Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B

Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly Gq- and to a lesser extent Gs-mediated; p38 activation even had a small Gi-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules.

Cathodic protection of reinforced concrete:anodic processes in cements and related electrolytes

The nature and kinetics of electrode reactions and processes occurring for four lightweight anode systems which have been utilised in reinforced concrete cathodic protection systems have been studied. The anodes investigated were flame sprayed zinc, conductive paint and two activated titanium meshes. The electrochemical properties of each material were investigated in rapidly stirred de-oxygenated electrolytes using anodic potentiodynamic polarisation. Conductive coating electrodes were formed on glass microscope slides, whilst mesh strands were immersed directly. Oxygen evolution occurred preferentially for both mesh anodes in saturated Ca (OH)2/CaC12 solutions but was severely inhibited in less alkaline solutions and significant current only passed in chloride solutions. The main reactions for conductive paint was based on oxygen evolution in all electrolytes, although chlorides increased the electrical activity. Self-corrosion of zinc was controlled by electrolyte composition and the experimental set-up, chlorides increasing the electrical activity. Impressed current cathodic protection was applied to 25 externally exposed concrete slabs over a period of 18 months to investigate anode degradation mechanisms at normal and high current densities. Specimen chloride content, curing and reinforcement depth were also variables. Several destructive and non-destructive methods for assessing the performance of anodes were evaluated including a site instrument for quantitative "instant-off- potential measurements. The impact of cathodic protection on the concrete substrate was determined for a number of specimens using appropriate methods. Anodic degradation rates were primarily influenced by current density, followed by cemendtious alkalinity, chloride levels and by current distribution. Degradation of cementitious overlays and conductive paint substrates proceeded by sequential neutralisation of cement phases, with some evidence of paint binder oxidation. Sprayed zinc progressively formed an insulating layer of hydroxide complexes, which underwent pitting_ attack in the presence of sufficient chlorides, whilst substrate degradation was minimal. Adhesion of all anode systems decreased with increasing current density. The influence of anode material on the ionic gradients which can develop during cathodic protection was investigated. A constant current was passed through saturated cement paste prisms containing calcium chloride to central cathodes via anodes applied or embedded at each end. Pore solution was obtained from successive cut paste slices for anion and cation analyses. Various experimental errors reduced the value of the results. Characteristic S-shaped profiles were not observed and chloride ion profiles were ambiguous. Mesh anode specimens were significantly more durable than the conductive coatings in the high humidity environment. Limited results suggested zinc ion migration to the cathode region. Electrical data from each investigation clearly indicated a decreasing order of anode efficiency by specific anode material.

The prevalence and phenotype of activated microglia/macrophages within the spinal cord of the hyperostotic mouse (twy/twy) changes in response to chronic progressive spinalcord compression:implications for human cervical compressive myelopathy

Background:Cervical compressive myelopathy, e.g. due to spondylosis or ossification of the posterior longitudinal ligament is a common cause of spinal cord dysfunction. Although human pathological studies have reported neuronal loss and demyelination in the chronically compressed spinal cord, little is known about the mechanisms involved. In particular, the neuroinflammatory processes that are thought to underlie the condition are poorly understood. The present study assessed the localized prevalence of activated M1 and M2 microglia/macrophages in twy/twy mice that develop spontaneous cervical spinal cord compression, as a model of human disease.Methods:Inflammatory cells and cytokines were assessed in compressed lesions of the spinal cords in 12-, 18- and 24-weeks old twy/twy mice by immunohistochemical, immunoblot and flow cytometric analysis. Computed tomography and standard histology confirmed a progressive spinal cord compression through the spontaneously development of an impinging calcified mass.Results:The prevalence of CD11b-positive cells, in the compressed spinal cord increased over time with a concurrent decrease in neurons. The CD11b-positive cell population was initially formed of arginase-1- and CD206-positive M2 microglia/macrophages, which later shifted towards iNOS- and CD16/32-positive M1 microglia/macrophages. There was a transient increase in levels of T helper 2 (Th2) cytokines at 18 weeks, whereas levels of Th1 cytokines as well as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and macrophage antigen (Mac) -2 progressively increased.Conclusions:Spinal cord compression was associated with a temporal M2 microglia/macrophage response, which may act as a possible repair or neuroprotective mechanism. However, the persistence of the neural insult also associated with persistent expression of Th1 cytokines and increased prevalence of activated M1 microglia/macrophages, which may lead to neuronal loss and demyelination despite the presence of neurotrophic factors. This understanding of the aetiopathology of chronic spinal cord compression is of importance in the development of new treatment targets in human disease. © 2013 Hirai et al.

Predicting the effect of mixing on oxygen transfer and nutrient removal in activated sludge basins

Activated sludge basins (ASBs) are a key-step in wastewater treatment processes that are used to eliminate biodegradable pollution from the water discharged to the natural environment. Bacteria found in the activated sludge consume and assimilate nutrients such as carbon, nitrogen and phosphorous under specific environmental conditions. However, applying the appropriate agitation and aeration regimes to supply the environmental conditions to promote the growth of the bacteria is not easy. The agitation and aeration regimes that are applied to activated sludge basins have a strong influence on the efficacy of wastewater treatment processes. The major aims of agitation by submersible mixers are to improve the contact between biomass and wastewater and the prevention of biomass settling. They induce a horizontal flow in the oxidation ditch, which can be quantified by the mean horizontal velocity. Mean values of 0.3-0.35 m s-1 are recommended as a design criteria to ensure best conditions for mixing and aeration (Da Silva, 1994). To give circulation velocities of this order of magnitude, the positioning and types of mixers are chosen from the plant constructors' experience and the suppliers' data for the impellers. Some case studies of existing plants have shown that measured velocities were not in the range that was specified in the plant design. This illustrates that there is still a need for design and diagnosis approach to improve process reliability by eliminating or reducing the number of short circuits, dead zones, zones of inefficient mixing and poor aeration. The objective of the aeration is to facilitate the quick degradation of pollutants by bacterial growth. To achieve these objectives a wastewater treatment plant must be adequately aerated; thus resulting in 60-80% of all energetic consummation being dedicated to the aeration alone (Juspin and Vasel, 2000). An earlier study (Gillot et al., 1997) has illustrated the influence that hydrodynamics have on the aeration performance as measure by the oxygen transfer coefficient. Therefore, optimising the agitation and aeration systems can enhance the oxygen transfer coefficient and consequently reduce the operating costs of the wastewater treatment plant. It is critically important to correctly estimate the mass transfer coefficient as any errors could result in the simulations of biological activity not being physically representative. Therefore, the transfer process was rigorously examined in several different types of process equipment to determine the impact that different hydrodynamic regimes and liquid-side film transfer coefficients have on the gas phase and the mass transfer of oxygen. To model the biological activity occurring in ASBs, several generic biochemical reaction models have been developed to characterise different biochemical reaction processes that are known as Activated Sludge Models, ASM (Henze et al., 2000). The ASM1 protocol was selected to characterise the impact of aeration on the bacteria consuming and assimilating ammonia and nitrate in the wastewater. However, one drawback of ASM protocols is that the hydrodynamics are assumed to be uniform by the use of perfectly mixed, plug flow reactors or as a number of perfectly mixed reactors in series. This makes it very difficult to identify the influence of mixing and aeration on oxygen mass transfer and biological activity. Therefore, to account for the impact of local gas-liquid mixing regime on the biochemical activity Computational Fluid Dynamics (CFD) was used by applying the individual ASM1 reaction equations as the source terms to a number of scalar equations. Thus, the application of ASM1 to CFD (FLUENT) enabled the investigation of the oxygen transfer efficiency and the carbon & nitrogen biological removal in pilot (7.5 cubic metres) and plant scale (6000 cubic metres) ASBs. Both studies have been used to validate the effect that the hydrodynamic regime has on oxygen mass transfer (the circulation velocity and mass transfer coefficient) and the effect that this had on the biological activity on pollutants such as ammonia and nitrate (Cartland Glover et al., 2005). The work presented here is one part to of an overall approach for improving the understanding of ASBs and the impact that they have in terms of the hydraulic and biological performance on the overall wastewater treatment process. References CARTLAND GLOVER G., PRINTEMPS C., ESSEMIANI K., MEINHOLD J., (2005) Modelling of wastewater treatment plants ? How far shall we go with sophisticated modelling tools? 3rd IWA Leading-Edge Conference & Exhibition on Water and Wastewater Treatment Technologies, 6-8 June 2005, Sapporo, Japan DA SILVA G. (1994). Eléments d'optimisation du transfert d'oxygène par fines bulles et agitateur séparé en chenal d'oxydation. PhD Thesis. CEMAGREF Antony ? France. GILLOT S., DERONZIER G., HEDUIT A. (1997). Oxygen transfer under process conditions in an oxidation ditch equipped with fine bubble diffusers and slow speed mixers. WEFTEC, Chicago, USA. HENZE M., GUJER W., MINO T., van LOOSDRECHT M., (2000). Activated Sludge Models ASM1, ASM2, ASM2D and ASM3, Scientific and Technical Report No. 9. IWA Publishing, London, UK. JUSPIN H., VASEL J.-L. (2000). Influence of hydrodynamics on oxygen transfer in the activated sludge process. IWA, Paris - France.