Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome

The voltage-dependent K+ channel responsible for the slowly activating delayed K+ current IKs is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because KCNQ1 and KCNE1 genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K+ and Na+ intakes. On a normal K+ diet, homozygous kcne1−/− mice exhibit signs of chronic volume depletion associated with fecal Na+ and K+ wasting and have lower plasma K+ concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K+ diets or stimulated by low Na+ diets, a high K+ diet provokes a tremendous increase of plasma aldosterone levels in kcne1−/− mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K+ in kcne1−/− mice. This exacerbated aldosterone production in kcne1−/− mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where IKs may directly participate in the control of aldosterone production by plasma K+. These results, which show that KCNE1 and IKs are involved in K+ homeostasis, might have important implications for patients with IKs-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.

Glutamate is required to maintain the steady-state potassium pool in Salmonella typhimurium.

In many bacteria, accumulation of K+ at high external osmolalities is accompanied by accumulation of glutamate. To determine whether there is an obligatory relationship between glutamate and K+ pools, we studied mutant strains of Salmonella typhimurium with defects in glutamate synthesis. Enteric bacteria synthesize glutamate by the combined action of glutamine synthetase and glutamate synthase (GS/GOGAT cycle) or the action of biosynthetic glutamate dehydrogenase (GDH). Activity of the GS/GOGAT cycle is required under nitrogen-limiting conditions and is decreased at high external ammonium/ammonia ((NH4)+) concentrations by lowered synthesis of GS and a decrease in its catalytic activity due to covalent modification (adenylylation by GS adenylyltransferase). By contrast, GDH functions efficiently only at high external (NH4)+ concentrations, because it has a low affinity for (NH4)+. When grown at low concentrations of (NH4)+ (< or = 2 mM), mutant strains of S. typhimurium that lack GOGAT and therefore are dependent on GDH have a low glutamate pool and grow slowly; we now demonstrate that they have a low K+ pool. When subjected to a sudden (NH4)+ upshift, strains lacking GS adenylyltransferase drain their glutamate pool into glutamine and grow very slowly; we now find that they also drain their K+ pool. Restoration of the glutamate pool in these strains at late times after shift was accompanied by restoration of the K+ pool and a normal growth rate. Taken together, the results indicate that glutamate is required to maintain the steady-state K+ pool -- apparently no other anion can substitute as a counter-ion for free K+ -- and that K+ glutamate is required for optimal growth.

Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation.

To probe the protein environment of an ion channel, we have perturbed the structure of a transmembrane domain by substituting side chains with those of two different sizes by using site-specific mutagenesis. We have used Trp and Ala as a high- and a low-impact perturbation probe, respectively, to replace each of 18 consecutive residues within the putative second transmembrane segment, M2, of an inwardly rectifying potassium channel, ROMK1. Our rationale is that a change in the channel function as a consequence of these mutations at a particular position will reflect the structural environment of the altered side chain. Each position can then be assigned to one of three classes of environments, as grated by different levels of perturbation: very tolerant (channel functions with both Trp and Ala substitutions), tolerant (function preserved with Ala but not with Trp substitution), and intolerant (either Ala or Trp substitution destroys function). We identify the very tolerant environment as being lipid-facing, tolerant as protein-interior-facing, and intolerant as pore-facing. We observe a strikingly ordered pattern of perturbation of all three environmental classes. This result indicates that M2 is a straight alpha-helix.

Role of potassium orbitals in the metallic behavior of K3picene

Detailed electronic structure calculations of picene clusters doped by potassium modeling the crystalline K3picene structure show that while two electrons are completely transferred from potassium atoms to the lowest-energy unoccupied molecular orbital of pristine picene, the third one remains closely attached to both material components. Multiconfigurational analysis is necessary to show that many structures of almost degenerate total energies compete to define the cluster ground state. Our results prove that the 4s orbital of potassium should be included in any interaction model describing the material. We propose a quarter-filled two-orbital model as the most simple model capable of describing the electronic structure of K-intercalated picene. Precise solutions obtained by a development of the Lanczos method show low-energy electronic excitations involving orbitals located at different positions. Consequently, metallic transport is possible in spite of the clear dominance of interaction over hopping.

Independent roles of calcium and voltage-dependent potassium currents in controlling spike frequency adaptation in lateral amygdala pyramidal neurons

The calcium-dependent afterhyperpolarization (AHP) that follows trains of action potentials is responsible for controlling action potential firing patterns in many neuronal cell types. We have previously shown that the slow AHP contributes to spike frequency adaptation in pyramidal neurons in the rat lateral amygdala. In addition, a dendritic voltage-gated potassium current mediated by Kv1.2-containing channels also suppresses action potential firing in these neurons. In this paper we show that this voltage-gated potassium current and the slow AHP act together to control spike frequency adaptation in lateral amygdala pyramidal neurons. The two currents have similar effects on action potential number when firing is evoked either by depolarizing current injections or by synaptic stimulation. However, they differ in their control of firing frequency, with the voltage-gated potassium current but not the slow AHP determining the initial frequency of action potential firing. This dual mechanism of controlling firing patterns is unique to lateral amygdala neurons and is likely to contribute to the very low levels of firing seen in lateral amygdala neurons in vivo.

Effects of sodium on potassium nutrition in three tropical root crop species

The potassium (K) nutrition and high K requirement of tropical root crops may be affected by their sodium (Na) status, as has been observed in a number of plant species. Solution culture was used to study the effects of K and Na supplies in tannia [Xanthosoma sagittifolium (L.) Schott.], sweetpotato [Ipomoea batatas (L.) Lam.] and taro [Colocasia esculenta (L.) Schott]. At low K supply, Na ameliorated symptoms of K deficiency and increased growth in tannia, and to a lesser extent in sweetpotato, but not in taro. None of the species responded to Na at adequate K supply. Differences in response to Na were attributed to differences in Na translocation to plant tops. At maximum Na supply, the Na concentration in index leaves averaged 1.82% in tannia, 0.205% in sweetpotato, and 0.0067% in taro. An increase in the supply of Na resulted in a shift in the critical K concentration for deficiency (i.e., 90% of maximum yield) in index leaves from 2.9% to 1.2% in tannia, and from 4.8% to 2.5% in sweetpotato. The critical K concentration in taro was 3.3%, irrespective of Na supply. To overcome the problem in tannia and sweetpotato of determining the critical concentration relevant to a leaf sample of unknown K status, a relationship was established for each species relating the critical K concentration to the concentration of Na in the index leaves.

Expression of multilectin receptors and comparative FITC-dextran uptake by human dendritic cells

Dendritic cells (DC) are potent antigen-presenting cells and understanding their mechanisms of antigen uptake is important for loading DC with antigen for immunotherapy. The multilectin receptors, DEC-205 and macrophage mannose receptor (MMR), are potential antigen-uptake receptors; therefore, we examined their expression and FITC-dextran uptake by various human DC preparations. The RT-PCR analysis detected low levels of DEC-205 mRNA in immature blood DC, Langerhans cells (LC) and immature monocyte-derived DC (Mo-DC), Its mRNA expression increased markedly upon activation, indicating that DEC-205 is an activation-associated molecule. In Mo-DC, the expression of cell-surface DEC-205 increased markedly during maturation. In blood DC, however, the cell-surface expression of DEC-205 did not change during activation, suggesting the presence of a large intracellular pool of DEC-205 or post-transcriptional regulation. Immature Mo-DC expressed abundant MMR, but its expression diminished upon maturation. Blood DC and LC did not express detectable levels of the MMR, FITC-dextran uptake by both immature and activated blood DC was 30- to 70-fold less than that of LC, immature Mo-DC and macrophages. In contrast to immature Mo-DC, the FITC-dextran uptake by LC was not inhibited effectively by mannose, an inhibitor for MMR-mediated FITC-dextran uptake. Thus, unlike Mo-DC, blood DC and LC do not use the MMR for carbohydrate-conjugated antigen uptake and alternative receptors may yet be defined on these DC. Therefore, DEC-205 may have a different specificity as an antigen uptake receptor or contribute to an alternative DC function.

Biosynthesis and separation of dextran fructose mixtures in a chromatographic reactor

A literature review of work carried out on batch and continuous chromatographic biochemical reactor-separators has been made. The major part of this work has involved the development of a batch chromatographic reactor-separator for the production of dextran and fructose by the enzymatic action of the enzyme dextransucrase on sucrose. In this reactor, simultaneous reaction and separation occurs thus reducing downstream processing and isolation of products as compared to the existing industrial process. The chromatographic reactor consisted of a glass column packed with a stationary phase consisting of cross linked polysytrene resin in the calcium form. The mobile phase consisted of diluted dextransucrase in deionised water. Initial experiments were carried out on a reactor separtor which had an internal diameter of 0.97cm and length of 1.5m. To study the effect of scale up the reactor diameter was doubled to 1.94cm and length increased to 1.75m. The results have shown that the chromatographic reactor uses more enzyme than a conventional batch reactor for a given conversion of sucrose and that an increase in void volume results in higher conversions of sucrose. A comparison of the molecular weight distribution of dextran produced by the chromatographic reactor was made with that from a conventional batch reactor. The results have shown that the chromatographic reactor produces 30% more dextran of molecular weight greater than 150,000 daltons at 20% w/v sucrose concentration than conventional reactors. This is because some of the fructose molecules are prevented as acting as acceptors in the chromatographic reactor due to their removal from the reaction zone. In the conventional reactor this is not possible and therefore a greater proportion of low molecular weight dextran is produced which does not have much clinical use. A theoretical model was developed to describe the behaviour of the reactor separator and this model was simulated using a computer. The simulation predictions showed good agreement with experimental results at high eluent flowrates and low conversions.

The fractionation of dextran polymer by ultrafiltration to yield clinical products

A review of ultrafiltration (UF) theory and equipment has been made. Dextran is fractionated industrially by ethanol precipitation, which is a high energy intensive process. The aims of this work were to investigate the fractionation of dextran using UF and to compare the efficiency and costs of UF fractionation with ethanol fractionation. This work is the continuation of research conducted at Aston, which was concerned with the fractionation of dextran using gel permeation chromatography (GPC) and hollow fibre UF membranes supplied by Amicon Ltd. Initial laboratory work centred on determining the most efficient make and configuration of membrane. UF membranes of the Millipore cassette configuration, and the DDS flat-sheet configuration, were examined for the fracationation of low molecular weight (MW) dextran. When compared to Amicon membranes, these membranes were found to be inferior. DDS membranes of 25 000 and 50 000 MW cut-offs were shown to be capable of fractionating high MW dextran with the same efficiency as GPC. The Amicon membranes had an efficiency comparable to that of ethanol fractionation. To increase this efficiency a theoretical UF membrane cascade was adopted to utilize favourable characteristics encountered in batch mode membrane experiments. The four stage cascade used recycled permeates in a counter- current direction to retentate flow, and was operated 24 hours per day controlled by a computer. Using 5 000 MW cut-off membranes the cascade improved the batch efficiency by at least 10% for a fractionation at 6 000 MW. Economic comparisons of ethanol fractionation, combined GPC and UF fractionation, and UF fractionation of dextran were undertaken. On an economic basis GPC was the best method for high MW dextran fractionation. When compared with a plant producing 100 tonnes pa of clinical dextran, by ethanol fractionation, a combined GPC and UF cascade fractionation could produce savings on operating costs and an increased dextran yield of 5%.

Uncatalysed and potassium-catalysed pyrolysis of the cell-wall constituents of biomass and their model compounds

Cell-wall components (cellulose, hemicellulose (oat spelt xylan), lignin (Organosolv)), and model compounds (levoglucosan (an intermediate product of cellulose decomposition) and chlorogenic acid (structurally similar to lignin polymer units)) have been investigated to probe in detail the influence of potassium on their pyrolysis behaviours as well as their uncatalysed decomposition reaction. Cellulose and lignin were pretreated to remove salts and metals by hydrochloric acid, and this dematerialized sample was impregnated with 1% of potassium as potassium acetate. Levoglucosan, xylan and chlorogenic acid were mixed with CHCOOK to introduce 1% K. Characterisation was performed using thermogravimetric analysis (TGA) and differential thermal analysis (DTA). In addition to the TGA pyrolysis, pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS) analysis was introduced to examine reaction products. Potassium-catalysed pyrolysis has a huge influence on the char formation stage and increases the char yields considerably (from 7.7% for raw cellulose to 27.7% for potassium impregnated cellulose; from 5.7% for raw levoglucosan to 20.8% for levoglucosan with CHCOOK added). Major changes in the pyrolytic decomposition pathways were observed for cellulose, levoglucosan and chlorogenic acid. The results for cellulose and levoglucosan are consistent with a base catalysed route in the presence of the potassium salt which promotes complete decomposition of glucosidic units by a heterolytic mechanism and favours its direct depolymerization and fragmentation to low molecular weight components (e.g. acetic acid, formic acid, glyoxal, hydroxyacetaldehyde and acetol). Base catalysed polymerization reactions increase the char yield. Potassium-catalysed lignin pyrolysis is very significant: the temperature of maximum conversion in pyrolysis shifts to lower temperature by 70 K and catalysed polymerization reactions increase the char yield from 37% to 51%. A similar trend is observed for the model compound, chlorogenic acid. The addition of potassium does not produce a dramatic change in the tar product distribution, although its addition to chlorogenic acid promoted the generation of cyclohexane and phenol derivatives. Postulated thermal decomposition schemes for chlorogenic acid are presented. © 2008 Elsevier B.V. All rights reserved.

New insights in the deactivation of sulfonic modified SBA-15 catalysts for biodiesel production from low-grade oleaginous feedstock

Arenesulfonic-acid functionalized SBA-15 materials have been used in the production of biodiesel from low grade oleaginous feedstock. These materials display an outstanding catalytic activity, being able to promote the transformation of crude palm oil with methanol into fatty acid methyl esters with high yield (85%) under mild reaction conditions. However, high sensitivity of the catalyst against poisoning by different substances has also been detected. Thus, alkaline metal cations, such as sodium or potassium exert a negative influence on the catalytic activity of these materials, being necessary amounts around 500 ppm of sodium in the reaction media to decrease the catalytic activity of these materials to a half of its initial value in just two reaction runs. The deactivation of arenesulfonic acid functionalized SBA-15 materials seems to occur in this case by ion exchange of the acid protons at the sulfonic groups. Organic unsaponifiable compounds like lecithin or retinol also induce a negative influence in the catalytic activity of these sulfonic acid-based materials, though not so intense as in the case of alkaline metals. The deactivating mechanism associated to the influence of the organic compounds seems to be linked to the adsorption of such substances onto the catalytic acid sites as well as on the silica surface. The accumulation of lecithin in the surface of catalyst, observed by means of thermogravimetric analysis, suggest the creation of a strong interaction, probably by ion pair, between this compound and the sulfonic acid group.

Modified lipids from LDL in the blood during mid-life increase blood brain barrier permeability

Low density lipoprotein levels (LDL) are consistently elevated in cardiovascular disease. It has been suggested that those with high circulating LDL levels in mid-life may be susceptible to develop neurodegenerative diseases in later life. In the circulation, high levels of LDL are associated with increased oxidative modification (oxLDL) and nitration. We have investigated the hypothesis that disruption of blood brain barrier function by oxLDL and their lipids may increase risk of neurodegeneration in later life and that statin intervention in mid-life can mitigate the neurodegenerative effects of hyperlipidaemia. Blood from statin-naïve, normo- and hyperlipidaemic subjects (n=10/group) was collected at baseline. Hyperlipidaemic subjects received statin-intervention whereas normolipidaemic subjects did not prior to a second blood sampling, taken after 3 months. The intervention will be completed in June 2013. Plasma was separated by centrifugation (200g, 30min) and LDL was isolated by potassium bromide density gradient ultracentrifugation. Total homocysteine, LDL cholesterol, 8-isoprostane F2α levels were measured in plasma using commercial kits. LDL were analysed by agarose gel electrophoresis. LDL-lipids were extracted by partitioning in 1:1 chloroform:methanol (v/v) and conjugated to fatty acid free-BSA in serum-free EGM-2 medium (4hrs, 370C) for co-culture with human microvascular endothelial cells (HMVEC). HMVEC were maintained on polycarbonate inserts for two weeks to create a microvascular barrier. Change in barrier permeability was measured by trans-endothelial electrical resistance (TER), FITC-dextran permeability and immunohistochemistry. HMVEC glutathione (GSH) levels were measured after 2 hours by GSH-glo assay. LDL isolated from statin-naïve hyperlipidaemic subjects had higher mobility by agarose gel electrophoresis (Rf;0.53±0.06) and plasma 8-isoprostane F2α (43.5±8.42 pg/ml) compared to control subjects (0.46±0.05 and 24.2±5.37 pg/ml; p<0.05). Compared to HMVEC treatment with the LDL-lipids (5μM) from normolipidaemic subjects, LDL-lipids from hyperlipidaemic subjects increased barrier permeability (103.4±12.5 Ωcm2 v 66.7±7.3 Ωcm2,P<0.01) and decreased GSH (18.5 nmol/mg v 12.3 nmol/mg; untreated cells 26.2±3.6 nmol/mg).

Design of a Low Power – High Temperature Heated Ceramic Sensor to Detect Halogen Gases

The design, construction and optimization of a low power-high temperature heated ceramic sensor to detect leaking of halogen gases in refrigeration systems are presented. The manufacturing process was done with microelectronic assembly and the Low Temperature Cofire Ceramic (LTCC) technique. Four basic sensor materials were fabricated and tested: Li2SiO3, Na2SiO3, K2SiO3, and CaSiO3. The evaluation of the sensor material, sensor size, operating temperature, bias voltage, electrodes size, firing temperature, gas flow, and sensor life was done. All sensors responded to the gas showing stability and reproducibility. Before exposing the sensor to the gas, the sensor was modeled like a resistor in series and the calculations obtained were in agreement with the experimental values. The sensor response to the gas was divided in surface diffusion and bulk diffusion; both were analyzed showing agreement between the calculations and the experimental values. The sensor with 51.5%CaSiO3 + 48.5%Li2SiO3 shows the best results, including a stable current and response to the gas.