DAILY VARIATION OF CONSTITUTIVELY ACTIVATED NUCLEAR FACTOR KAPPA B (NFKB) IN RAT PINEAL GLAND

In mammals, the production of melatonin by the pineal gland is mainly controlled by the suprachiasmatic nuclei (SCN), the master clock of the circadian system. We have previously shown that agents involved in inflammatory responses, such as cytokines and corticosterone, modulate pineal melatonin synthesis. The nuclear transcription factor NFKB, detected by our group in the rat pineal gland, modulates this effect. Here, we evaluated a putative constitutive role for the pineal gland NFKB pathway. Male rats were kept under 12 h: 12 h light-dark (LD) cycle or under constant darkness (DD) condition. Nuclear NFKB was quantified by electrophoretic mobility shift assay on pineal glands obtained from animals killed throughout the day at different times. Nuclear content of NFKB presented a daily rhythm only in LD-entrained animals. During the light phase, the amount of NFKB increased continuously, and a sharp drop occurred when lights were turned off. Animals maintained in a constant light environment until ZT 18 showed diurnal levels of nuclear NFKB at ZT15 and ZT18. Propranolol (20 mg/kg, i.p., ZT 11) treatment, which inhibits nocturnal sympathetic input, impaired nocturnal decrease of NFKB only at ZT18. A similar effect was observed in free-running animals, which secreted less nocturnal melatonin. Because melatonin reduces constitutive NFKB activation in cultured pineal glands, we propose that this indolamine regulates this transcription factor pathway in the rat pineal gland, but not at the LD transition. The controversial results regarding the inhibition of pineal function by constant light or blocking sympathetic neurotransmission are discussed according to the hypothesis that the prompt effect of lights-off is not mediated by noradrenaline, which otherwise contributes to maintaining low levels of nuclear NFKB at night. In summary, we report here a novel transcription factor in the pineal gland, which exhibits a constitutive rhythm dependent on environmental photic information. (Author correspondence: rpmarkus@usp.br)

Glucocorticoids Exacerbate Lipopolysaccharide-Induced Signaling in the Frontal Cortex and Hippocampus in a Dose-Dependent Manner

Although the anti-inflammatory actions of glucocorticoids (GCs) are well established, evidence has accumulated showing that proinflammatory GC effects can occur in the brain, in a poorly understood manner. Using electrophoretic mobility shift assay, real-time PCR, and immunoblotting, we investigated the ability of varying concentrations of corticosterone (CORT, the GC of rats) to modulate lipopolysaccharide (LPS)-induced activation of NF-kappa B (nuclear factor kappa B), expression of anti- and proinflammatory factors and of the MAP (mitogen-activated protein) kinase family [ERK (extracellular signal-regulated kinase), p38, and JNK/ SAPK (c-Jun N-terminal protein kinase/ stress-activated protein kinase)], and AKT. In the frontal cortex, elevated CORT levels were proinflammatory, exacerbating LPS effects on NF-kappa B, MAP kinases, and proinflammatory gene expression. Milder proinflammatory GCs effects occurred in the hippocampus. In the absence of LPS, elevated CORT levels increased basal activation of ERK1/ 2, p38, SAPK/ JNK, and AKT in both regions. These findings suggest that GCs do not uniformly suppress neuroinflammation and can even enhance it at multiple levels in the pathway linking LPS exposure to inflammation.

Effects of isoproterenol treatment for 7 days on inflammatory mediators in the rat aorta

The aim of the present study was to evaluate the effect of overstimulation of beta-adrenoceptors on vascular inflammatory mediators. Wistar rats were treated with the beta-adrenoceptor agonist isoproterenol (0.3 mg(.)kg(-1.)day(-1) sc) or vehicle (control) for 7 days. At the end of treatment, the right carotid artery was catheterized for arterial and left ventricular (LV) hemodynamic evaluation. Isoproterenol treatment increased LV weight but did not change hemodynamic parameters. Aortic mRNA and protein expression were quantified by real-time RT-PCR and Western blot analysis, respectively. Isoproterenol enhanced aortic mRNA and protein expression of IL-1 beta (124% and 125%) and IL-6 (231% and 40%) compared with controls but did not change TNF-alpha expression. The nuclear-to-cytoplasmatic protein expression ration of the NF-beta B p65 subunit was increased by isoproterenol treatment (51%); in addition, it reduced the cytoplasmatic expression of I kappa B-alpha (52%) in aortas. An electrophoretic mobility shift assay was performed using the aorta, and increased NF-kappa B DNA binding (31%) was observed in isoproterenol-treated rats compared with controls (P < 0.05). Isoproterenol treatment increased phenylephrine-induced contraction in aortic rigs (P < 0.05), which was significantly reduced by superoxide dismutase (150 U/ml) and sodium salicylate (5 mM). Cotreatment with thalidomide (150 mg(.)kg(-1.)day(-1) for 7 days) also reduced hyperreactivity to phenylephrine induced by isoproterenol. In conclusion, overstimulation of beta-adrenoceptors increased proinflammatory cytokines and upregulated NF-kappa B in the rat aorta. Moreover, local oxidative stress and the proinflammatory state seem to play key roles in the altered vascular reactivity of the rat aorta induced by chronic beta-adrenergic stimulation.

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H(2)O(2) and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

Ionization and Structural Changes of the DMPG Vesicle along Its Anomalous Gel-Fluid Phase Transition: A Study with Different Lipid Concentrations

Dispersions of saturated anionic phospholipid dimyristoyl phosphatidylglycerol (DMPG) have been extensively studied regarding their peculiar thermostructural behavior. At low ionic strength, the gel-fluid transition is spread along nearly 17 degrees C, displaying several thermal events in the calorimetric profile that is quite different from the single sharp peak around 23 degrees C found for higher ionic strength DMPG dispersions. To investigate the role of charge in the bilayer transition, we carefully examine the temperature dependence of the electrical conductivity of DMPG dispersions at different concentrations, correlating the data with the corresponding differential scanning calorimetry (DSC) traces. Electrical conductivity together with electrophoretic mobility measurements allowed the calculation of the dependence of the degree of ionization of DMPG vesicles on lipid concentration and temperature. It was shown that there is a decrease in vesicle charge as the lipid concentration increases, which is probably correlated with the increase in the concentration of bulk Na(+). Apart from the known increase in the electrical conductivity along the DMPG temperature transition region, a sharp rise was observed at the bilayer pretransition for all lipid concentrations studied, possibly indicating that the beginning of the chain melting process is associated with an increase in bilayer ionization. It is confirmed here that the gel-fluid transition of DMPG at low ionic strength is accompanied by a huge increase in the dispersion viscosity. However, it is shown that this measured macroviscosity is distinct from the local viscosity felt by either charged ions or DMPG charged aggregates in measurements of electrical conductivity or electrophoretic mobility, Data presented here give support to the idea that DMPG vesicles, at low ionic strength, get more ionized along the temperature transition region and could be perforated and/or deformed vesicle structures.

The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2`-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 mu M ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.

Sample stacking in CZE using dynamic thermal junctions II: Analytes with high dpK(a)/dT crossing a single thermal junction in a BGE with low dpH/dT

In a previous work [M. Mandaji, et al., this issue] a sample stacking method was theoretically modeled and experimentally demonstrated for analytes with low dpK(a)/dT (analytes carrying carboxylic groups) and BGEs with high dpH/dT (high pH-temperature-coefficients). In that work, buffer pH was modulated with temperature, inducing electrophoretic mobility changes in the analytes. In the present work, the opposite conditions are studied and tested, i.e. analytes with high dpK(a)/dT and BGEs that exhibit low dpH/dT. It is well known that organic bases such as amines, imidazoles, and benzimidazoles exhibit high dpK(a)/dT. Temperature variations induce instantaneous changes on the basicity of these and other basic groups. Therefore, the electrophoretic velocity of some analytes changes abruptly when temperature variations are applied along the capillary. This is true only if BGE pH remains constant or if it changes in the opposite direction of pK(a) of the analyte. The presence of hot and cold sections along the capillary also affects local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band stacking efficacy was also taken into account in the theoretical model presented. Finally, this stacking method is demonstrated for lysine partially derivatized with naphthalene-2,3-dicarboxaldehyde. In this case, the amino group of the lateral chain was left underivatized and only the alpha amino group was derivatized. Therefore, the basicity of the lateral amino group, and consequently the electrophoretic mobility, was modulated with temperature while the pH of the buffer used remained unchanged.

Characterisation of anthracyclines from a cosmomycin D-producing species of Streptomyces by collisionally-activated dissociation and ion mobility mass spectrometry

Cultures of cosmomycin D-producing Streptomyces olindensis ICB20 that were propagated for many generations underwent mutations that resulted in production of a range of related anthracyclines by the bacteria. The anthracyclines that retained the two trisaccharide chains of the parent compound were separated by HPLC. Exact mass determination of these compounds revealed that they differed from cosmomycin D (CosD) in that they contained one to three fewer oxygen atoms (loss of hydroxyl groups). Some of the anthracyclines that were separated by HPLC had the same mass. The location from which the hydroxyl groups had been lost relative to CosD (on the aglycone and/or on the sugar residues) was probed by collisionally-activated dissociation using an electrospray ionisation linear quadrupole ion trap mass spectrometer. The presence of anthracyclines with the same mass, but different structure, was confirmed using an electrospray ionisation travelling wave ion mobility mass spectrometer.

Integer and fractional microwave induced resistance oscillations in a 2D system with moderate mobility

We report on integer and fractional microwave-induced resistance oscillations in a 2D electron system with high density and moderate mobility, and present results of measurements at high microwave intensity and temperature. Fractional microwave-induced resistance oscillations occur up to fractional denominator 8 and are quenched independently of their fractional order. We discuss our results and compare them with existing theoretical models. (C) 2009 Elsevier B.V. All rights reserved.

Electrophoretic deposition of Ba0.77Ca0.23TiO3 nanopowders

Ba0.77Ca0.23TiO3 (BCT23) nanometric powders, synthesized by the modified Pechini method, were used as precursor to produce thick films (50-130 mu m) employing the electrophoretic deposition (EPD) technique. The BCT23 powder presented a single crystalline phase with an average particle size and a crystallite size of similar to 60 nm and similar to 20 nm, respectively, when calcined at 800 degrees C/2h. BCT23 thick films were deposited on platinum substrates starting from different suspensions prepared by dispersion of the powder into: isopropyl alcohol (IPA) or a mixture of acetylacetone (Acac) and ethanol (EtOH) (1:1, volumetric ratio). A milling process was used to deagglomerate the powders in order to increase the suspension stability and improving the deposition. Dense and crack free thick films with uniform microstructure were obtained after sintering at 1300 degrees C/2 h from Acac+EtOH solution. (C) 2007 Elsevier B.V. All rights reserved.

The Mobility of Chondroitin Sulfate in Articular and Artificial Cartilage Characterized by (13)C Magic-Angle Spinning NMR Spectroscopy

We have studied the molecular dynamics of one of the major macromolecules in articular cartilage, chondroitin sulfate. Applying (13)C high-resolution magic-angle spinning NMR techniques, the NMR signals of all rigid macromolecules in cartilage can be suppressed, allowing the exclusive detection of the highly mobile chondroitin sulfate. The technique is also used to detect the chondroitin sulfate in artificial tissue-engineered cartilage. The tissue-engineered material that is based on matrix producing chondrocytes cultured in a collagen gel should provide properties as close as possible to those of the natural cartilage. Nuclear relaxation times of the chondroitin sulfate were determined for both tissues. Although T(1) relaxation times are rather similar, the T(2) relaxation in tissue-engineered cartilage is significantly shorter. This suggests that the motions of chondroitin sulfate in data:rat and artificial cartilage different. The nuclear relaxation times of chondroitin sulfate in natural and tissue-engineered cartilage were modeled using a broad distribution function for the motional correlation times. Although the description of the microscopic molecular dynamics of the chondroitin sulfate in natural and artificial cartilage required the identical broad distribution functions for the correlation times of motion, significant differences in the correlation times of motion that are extracted from the model indicate that the artificial tissue does not fully meet the standards of the natural ideal. This could also be confirmed by macroscopic biomechanical elasticity measurements. Nevertheless, these results suggest that NMR is a useful tool for the investigation of the quality of artificially engineered tissue. (C) 2010 Wiley Periodicals, Inc. Biopolymers 93: 520-532, 2010.

Electrostatic layer-by-layer and electrophoretic depositions as methods for electrochromic nanoparticle immobilization

The present paper describes the immobilization of nanoparticles onto conducting substrates by using both electrostatic layer-by-layer and electrophoretic deposition (EPD) methods. These two techniques were compared in high-performance electrochromic electrodes based on mixed nickel hydroxide nanoparticles. In addition to easy handling, EPD seems to be the most suitable method for the immobilization of nanoparticles, leading to higher electrochromic efficiencies, lower response times and higher stability upon coloration and bleaching cycling. (C) 2008 Elsevier Ltd. All rights reserved.

Environmental formaldehyde analysis by active diffusive sampling with a bundle of polypropylene porous capillaries followed by capillary zone electrophoretic separation and contactless conductivity detection

Compared to other volatile carbonylic compounds present in outdoor air, formaldehyde (CH2O) is the most toxic, deserving more attention in terms of indoor and outdoor air quality legislation and control. The analytical determination of CH2O in air still presents challenges due to the low-level concentration (in the sub-ppb range) and its variation with sampling site and time. Of the many available analytical methods for carbonylic compounds, the most widespread one is the time consuming collection in cartridges impregnated with 2,4-dinitrophenylhydrazine followed by the analysis of the formed hydrazones by HPLC. The present work proposes the use of polypropylene hollow porous capillary fibers to achieve efficient CH2O collection. The Oxyphan (R) fiber (designed for blood oxygenation) was chosen for this purpose because it presents good mechanical resistance, high density of very fine pores and high ratio of collection area to volume of the acceptor fluid in the tube, all favorable for the development of air sampling apparatus. The collector device consists of a Teflon pipe inside of which a bundle of polypropylene microporous capillary membranes was introduced. While the acceptor passes at a low flow rate through the capillaries, the sampled air circulates around the fibers, impelled by a low flow membrane pump (of the type used for aquariums ventilation). The coupling of this sampling technique with the selective and quantitative determination of CH2O, in the form of hydroxymethanesulfonate (HMS) after derivatization with HSO3-, by capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-(CD)-D-4) enabled the development of a complete analytical protocol for the CH2O evaluation in air. (C) 2008 Published by Elsevier B.V.