Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

The sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

Residential heat pump installations: the role of vocational education and training

Meeting European emissions targets is reliant on innovative renewable technologies, particularly ‘renewable heat’ from heat pumps. Heat pump performance is driven by Carnot efficiency and optimum performance requires the lowest possible space heating flow temperatures leading to greater sensitivity to poor design, installation and operation. Does sufficient training and installer capacity exist for this technology? This paper situates the results of heat pump field trial performance in a socio-technical context, identifying how far installer competence requirements are met within the current vocational education and training (VET) system and considers possible futures. Few UK installers have formal heat pump qualifications at National Vocational Qualification (NVQ) level 3 and heat pump VET is generally through short-course provision where the structure of training is largely unregulated with no strict adherence to a common syllabus or a detailed training centre specification. Prerequisites for short-course trainees, specifically the demand for heating system knowledge based on metric design criteria, is limited and proof of ‘experience’ is an accepted alternative to formal educational qualifications. The lack of broader educational content and deficiencies in engineering knowledge will have profound negative impacts on both the performance and market acceptance of heat pumps. Possible futures to address this problem are identified.

L-Glutamate uptake, decarboxylation to -aminobutyric acid and GABA efflux in isolated Asparagus sprengeri mesophyll cells

Addition of L-glutamate caused alkalinization of the medium surrounding Asparagus spreng.ri mesophyll cells. This suggests a H+/L-glutmate symport uptake system for L-glutamate. However stoichiometries of H+/L-glutamate symport into Asparagus cells were much higher than those in other plant systems. Medium alkalinization may also result from a metabolic decarboxylation process. Since L-glutmate is decarboxylated to r-amino butyric acid (SABA) in this system, the origin of medium alkalinization was reconsidered. Suspensions of mechanically isolated and photosyntheically competent Asparagus sprengeri mesophyll cells were used to investigate the H+/L-glutamate symport system, SABA production, GABA transport, and the origin of L-glutamate dependent medium alkalinization. The major results obtained are summarized as follows: 1. L-Glutamate and GABA were the second or third most abundant amino acids in these cells. Cellular concentrations of L-glutamate were 1.09 mM and 1.31 mM in the light and dark, respectively. Those of SABA were 1.23 mM and 1.17 mM in the light and dark, respectively. 2. Asparagine was the most abundant amino acid in xylem sap and comprised 54 to 68 1. of the amino acid pool on a molar basis. GABA was the second most abundant amino acid and represented 10 to 11 1. of the amino acid pool. L-Slutamate was a minor component. 3. A 10 minute incubation with 1 mM L-glutamate increased the production of GABA in the medium by 2,743 7. and 2,241 7. in the light and dark, respectively. 4. L-Glutamate entered the cells prior to decarboxylation. 5. There was no evidence for a H+/GABA symport process • 6. GABA was produced by loss of carbon-1 of L-glutamate. 7. The specific activity of newly synthesized labeled GABA suggests that it is not equilibrated with a storage pool of GABA. 8. The mechanism of GABA efflux appears to be a passive process. 9. The evidence indicates that the origin of L-glutamate dependent medium alkalinization is a H+/L-glutamate symport not an extracellular decarboxylation. The possible role of GABA production in regulating cytoplasmic pH and L-glutamate levels during rapid electrogenic H+/L-glutamate symport is discussed.

Femtosecond pump-probe study of the spreading and recurrence of a vibrational wave packet in Na_2

The motion of a vibrational wave packet in the bound A(^1 \summe^+_u) electronic state of the sodium dimer is detected in a femtosecond pump/probe molecular beam experiment. For short times harmonic motion is seen in the total ion yield of Na^+_2 as a function of delay time between the two laser pulses. The spreading of the wave packet results in the loss of the periodic variation of the ion signal. For longer delay times (47 ps) the wave packet regains its initial form which is reflected in the revival structure of the Na^+_2 signal. Time-dependent quantum calculations reproduce the measured effects.

Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) A1 from human macrophage foam cells

High circulating levels of triglyceride-rich lipoproteins (TGRL) represent an independent risk factor for coronary artery disease. Here, we show that TGRL inhibit the efflux of cholesterol from 'foam cell' macrophages to lipid-poor apolipoprotein (apo) A1, and may thereby inhibit arterial reverse cholesterol transport and promote the formation of atherosclerotic lesions. Human (THP-1) monocyte-derived macrophages were pre-incubated (48h) with acetylated low-density lipoprotein (AcLDL) to provide a foam cell model of cholesterol efflux to apoA1. Pre-incubation of macrophage 'foam cells' with TGRL (0-200 mug/ml, 0-24 h) inhibited the efflux of exogenously radiolabelled ([H-3]), endogenously synthesised ([C-14]) and cellular cholesterol mass to lipid-poor apoA1, but not control medium, during a (subsequent) efflux period. This inhibition is dependent upon the length of prior exposure to, and concentration of, TGRL employed, but is independent of changes in intracellular triglyceride accumulation or turnover of the cholesteryl ester pool. Despite the negative impact of TGRL on cholesterol efflux, major proteins involved in this process-namely apoE, ABCA1, SR-B1 and caveolin-1-were unaffected by TGRL pre-incubation, suggesting that exposure to these lipoproteins inhibits an alternate, and possibly novel, anti-atherogenic pathway. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Discrete demand side control performance under dynamic building simulation: a heat pump application

This study presents the findings of applying a Discrete Demand Side Control (DDSC) approach to the space heating of two case study buildings. High and low tolerance scenarios are implemented on the space heating controller to assess the impact of DDSC upon buildings with different thermal capacitances, light-weight and heavy-weight construction. Space heating is provided by an electric heat pump powered from a wind turbine, with a back-up electrical network connection in the event of insufficient wind being available when a demand occurs. Findings highlight that thermal comfort is maintained within an acceptable range while the DDSC controller maintains the demand/supply balance. Whilst it is noted that energy demand increases slightly, as this is mostly supplied from the wind turbine, this is of little significance and hence a reduction in operating costs and carbon emissions is still attained.

Interactions between the physical soil environment and a horizontal ground coupled heat pump, for a domestic site in the UK

There is currently an increased interest of Government and Industry in the UK, as well as at the European Community level and International Agencies (i.e. Department of Energy, American International Energy Agency), to improve the performance and uptake of Ground Coupled Heat Pumps (GCHP), in order to meet the 2020 renewable energy target. A sound knowledge base is required to help inform the Government Agencies and advisory bodies; detailed site studies providing reliable data for model verification have an important role to play in this. In this study we summarise the effect of heat extraction by a horizontal ground heat exchanger (installed at 1 m depth) on the soil physical environment (between 0 and 1 m depth) for a site in the south of the UK. Our results show that the slinky influences the surrounding soil by significantly decreasing soil temperatures. Furthermore, soil moisture contents were lower for the GCHP soil profile, most likely due to temperature-gradient related soil moisture migration effects and a decreased hydraulic conductivity, the latter as a result of increased viscosity (caused by the lower temperatures for the GCHP soil profile). The effects also caused considerable differences in soil thermal properties. This is the first detailed mechanistic study conducted in the UK with the aim to understand the interactions between the soil, horizontal heat exchangers and the aboveground environment. An increased understanding of these interactions will help to achieve an optimum and sustainable use of the soil heat resources in the future. The results of this study will help to calibrate and verify a simulation model that will provide UK-wide recommendations to improve future GCHP uptake and performance, while safeguarding the soil physical resources.

Intracellular accumulation of high levels of gamma-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: uncoupling of gamma-aminobutyrate synthesis from efflux in a chemically defined medium

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular gamma-aminobutyrate (GABA(i)). The glutamate is then decarboxylated to GABA(i), a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA(e)) in response to acidification. In addition, high levels of GABA(i) (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA(i) in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA(i). These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.

Simulation of a domestic ground source heat pump system using a three-dimensional numerical borehole heat exchanger model

Common approaches to the simulation of borehole heat exchangers (BHEs) assume heat transfer in circulating fluid and grout to be in a quasi-steady state and ignore fluctuations in fluid temperature due to transport of the fluid around the loop. However, in domestic ground source heat pump (GSHP) systems, the heat pump and circulating pumps switch on and off during a given hour; therefore, the effect of the thermal mass of the circulating fluid and the dynamics of fluid transport through the loop has important implications for system design. This may also be important in commercial systems that are used intermittently. This article presents transient simulation of a domestic GSHP system with a single BHE using a dynamic three-dimensional (3D) numerical BHE model. The results show that delayed response associated with the transit of fluid along the pipe loop is of some significance in moderating swings in temperature during heat pump operation. In addition, when 3D effects are considered, a lower heat transfer rate is predicted during steady operations. These effects could be important when considering heat exchanger design and system control. The results will be used to develop refined two-dimensional models.

Photovoltaic and solar-assisted ground-source heat pump systems

This review investigates the performance of photovoltaic and solar-assisted ground-source heat pumps in which solar heat is transferred to the ground to improve the coefficient of performance. A number of studies indicate that, for systems with adequately sized ground heat exchangers, the effect on system efficiency is small: about 1% improvement if the heat source is photovoltaic, a 1–2% decline if the source is solar thermal. With possible exceptions for systems in which the ground heat exchanger is undersized, or natural recharge from ground water is insufficient, solar thermal energy is better used for domestic hot water than to recharge ground heat. This appears particularly true outside the heating season, as although much of the heat extracted from the ground can be replaced, it seems to have little effect on the coefficient of performance. Any savings in electrical consumption that do result from an improved coefficient can easily be outweighed by an inefficient control system for the circulation pumps.

The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis

The ability of an isogenic set of mutants of Salmonella enterica serovar Typhimurium L354 (SL1344) with defined deletions in genes encoding components of tripartite efflux pumps, including acrB, acrD, acrF and tolC, to colonize chickens was determined in competition with L354. In addition, the ability of L354 and each mutant to adhere to, and invade, human embryonic intestine cells and mouse monocyte macrophages was determined in vitro. The tolC and acrB knockout mutants were hyper-susceptible to a range of antibiotics, dyes and detergents; the tolC mutant was also more susceptible to acid pH and bile and grew more slowly than L354. Complementation of either gene ablated the phenotype. The tolC mutant poorly adhered to both cell types in vitro and was unable to invade macrophages. The acrB mutant adhered, but did not invade macrophages. In vivo, both the acrB mutant and the tolC mutant colonized poorly and did not persist in the avian gut, whereas the acrD and acrF mutant colonized and persisted as well as L354. These data indicate that the AcrAB-TolC system is important for the colonization of chickens by S. Typhimurium and that this system has a role in mediating adherence and uptake into target host cells.

Simulations of a photovoltaic-thermal ground source heat pump system

A ground source heat pump assisted by an array of photovoltaic (PV)-thermal modules was studied in this work. Extracting heat from an array of PV modules should improve the performance of both the PV cells and the heat pump. A series of computer simulations compare the performance of a ground source heat pump with a short ground circuit, used to provide space heating and domestic hot water at a house in southern England. The results indicate that extracting heat from an array of PV-thermal modules would improve the performance of a ground source heat pump with an undersized ground loop. Nevertheless, open air thermal collectors could be more effective, especially during winter. In one model more electricity was saved in ohmic heating than was generated by cooling the PV cells. Cooling the PV modules was found to increase their electrical output up to 4%, but much of the extra electricity was consumed by the cooling pumps.

Subsurface urban heat island and its effects on horizontal ground-source heat pump potential under climate change

Recent urban air temperature increase is attributable to the climate change and heat island effects due to urbanization. This combined effects of urbanization and global warming can penetrate into the underground and elevate the subsurface temperature. In the present study, over-100 years measurements of subsurface temperature at a remote rural site were analysed, and an increasing rate of 0.17⁰C per decade at soil depth of 30cm due to climate change was identified in the UK, but the subsurface warming in an urban site showed a much higher rate of 0.85⁰C per decade at a 30cm depth and 1.18⁰C per decade at 100cm. The subsurface urban heat island (SUHI) intensity obtained at the paired urban-rural stations in London showed an unique 'U-shape', i.e. lowest in summer and highest during winter. The maximum SUHII is 3.5⁰C at 6:00 AM in December, and the minimum UHII is 0.2⁰C at 18:00PM in July. Finally, the effects of SUHI on the energy efficiency of the horizontal ground source heat pump (GSHP) were determined. Provided the same heat pump used, the installation at an urban site will maintain an overall higher COP compared with that at a rural site in all seasons, but the highest COP improvement can be achieved in winter.