen. ABA caused also a significant increase in MDA content in the spleen, lungs, heart and kidney. The GSH levels were significantly depleted in the spleen, lungs and stomach of rats treated with ABA without any change in the tissues of rats treated with GA₃ except the kidney where it increased. Antioxidant enzyme activities such as SOD significantly increased in the lungs and stomach and decreased in the spleen and heart tissues of rats treated with GA₃. Meanwhile, SOD significantly decreased in the spleen, heart and kidney and increased in the lungs of rats treated with ABA. While CAT activity significantly decreased in the lungs of rats treated with GA₃, a significant increase occurred in the heart of rats treated with both PGRs. On the other hand, the ancillary enzyme GR activity in the tissues were either significantly depleted or not changed with PGRs treatment. The drug metabolizing enzyme GST activity significantly decreased in the lungs of rats treated with ABA but increased in the stomach of rats treated with both PGRs.

As a conclusion, the rats resisted oxidative stress via the antioxidant mechanism. But the antioxidant mechanism could not prevent the increases in lipid peroxidation in rat's tissues. This data, along with changes, suggests that PGRs produced substantial systemic organ toxicity in the spleen, lungs, stomach, heart and kidney during a 50-day period of subchronic exposure.     

The effect of temperature on photosynthetic induction under fluctuating light in Chrysanthemum morifolium

The photosynthetic response was investigated on Chrysanthemum morifolium under dynamic light conditions in the 20–35 °C temperature range to evaluate the effect of climatic variables on photosynthetic induction. The plant material was grown under uniform, controlled conditions and its gas exchange was analyzed. The gas exchange measurements were used to investigate the rate of induction, momentary induction state, and the opening of stomata. At the varying temperature ranges and under dynamic light conditions, C. morifolium reached a quasi-steady-state induction equilibrium (IS_eq(PAR,T)) within 14–45 min. For the same level of photosynthetically active radiation (PAR), the equilibrated level of steady-state induction increased as the temperature increased. It was highest approximately at 30 °C. The induction state was equilibrated at a lower level as the temperature increased to 35 °C. The interaction effect of PAR and temperature on induction state was not significant. The rate of photosynthetic induction and the time required at which the induction reached its 90 % value (t ₉₀) was influenced by PAR significantly. The light history of a leaf had a significant effect on t ₉₀, indicating that the time to reach a steady-state induction is different depending on the light environment and the period at which the leaf was exposed to light. The velocity of the photosynthetic induction was not affected by the temperature. It was associated with stomatal conductance of the leaf prior to the onset of light (g _Sini).     

Anion inhibition studies of the α-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae

An α-carbonic anhydrase (CA, EC 4.2.1.1) has been recently cloned and characterized in the human pathogenic bacterium Vibrio cholerae, denominated VchCA (Del Prete et al. J. Med. Chem. 2012, 55, 10742). This enzyme shows a good catalytic activity for the CO₂ hydration reaction, comparable to that of the human (h) isoform hCA I. Many inorganic anions and several small molecules were investigated as VchCA inhibitors. Inorganic anions such as cyanate, cyanide, hydrogen sulfide, hydrogen sulfite, and trithiocarbonate were effective VchCA inhibitors with inhibition constants in the range of 33–88 μM. Other effective inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with K_Is of 7–43 μM. Halides (bromide, iodide), bicarbonate and carbonate were much less effective VchCA inhibitors, with K_Is in the range of 4.64–28.0 mM. The resistance of VchCA to bicarbonate inhibition may represent an evolutionary adaptation of this enzyme to living in an environment rich in this ion, such as the gastrointestinal tract, as bicarbonate is a virulence enhancer of this bacterium.     

Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis.     

Cutting edge: identification of a pre-ligand assembly domain (PLAD) and ligand binding site in the IL-17 receptor

IL-17 is the hallmark cytokine of the newly described "Th17" lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a "pre-ligand assembly domain" (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.