No description available.

Microglia are immunocompetent cells in the central nervous system that have many similarities with macrophages of peripheral tissues. Their activation protects local cells from foreign microbial infection in the CNS. However, “over-activated“ microglia become a “Double-edged sword” which show neuronal toxicity and are implicated in a variety of neurodegenerative diseases. Previous studies have suggested that potassium channels play a role in regulating microglial activation, migration and proliferation. However what kinds of potassium channel subunits are expressed in microglia, whether their expression changes after microglial activation and the functional role of most potassium channels expressed in microglia are still not fully characterized. To address these questions, we used the N9 mouse microglial cell line as a cell model for experiments in vitro. We first optimized the cell culture and lipopolysaccharide (LPS), the endotoxin of gram-negative bacteria, mediated stimulation of microglial activation that results in subsequent nitric oxide (NO) release. Using qRT-PCR, we analyzed mRNA expression of >80 potassium channel pore-forming subunits and their regulatory subunits in both LPS-treated (1μg/ml, 24hr) and untreated microglia. The subunits which displayed the highest mRNA expression in resting N9 cells included Kcnma1 (KCa1.1), Kcnk6 (K2p6.1), Kcnc3 (Kv3.3) and Abcc8 (SUR1). In addition, N9 cells also expressed the mRNAs for other channel subunits previously reported in microglia such as Kcnn4 (KCa3.1), Kcna3 (Kv1.3) and Kcna5 (Kv1.5) subunits. Of these channel subunits LPS had no significant effect on mRNA expression except for Kcnk6 which was significantly reduced. We then examined whether pharmacological manipulation of these channels controlled LPS-induced NO release. It was found out that the KCa3.1 selective blocker Tram34 and the Kv1.5 inhibitor propafenone (PPF) significantly decreased LPS-induced NO in agreement with data in primary microglia. Ba2+ that inhibits inwardly rectifying potassium channels as well as K2p6.1 also significantly attenuated LPS-induced microglial activation. Inhibition or activation of KCa1.1 channels by paxilline and NS1619 respectively had no significant effect. However, paxilline significantly attenuated the effect of Tram34, PPF and Ba2+ to control LPSinduced NO release while NS1619 significantly facilitated the effect of Tram34 and PPF. To investigate the major ionic currents expressed in N9 microglia with and without LPS application, we examined whole-cell ionic currents using the patch-clamp technique. Resting N9 cells display a small outward current at positive potentials but a large inwardly rectifying component at negative potentials in physiological potassium gradients. The outward current was dramatically increased by LPS application that was dependent upon the intracellular free calcium concentration. Paxilline or Tram34 was then applied to acutely block this apparent outward KCa current. The result indicated that the LPS triggered KCa current was mainly paxilline sensitive supporting a role for an LPS-induced increase in KCa1.1 channel current. In addition, by using current clamp the mean resting membrane potential of N9 cells was -50.6±6.6mV (N=7) determined in the presence of 1μM [Ca2+]i and -59.4±8.5mV (N=10) with 10nM [Ca2+]i. N9 cells did not display any spontaneous action potentials and the resting membrane potential was not significantly affected by LPS. To conclude, the work presented in this thesis extends the current knowledge regarding potassium channel mRNA expression in microglia and their function in microglial NO release. What is more, it was found that KCa1.1 current expression was increased in LPS-activated N9 cells and revealed KCa1.1 channels as a modulator of NO release by activated microglia.

ABSTRACT The voltage-gated potassium channel Kv1.2 is a key determinant of cellular excitability in the nervous and cardiovascular systems. In the brain, Kv1.2 is strongly expressed in neurons of the hippocampus, a structure essential for learning and memory, and the cerebellum, a structure essential for motor control and cognition. In the vasculature, Kv1.2 is expressed in smooth muscle cells where it contributes to the regulation of blood flow. Dynamic regulation of Kv1.2 is fundamental to its role in these tissues. Disruption of this regulation can manifest in a range of pathological conditions, including seizure, hypertension and neuropathic pain. Thus, elucidating the mechanisms by which Kv1.2 is regulated addresses fundamental aspects of human physiology and disease. Kv1.2 was the first voltage gated ion channel found to be regulated by tyrosine phosphorylation. The ionic current of Kv1.2 is suppressed following tyrosine phosphorylation by a process involving channel endocytosis. Movement of channel away from the plasma membrane involves many proteins associated with the cytoskeleton, including dynamin, cortactin and RhoA. Because trafficking of Kv1.2 away from the cell surface has emerged as the primary mechanism for its negative regulation, we hypothesized that trafficking of the channel to the cell surface could be a mechanism for positive regulation of the Kv1.2 ionic current. Activation of the cAMP/PKA pathway enhances the ionic current of Kv1.2. We hypothesized that a mechanism for this positive regulation is an increase in the amount of channel protein present at the cell surface. Our data show that cAMP can regulate Kv1.2 surface levels by two opposing trafficking pathways, one PKA-dependent and one PKA-independent. Channel homeostasis is preserved by the dynamic balance between these two pathways. Accordingly, any change in the levels of cAMP causes a net increase in the amount of Kv1.2 present at the cell surface. Specific C-terminal phosphorylation sites of Kv1.2 were identified and shown to have a role in maintaining basal surface channel levels. These findings demonstrate channel trafficking as a mechanism for the positive regulation of the Kv1.2 ionic current. In addition to Kv1.2 trafficking at the plasma membrane, movement of the channel from the biosynthetic pathway to the cell surface is another checkpoint for its regulation. Here we show that the protein arginine methyltransferase 8 (PRMT8) is able to promote the ER exit of Kv1.2, resulting in an increase in Kv1.2 surface expression. PRMT8 not only promoted surface expression of the high mannose glycosylated form of Kv1.2, characteristic of immature, ER-localized channels, but also enhanced Kv1.2 total protein levels, most likely by decreasing the amount of channel protein available for ER-associated degradation (ERAD). These findings highlight biosynthetic trafficking of Kv1.2 as a crucial part of its regulation and identify a novel role for PRMT8, as a regulator of biosynthetic protein trafficking.

No description available.

The effects of foliar applied urea and potassium chloride on the severity of leaf diseases of cereals were investigated in the laboratory, glasshouse and field between 1992 and 1995. Field studies with urea gave inconsistent results with respect to severity of Erysiphe graminis and consistently increased the leaf area affected by Septaria tritici. However, potassium chloride applied as a foliar spray consistently decreased the leaf area of wheat affected by E. graminis and S. trifid compared with equivalent applications of soil applied fertiliser. Disease control was achieved at early stem extension and after flag leaf emergence but yield responses were not detected. Laboratory investigations were undertaken to determine the mechanism by which foliar applied potassium chloride reduced the leaf area affected by E. graminis. The timing of application, within seven days pre or post inoculation, had no consistent effect on the efficacy of the fertiliser. Investigations using polyethylene glycol as a control showed that the percentage leaf area affected declined linearly as the osmotic potential of the solutions were increased. Light microscopy revealed that the germination of spores in solution and on treated leaves was reduced as the osmotic potential of the solutions were increased. Spores which did germinate developed normally but those on leaves treated with solutions of high osmotic potential rarely formed haustoria. This suggested a second mechanism acting inside the leaf. Multiple regression analysis of experimental data indicated that the inhibition of spore germination was the major response reducing the area of the leaf affected. Although the data were not conclusive it appeared that the increase in leaf water potential, following the foliar application of potassium chloride, was involved in the control of E. graminis.

Digitized by Kansas Correctional Industries

No description available.

Thesis (Ph. D.)--University of Chicago, Dept. of Neurobiology, Pharmacology and Physiology, August 2003. / Includes bibliographical references. Also available on the Internet.

The objective of this study was to evaluate the effects of non-meat ingredients, blade tenderization and vacuum-tumbling on the textural, visual and sensory characteristics of steaks from Biceps femoris, Supraspinatus, Triceps brachii long head, and Longissimus dorsi muscles packaged in high oxygen, modified atomosphere (MAP) system. United Department of Agriculture (USDA) Select muscles (n=72) from each cut were obtained from a commercial processor over three processing days. Muscles were aged for five days at 4ºC. Denuded muscles within a processing day were randomly assigned to one of 24 treatments. This study was a 2 x 4 x 3 factorial arrangement where treatments were control, injection (injected or non-injected), blade tenderization (0, 1, or 2 passes) and vacuum-tumbling (0, 5, 10 or 20 minutes). Injected muscles contained up to 10% of a brine containing 1.55% potassium lactate, 0.1% sodium diacetate, 0.3% sodium tripolyphosphate blend and 0.4% salt in the final product. Muscles were vacuumtumbled and blade tenderized sequentially after injection. Steaks from the muscles were stored in a high oxygen (80% O2, 20% CO2) MAP system for 0, 3, 7, 10 and 14 days at 2ºC. Steaks were evaluated for package purge (%), Warner-Bratzler shear force (kg), cook loss (%), cook time (min), pH, CIE L* a* b* color space values and trained color panel scores on each storage day. A trained descriptive attribute sensory panel evaluated steaks on day 1 only. Warner-Bratzler shear force (P<0.01) and trained sensory panel results (P<0.05) showed that the addition of non-meat ingredients improved tenderness in all four muscles. Sensory detectable connective tissue was lower (P<0.01) in injected steaks for all muscles except in Biceps femoris steaks. Injected steaks had higher pH (P<0.01) measurements than non-injected treatments in all muscles except the Triceps brachii long head. Neither blade tenderization nor vacuum-tumbling had consistent effects in all four muscles. Vacuum-tumbled Biceps femoris steaks had lower bitter flavor aromatics (P<0.05). In conclusion, enhancing beef with non-meat ingredients had the greatest impact on the quality attributes of high connective tissue cuts and Longissimus dorsi steaks.

Academic dissertation--Department of biology, Faculty of mathematics and natural sciences--Turku--Åbo akademi University, 2007. / Bibliogr. p. 47-59.